Qualification of serological infectious disease assays for the screening of samples from deceased tissue donors.

PubMed

Kitchen, A D; Newham, J A

2011-05-01

Whilst some of the assays used for serological screening of post-mortem blood samples from deceased tissue donors in some countries have been specifically validated by the manufacturer for this purpose, a significant number of those currently in use globally have not. Although specificity has previously been considered a problem in the screening of such samples, we believe that ensuring sensitivity is more important. The aim of this study was to validate a broader range of assays for the screening of post-mortem blood samples from deceased tissue donors. Six microplate immunoassays currently in use within National Health Service Blood and Transplant (NHSBT) for the screening of blood, tissue and stem cell donations were included. Representative samples from confirmed positive donors were titrated in screen negative post-mortem samples in parallel with normal pooled negative serum to determine if there was any inhibition with the post-mortem samples. There were no significant differences seen (P < 0.005) between the dilution curves obtained for the positive samples diluted in post-mortem samples and normal pooled sera. Although small numbers of samples were studied, it can be surmised that the post-mortem blood samples from deceased tissue donors, collected according to United Kingdom guidelines, are a suitable substrate for the assays evaluated. No diminution of reactivity was seen when dilution with sera from deceased donors was compared to dilution using pooled serum from live donors. In the absence of genuine low titre positive post-mortem samples, the use of samples spiked with various levels of target material provides a means of qualifying serological screening assays used by NHSBT for the screening of post-mortem blood samples from deceased tissue donors.

Organic compounds and trace elements in fish tissue and bed sediment from streams in the Yellowstone River basin, Montana and Wyoming, 1998

USGS Publications Warehouse

Peterson, David A.; Boughton, Gregory K.

2000-01-01

A comprehensive water-quality investigation of the Yellowstone River Basin began in 1997, under the National Water-Quality Assessment (NAWQA) Program. Twenty-four sampling sites were selected for sampling of fish tissue and bed sediment during 1998. Organic compounds analyzed included organochlorine insecticides and their metabolites and total polychlorinated biphenyls (PCBs) from fish-tissue and bed-sediment samples, and semivolatile organic compounds from bed-sediment samples. A broad suite of trace elements was analyzed from both fish-tissue and bed-sediment samples, and a special study related to mercury also was conducted. Of the 12 organochlorine insecticides and metabolites detected in the fish-tissue samples, the most compounds per site were detected in samples from integrator sites which represent a mixture of land uses. The presence of DDT, and its metabolites DDD and DDE, in fish collected in the Yellowstone Park area likely reflects long-term residual effects from historical DDT-spraying programs for spruce budworm. Dieldrin, chlordane, and other organic compounds also were detected in the fish-tissue samples. The compound p, p'-DDE was detected at 71 percent of the sampling sites, more than any other compound. The concentrations of total DDT in fish samples were low, however, compared to concentrations from historical data from the study area, other NAWQA studies in the Rocky Mountains, and national baseline concentrations. Only 2 of the 27 organochlorine insecticides and metabolites and total PCBs analyzed in bed sediment were detected. Given that 12 of the compounds were detected in fish-tissue samples, fish appeared to be more sensitive indicators of contamination than bed sediment.Concentrations of some trace elements in fish and bed sediment were higher at sites in mineralized areas than at other sites. Concentrations of selenium in fish tissue from some sites were above background levels. Concentrations of arsenic, chromium, copper, and lead in some of the bed-sediment samples potentially exceeded criteria for the protection of aquatic life.

Variation in glycogen concentrations within mantle and foot tissue in Amblema plicata plicata: Implications for tissue biopsy sampling

USGS Publications Warehouse

Naimo, T.J.; Monroe, E.M.

1999-01-01

With the development of techniques to non-lethally biopsy tissue from unionids, a new method is available to measure changes in biochemical, contaminant, and genetic constituents in this imperiled faunal group. However, before its widespread application, information on the variability of biochemical components within and among tissues needs to be evaluated. We measured glycogen concentrations in foot and mantle tissue in Amblema plicata plicata (Say, 1817) to determine if glycogen was evenly distributed within and between tissues and to determine which tissue might be more responsive to the stress associated with relocating mussels. Glycogen was measured in two groups of mussels: those sampled from their native environment (undisturbed mussels) and quickly frozen for analysis and those relocated into an artificial pond (relocated mussels) for 24 months before analysis. In both undisturbed and relocated mussels, glycogen concentrations were evenly distributed within foot, but not within mantle tissue. In mantle tissue, concentrations of glycogen varied about 2-fold among sections. In addition, glycogen varied significantly between tissues in undisturbed mussels, but not in relocated mussels. Twenty-four months after relocation, glycogen concentrations had declined by 80% in mantle tissue and by 56% in foot tissue relative to the undisturbed mussels. These data indicate that representative biopsy samples can be obtained from foot tissue, but not mantle tissue. We hypothesize that mantle tissue could be more responsive to the stress of relocation due to its high metabolic activity associated with shell formation.

Organic Compounds and Trace Elements in Fish Tissue and Bed Sediment in the Delaware River Basin, New Jersey, Pennsylvania, New York, and Delaware, 1998-2000

USGS Publications Warehouse

Romanok, Kristin M.; Fischer, Jeffrey M.; Riva-Murray, Karen; Brightbill, Robin; Bilger, Michael

2006-01-01

As part of the National Water-Quality Assessment (NAWQA) program activities in the Delaware River Basin (DELR), samples of fish tissue from 21 sites and samples of bed sediment from 35 sites were analyzed for a suite of organic compounds and trace elements. The sampling sites, within subbasins ranging in size from 11 to 600 square miles, were selected to represent 5 main land-use categories in the DELR -forest, low-agricultural, agricultural, urban, and mixed use. Samples of both fish tissue and bed sediment were also collected from 4 'large-river' sites that represented drainage areas ranging from 1,300 to 6,800 square miles, areas in which the land is used for a variety of purposes. One or more of the organochlorine compounds-DDT and chlordane metabolites, polychlorinated biphenyls (total PCBs), and dieldrin- were detected frequently in samples collected over a wide geographic area. One or more of these compounds were detected in fish-tissue samples from 92 percent of the sites and in bed-sediment samples from 82 percent of the sites. Concentrations of total DDT, total chlordanes, total PCBs, and dieldrin in whole white suckers and in bed sediment were significantly related to urban/industrial basin characteristics, such as percentage of urban land use and population density. Semi-volatile organic compounds (SVOCs)-total polycyclic aromatic hydrocarbons (PAHs), total phthalates, and phenols- were detected frequently in bed-sediment samples. All three types of SVOCs were detected in samples from at least one site in each land-use category. The highest detection rates and concentrations typically were in samples from sites in the urban and mixed land-use categories, as well as from the large-river sites. Concentrations of total PAHs and total phthalates in bed-sediment samples were found to be statistically related to percentages of urban land use and to population density in the drainage areas represented by the sampling sites. The samples of fish tissue and bed sediment collected throughout the DELR were analyzed for a large suite of trace elements, but results of the analyses for eight elements-arsenic, cadmium, chromium, copper, lead, nickel, mercury, and zinc- that are considered contaminants of concern are described in this report. One or more of the eight trace elements were detected in samples from every fish tissue and bed-sediment sampling site, and all of the trace elements were detected in samples from 97 percent of the bed-sediment sites. The concentrations of organic compounds and trace elements in the DELR samples were compared to applicable guidelines for the protection of wildlife and other biological organisms. Concentrations of total DDT, total chlordanes, total PCBs, and dieldrin in fish-tissue samples from 14 sites exceeded one or more of the Wildlife Protective Guidelines established by the New York State Department of Environmental Conservation. Concentrations of one or more organic compounds in samples from 16 bed-sediment sites exceeded the Threshold Effects Concentrations (TEC) of the Canadian Sediment Quality Guidelines, and concentrations of one or more of the eight trace elements in samples from 38 bed-sediment sites exceeded the TEC. (The TEC is the concentration below which adverse biological effects in freshwater ecosystems are expected to be rare.) Concentrations of organic compounds in samples from some bed-sediment sites exceeded the Canadian Probable Effects Concentrations (PEC), and concentrations of trace elements in samples from 18 sites exceeded the PEC. (The PEC is the concentration above which adverse effects to biological organisms are expected to occur frequently). Concentrations of organic compounds and trace elements in samples from the DELR were compared to similar data from other NAWQA study units in the northeastern United States and also data from the Mobile River (Alabama) Basin and the Northern Rockies Intermontane Basin study units. Median concentrations of to

Towards High-Resolution Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry Coupled to Shear Force Microscopy

NASA Astrophysics Data System (ADS)

Nguyen, Son N.; Sontag, Ryan L.; Carson, James P.; Corley, Richard A.; Ansong, Charles; Laskin, Julia

2018-02-01

Constant mode ambient mass spectrometry imaging (MSI) of tissue sections with high lateral resolution of better than 10 μm was performed by combining shear force microscopy with nanospray desorption electrospray ionization (nano-DESI). Shear force microscopy enabled precise control of the distance between the sample and nano-DESI probe during MSI experiments and provided information on sample topography. Proof-of-concept experiments were performed using lung and brain tissue sections representing spongy and dense tissues, respectively. Topography images obtained using shear force microscopy were comparable to the results obtained using contact profilometry over the same region of the tissue section. Variations in tissue height were found to be dependent on the tissue type and were in the range of 0-5 μm for lung tissue and 0-3 μm for brain tissue sections. Ion images of phospholipids obtained in this study are in good agreement with literature data. Normalization of nano-DESI MSI images to the signal of the internal standard added to the extraction solvent allowed us to construct high-resolution ion images free of matrix effects.

Technical note: Alternatives to reduce adipose tissue sampling bias.

PubMed

Cruz, G D; Wang, Y; Fadel, J G

2014-10-01

Understanding the mechanisms by which nutritional and pharmaceutical factors can manipulate adipose tissue growth and development in production animals has direct and indirect effects in the profitability of an enterprise. Adipocyte cellularity (number and size) is a key biological response that is commonly measured in animal science research. The variability and sampling of adipocyte cellularity within a muscle has been addressed in previous studies, but no attempt to critically investigate these issues has been proposed in the literature. The present study evaluated 2 sampling techniques (random and systematic) in an attempt to minimize sampling bias and to determine the minimum number of samples from 1 to 15 needed to represent the overall adipose tissue in the muscle. Both sampling procedures were applied on adipose tissue samples dissected from 30 longissimus muscles from cattle finished either on grass or grain. Briefly, adipose tissue samples were fixed with osmium tetroxide, and size and number of adipocytes were determined by a Coulter Counter. These results were then fit in a finite mixture model to obtain distribution parameters of each sample. To evaluate the benefits of increasing number of samples and the advantage of the new sampling technique, the concept of acceptance ratio was used; simply stated, the higher the acceptance ratio, the better the representation of the overall population. As expected, a great improvement on the estimation of the overall adipocyte cellularity parameters was observed using both sampling techniques when sample size number increased from 1 to 15 samples, considering both techniques' acceptance ratio increased from approximately 3 to 25%. When comparing sampling techniques, the systematic procedure slightly improved parameters estimation. The results suggest that more detailed research using other sampling techniques may provide better estimates for minimum sampling.

Systematic bias in genomic classification due to contaminating non-neoplastic tissue in breast tumor samples.

PubMed

Elloumi, Fathi; Hu, Zhiyuan; Li, Yan; Parker, Joel S; Gulley, Margaret L; Amos, Keith D; Troester, Melissa A

2011-06-30

Genomic tests are available to predict breast cancer recurrence and to guide clinical decision making. These predictors provide recurrence risk scores along with a measure of uncertainty, usually a confidence interval. The confidence interval conveys random error and not systematic bias. Standard tumor sampling methods make this problematic, as it is common to have a substantial proportion (typically 30-50%) of a tumor sample comprised of histologically benign tissue. This "normal" tissue could represent a source of non-random error or systematic bias in genomic classification. To assess the performance characteristics of genomic classification to systematic error from normal contamination, we collected 55 tumor samples and paired tumor-adjacent normal tissue. Using genomic signatures from the tumor and paired normal, we evaluated how increasing normal contamination altered recurrence risk scores for various genomic predictors. Simulations of normal tissue contamination caused misclassification of tumors in all predictors evaluated, but different breast cancer predictors showed different types of vulnerability to normal tissue bias. While two predictors had unpredictable direction of bias (either higher or lower risk of relapse resulted from normal contamination), one signature showed predictable direction of normal tissue effects. Due to this predictable direction of effect, this signature (the PAM50) was adjusted for normal tissue contamination and these corrections improved sensitivity and negative predictive value. For all three assays quality control standards and/or appropriate bias adjustment strategies can be used to improve assay reliability. Normal tissue sampled concurrently with tumor is an important source of bias in breast genomic predictors. All genomic predictors show some sensitivity to normal tissue contamination and ideal strategies for mitigating this bias vary depending upon the particular genes and computational methods used in the predictor.

Detection of EWS/FLI-1 fusion in non-Ewing soft tissue tumors.

PubMed

Trancău, I O; Huică, R; Surcel, M; Munteanu, A; Ursaciuc, C

2015-01-01

EWS/FLI-1 fusion mainly appears in Ewing's sarcoma or the primitive neuroectodermal tumors and represents a genomic marker for these tumors. However, it can appear with lower frequency in other soft tissue tumors. The paper investigates the presence of EWS/FLI-1 fusion in clinically diagnosed sarcoma belonging to different non-Ewing connective tissue tumors in order to search for a possible new biomarker valuable for investigators. 20 patients with soft tissue tumors, who underwent surgery, were tested. Intra-operative samples of normal and tumor tissue were collected for histopathological diagnosis and genetics determinations. The patients' RNA from tumor and normal peritumoral tissue was extracted and EWS/FLI-1 fusion screened by quantitative real-time PCR. The relative expression of the fusion in the tumor sample was compared to the similar expression in normal tissue. The amplification in the threshold zone was shown by 5 samples (25%): 2 clear cell sarcoma, 1 fibrosarcoma, 1 malignant tumor of nerve sheath, 1 metastatic adenocarcinoma. We differentiated between the unspecific amplification and concluded that these are weak positive results. Genomic investigation may establish the tumor malignancy and its possible affiliation earlier than histopathology. It can support the screening of EWS/FLI-1 fusion in a larger variety of clinically diagnosed soft tissue tumors.

Determination of selected neurotoxic insecticides in small amounts of animal tissue utilizing a newly constructed mini-extractor.

PubMed

Seifertová, Marta; Čechová, Eliška; Llansola, Marta; Felipo, Vicente; Vykoukalová, Martina; Kočan, Anton

2017-10-01

We developed a simple analytical method for the simultaneous determination of representatives of various groups of neurotoxic insecticides (carbaryl, chlorpyrifos, cypermethrin, and α-endosulfan and β-endosulfan and their metabolite endosulfan sulfate) in limited amounts of animal tissues containing different amounts of lipids. Selected tissues (rodent fat, liver, and brain) were extracted in a special in-house-designed mini-extractor constructed on the basis of the Soxhlet and Twisselmann extractors. A dried tissue sample placed in a small cartridge was extracted, while the nascent extract was simultaneously filtered through a layer of sodium sulfate. The extraction was followed by combined clean-up, including gel permeation chromatography (in case of high lipid content), ultrasonication, and solid-phase extraction chromatography using C 18 on silica and aluminum oxide. Gas chromatography coupled with high-resolution mass spectrometry was used for analyte separation, detection, and quantification. Average recoveries for individual insecticides ranged from 82 to 111%. Expanded measurement uncertainties were generally lower than 35%. The developed method was successfully applied to rat tissue samples obtained from an animal model dealing with insecticide exposure during brain development. This method may also be applied to the analytical treatment of small amounts of various types of animal and human tissue samples. A significant advantage achieved using this method is high sample throughput due to the simultaneous treatment of many samples. Graphical abstract Optimized workflow for the determination of selected insecticides in small amounts of animal tissue including newly developed mini-extractor.

Elemental concentration analysis in prostate tissues using total reflection X-ray fluorescence

NASA Astrophysics Data System (ADS)

Leitão, R. G.; Palumbo, A.; Souza, P. A. V. R.; Pereira, G. R.; Canellas, C. G. L.; Anjos, M. J.; Nasciutti, L. E.; Lopes, R. T.

2014-02-01

Prostate cancer (PCa) currently represents the second most prevalent malignant neoplasia in men, representing 21% of all cancer cases. Benign Prostate Hyperplasia (BPH) is an illness prevailing in men above the age of 50, close to 90% after the age of 80. The prostate presents a high zinc concentration, about 10-fold higher than any other body tissue. In this work, samples of human prostate tissues with cancer, BPH and normal tissue were analyzed utilizing total reflection X-ray fluorescence spectroscopy using synchrotron radiation technique (SR-TXRF) to investigate the differences in the elemental concentrations in these tissues. SR-TXRF analyses were performed at the X-ray fluorescence beamline at Brazilian National Synchrotron Light Laboratory (LNLS), in Campinas, São Paulo. It was possible to determine the concentrations of the following elements: P, S, K, Ca, Fe, Cu, Zn and Rb. By using Mann-Whitney U test it was observed that almost all elements presented concentrations with significant differences (α=0.05) between the groups studied.

Integrative analysis for identification of shared markers from various functional cells/tissues for rheumatoid arthritis.

PubMed

Xia, Wei; Wu, Jian; Deng, Fei-Yan; Wu, Long-Fei; Zhang, Yong-Hong; Guo, Yu-Fan; Lei, Shu-Feng

2017-02-01

Rheumatoid arthritis (RA) is a systemic autoimmune disease. So far, it is unclear whether there exist common RA-related genes shared in different tissues/cells. In this study, we conducted an integrative analysis on multiple datasets to identify potential shared genes that are significant in multiple tissues/cells for RA. Seven microarray gene expression datasets representing various RA-related tissues/cells were downloaded from the Gene Expression Omnibus (GEO). Statistical analyses, testing both marginal and joint effects, were conducted to identify significant genes shared in various samples. Followed-up analyses were conducted on functional annotation clustering analysis, protein-protein interaction (PPI) analysis, gene-based association analysis, and ELISA validation analysis in in-house samples. We identified 18 shared significant genes, which were mainly involved in the immune response and chemokine signaling pathway. Among the 18 genes, eight genes (PPBP, PF4, HLA-F, S100A8, RNASEH2A, P2RY6, JAG2, and PCBP1) interact with known RA genes. Two genes (HLA-F and PCBP1) are significant in gene-based association analysis (P = 1.03E-31, P = 1.30E-2, respectively). Additionally, PCBP1 also showed differential protein expression levels in in-house case-control plasma samples (P = 2.60E-2). This study represented the first effort to identify shared RA markers from different functional cells or tissues. The results suggested that one of the shared genes, i.e., PCBP1, is a promising biomarker for RA.

Spatial mapping of metals in tissue-sections using combination of mass-spectrometry and histology through image registration

NASA Astrophysics Data System (ADS)

Anyz, Jiri; Vyslouzilova, Lenka; Vaculovic, Tomas; Tvrdonova, Michaela; Kanicky, Viktor; Haase, Hajo; Horak, Vratislav; Stepankova, Olga; Heger, Zbynek; Adam, Vojtech

2017-01-01

We describe a new procedure for the parallel mapping of selected metals in histologically characterized tissue samples. Mapping is achieved via image registration of digital data obtained from two neighbouring cryosections by scanning the first as a histological sample and subjecting the second to laser ablation inductively coupled plasma mass spectrometry. This computer supported procedure enables determination of the distribution and content of metals of interest directly in the chosen histological zones and represents a substantial improvement over the standard approach, which determines these values in tissue homogenates or whole tissue sections. The potential of the described procedure was demonstrated in a pilot study that analysed Zn and Cu levels in successive development stages of pig melanoma tissue using MeLiM (Melanoma-bearing-Libechov-Minipig) model. We anticipate that the procedure could be useful for a complex understanding of the role that the spatial distribution of metals plays within tissues affected by pathological states including cancer.

Spatial mapping of metals in tissue-sections using combination of mass-spectrometry and histology through image registration

PubMed Central

Anyz, Jiri; Vyslouzilova, Lenka; Vaculovic, Tomas; Tvrdonova, Michaela; Kanicky, Viktor; Haase, Hajo; Horak, Vratislav; Stepankova, Olga; Heger, Zbynek; Adam, Vojtech

2017-01-01

We describe a new procedure for the parallel mapping of selected metals in histologically characterized tissue samples. Mapping is achieved via image registration of digital data obtained from two neighbouring cryosections by scanning the first as a histological sample and subjecting the second to laser ablation inductively coupled plasma mass spectrometry. This computer supported procedure enables determination of the distribution and content of metals of interest directly in the chosen histological zones and represents a substantial improvement over the standard approach, which determines these values in tissue homogenates or whole tissue sections. The potential of the described procedure was demonstrated in a pilot study that analysed Zn and Cu levels in successive development stages of pig melanoma tissue using MeLiM (Melanoma-bearing-Libechov-Minipig) model. We anticipate that the procedure could be useful for a complex understanding of the role that the spatial distribution of metals plays within tissues affected by pathological states including cancer. PMID:28071735

Cancer Liquid Biopsy: Is It Ready for Clinic?

PubMed

Pan, Ying; Ji, John S; Jin, Jason Gang; Kuo, Winston Patrick; Kang, Hongjun

2017-01-01

The management of cancer relies on a combination of imaging and tissue biopsy for diagnosis, monitoring, and molecular classification-based patient stratification to ensure appropriate treatment. Conventional tissue biopsy harvests tumor samples with invasive procedures, which are often difficult for patients with advanced disease. Given the well-recognized intratumor genetic heterogeneity [1], the biopsy of small tumor fragments does not necessarily represent all the genetic aberrations in the tumor, but sampling the entire tumor in each patient is not realistic. Moreover, tumors evolve all the time from local to advanced disease and by adapting to selective pressure from treatment.

Electromechanical Coupling Factor of Breast Tissue as a Biomarker for Breast Cancer.

PubMed

Park, Kihan; Chen, Wenjin; Chekmareva, Marina A; Foran, David J; Desai, Jaydev P

2018-01-01

This research aims to validate a new biomarker of breast cancer by introducing electromechanical coupling factor of breast tissue samples as a possible additional indicator of breast cancer. Since collagen fibril exhibits a structural organization that gives rise to a piezoelectric effect, the difference in collagen density between normal and cancerous tissue can be captured by identifying the corresponding electromechanical coupling factor. The design of a portable diagnostic tool and a microelectromechanical systems (MEMS)-based biochip, which is integrated with a piezoresistive sensing layer for measuring the reaction force as well as a microheater for temperature control, is introduced. To verify that electromechanical coupling factor can be used as a biomarker for breast cancer, the piezoelectric model for breast tissue is described with preliminary experimental results on five sets of normal and invasive ductal carcinoma (IDC) samples in the 25-45 temperature range. While the stiffness of breast tissues can be captured as a representative mechanical signature which allows one to discriminate among tissue types especially in the higher strain region, the electromechanical coupling factor shows more distinct differences between the normal and IDC groups over the entire strain region than the mechanical signature. From the two-sample -test, the electromechanical coupling factor under compression shows statistically significant differences ( 0.0039) between the two groups. The increase in collagen density in breast tissue is an objective and reproducible characteristic of breast cancer. Although characterization of mechanical tissue property has been shown to be useful for differentiating cancerous tissue from normal tissue, using a single parameter may not be sufficient for practical usage due to inherent variation among biological samples. The portable breast cancer diagnostic tool reported in this manuscript shows the feasibility of measuring multiple parameters of breast tissue allowing for practical application.

Bacterial assemblages differ between compartments within the coral holobiont

NASA Astrophysics Data System (ADS)

Sweet, M. J.; Croquer, A.; Bythell, J. C.

2011-03-01

It is widely accepted that corals are associated with a diverse and host species-specific microbiota, but how they are organized within their hosts remains poorly understood. Previous sampling techniques (blasted coral tissues, coral swabs and milked mucus) may preferentially sample from different compartments such as mucus, tissue and skeleton, or amalgamate them, making comparisons and generalizations between studies difficult. This study characterized bacterial communities of corals with minimal mechanical disruption and contamination from water, air and sediments from three compartments: surface mucus layer (SML), coral tissue and coral skeleton. A novel apparatus (the `snot sucker') was used to separate the SML from tissues and skeleton, and these three compartments were compared to swab samples and milked mucus along with adjacent environmental samples (water column and sediments). Bacterial 16S rRNA gene diversity was significantly different between the various coral compartments and environmental samples (PERMANOVA, F = 6.9, df = 8, P = 0.001), the only exceptions being the complete crushed coral samples and the coral skeleton, which were similar, because the skeleton represents a proportionally large volume and supports a relatively rich microflora. Milked mucus differed significantly from the SML collected with the `snot sucker' and was contaminated with zooxanthellae, suggesting that it may originate at least partially from the gastrovascular cavity rather than the tissue surface. A common method of sampling the SML, surface swabs, produced a bacterial community profile distinct from the SML sampled using our novel apparatus and also showed contamination from coral tissues. Our results indicate that microbial communities are spatially structured within the coral holobiont, and methods used to describe these need to be standardized to allow comparisons between studies.

Hematopoietic Lineage Transcriptome Stability and Representation in PAXgene™ Collected Peripheral Blood Utilising SPIA Single-Stranded cDNA Probes for Microarray

PubMed Central

Kennedy, Laura; Vass, J. Keith; Haggart, D. Ross; Moore, Steve; Burczynski, Michael E.; Crowther, Dan; Miele, Gino

2008-01-01

Peripheral blood as a surrogate tissue for transcriptome profiling holds great promise for the discovery of diagnostic and prognostic disease biomarkers, particularly when target tissues of disease are not readily available. To maximize the reliability of gene expression data generated from clinical blood samples, both the sample collection and the microarray probe generation methods should be optimized to provide stabilized, reproducible and representative gene expression profiles faithfully representing the transcriptional profiles of the constituent blood cell types present in the circulation. Given the increasing innovation in this field in recent years, we investigated a combination of methodological advances in both RNA stabilisation and microarray probe generation with the goal of achieving robust, reliable and representative transcriptional profiles from whole blood. To assess the whole blood profiles, the transcriptomes of purified blood cell types were measured and compared with the global transcriptomes measured in whole blood. The results demonstrate that a combination of PAXgene™ RNA stabilising technology and single-stranded cDNA probe generation afforded by the NuGEN Ovation RNA amplification system V2™ enables an approach that yields faithful representation of specific hematopoietic cell lineage transcriptomes in whole blood without the necessity for prior sample fractionation, cell enrichment or globin reduction. Storage stability assessments of the PAXgene™ blood samples also advocate a short, fixed room temperature storage time for all PAXgene™ blood samples collected for the purposes of global transcriptional profiling in clinical studies. PMID:19578521

Expression profiling of microRNAs in human bone tissue from postmenopausal women.

PubMed

De-Ugarte, Laura; Serra-Vinardell, Jenny; Nonell, Lara; Balcells, Susana; Arnal, Magdalena; Nogues, Xavier; Mellibovsky, Leonardo; Grinberg, Daniel; Diez-Perez, Adolfo; Garcia-Giralt, Natalia

2018-01-01

Bone tissue is composed of several cell types, which express their own microRNAs (miRNAs) that will play a role in cell function. The set of total miRNAs expressed in all cell types configures the specific signature of the bone tissue in one physiological condition. The aim of this study was to explore the miRNA expression profile of bone tissue from postmenopausal women. Tissue was obtained from trabecular bone and was analyzed in fresh conditions (n = 6). Primary osteoblasts were also obtained from trabecular bone (n = 4) and human osteoclasts were obtained from monocyte precursors after in vitro differentiation (n = 5). MicroRNA expression profiling was obtained for each sample by microarray and a global miRNA analysis was performed combining the data acquired in all the microarray experiments. From the 641 miRNAs detected in bone tissue samples, 346 (54%) were present in osteoblasts and/or osteoclasts. The other 46% were not identified in any of the bone cells analyzed. Intersection of osteoblast and osteoclast arrays identified 101 miRNAs shared by both cell types, which accounts for 30-40% of miRNAs detected in these cells. In osteoblasts, 266 miRNAs were detected, of which 243 (91%) were also present in the total bone array, representing 38% of all bone miRNAs. In osteoclasts, 340 miRNAs were detected, of which 196 (58%) were also present in the bone tissue array, representing 31% of all miRNAs detected in total bone. These analyses provide an overview of miRNAs expressed in bone tissue, broadening our knowledge in the microRNA field.

Towards High-Resolution Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry Coupled to Shear Force Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, Son N.; Sontag, Ryan L.; Carson, James P.

Constant mode ambient mass spectrometry imaging (MSI) of tissue sections with high lateral resolution of better than 10 µm was performed by combining shear force microscopy with nanospray desorption electrospray ionization (nano-DESI). Shear force microscopy enabled precise control of the distance between the sample and nano-DESI probe during MSI experiments and provided information on sample topography. Proof-of-concept experiments were performed using lung and brain tissue sections representing spongy and dense tissues, respectively. Topography images obtained using shear force microscopy were comparable to the results obtained using contact profilometry over the same region of the tissue section. Variations in tissue heightmore » were found to be dependent on the tissue type and were in the range of 0-5 µm for lung tissue and 0-3 µm for brain tissue sections. Ion images of phospholipids obtained in this study are in good agreement with literature data. Normalization of nano-DESI MSI images to the signal of the internal standard added to the extraction solvent allowed us to construct high-resolution ion images free of matrix effects.« less

Telomere length in normal and neoplastic canine tissues.

PubMed

Cadile, Casey D; Kitchell, Barbara E; Newman, Rebecca G; Biller, Barbara J; Hetler, Elizabeth R

2007-12-01

To determine the mean telomere restriction fragment (TRF) length in normal and neoplastic canine tissues. 57 solid-tissue tumor specimens collected from client-owned dogs, 40 samples of normal tissue collected from 12 clinically normal dogs, and blood samples collected from 4 healthy blood donor dogs. Tumor specimens were collected from client-owned dogs during diagnostic or therapeutic procedures at the University of Illinois Veterinary Medical Teaching Hospital, whereas 40 normal tissue samples were collected from 12 control dogs. Telomere restriction fragment length was determined by use of an assay kit. A histologic diagnosis was provided for each tumor by personnel at the Veterinary Diagnostic Laboratory at the University of Illinois. Mean of the mean TRF length for 44 normal samples was 19.0 kilobases (kb; range, 15.4 to 21.4 kb), and the mean of the mean TRF length for 57 malignant tumors was 19.0 kb (range, 12.9 to 23.5 kb). Although the mean of the mean TRF length for tumors and normal tissues was identical, tumor samples had more variability in TRF length. Telomerase, which represents the main mechanism by which cancer cells achieve immortality, is an attractive therapeutic target. The ability to measure telomere length is crucial to monitoring the efficacy of telomerase inhibition. In contrast to many other mammalian species, the length of canine telomeres and the rate of telomeric DNA loss are similar to those reported in humans, making dogs a compelling choice for use in the study of human anti-telomerase strategies.

Experimental verification of stopping-power prediction from single- and dual-energy computed tomography in biological tissues

NASA Astrophysics Data System (ADS)

Möhler, Christian; Russ, Tom; Wohlfahrt, Patrick; Elter, Alina; Runz, Armin; Richter, Christian; Greilich, Steffen

2018-01-01

An experimental setup for consecutive measurement of ion and x-ray absorption in tissue or other materials is introduced. With this setup using a 3D-printed sample container, the reference stopping-power ratio (SPR) of materials can be measured with an uncertainty of below 0.1%. A total of 65 porcine and bovine tissue samples were prepared for measurement, comprising five samples each of 13 tissue types representing about 80% of the total body mass (three different muscle and fatty tissues, liver, kidney, brain, heart, blood, lung and bone). Using a standard stoichiometric calibration for single-energy CT (SECT) as well as a state-of-the-art dual-energy CT (DECT) approach, SPR was predicted for all tissues and then compared to the measured reference. With the SECT approach, the SPRs of all tissues were predicted with a mean error of (-0.84  ±  0.12)% and a mean absolute error of (1.27  ±  0.12)%. In contrast, the DECT-based SPR predictions were overall consistent with the measured reference with a mean error of (-0.02  ±  0.15)% and a mean absolute error of (0.10  ±  0.15)%. Thus, in this study, the potential of DECT to decrease range uncertainty could be confirmed in biological tissue.

Cellular and Molecular Changes in Orthodontic Tooth Movement

PubMed Central

Zainal Ariffin, Shahrul Hisham; Yamamoto, Zulham; Zainol Abidin, lntan Zarina; Megat Abdul Wahab, Rohaya; Zainal Ariffin, Zaidah

2011-01-01

Tooth movement induced by orthodontic treatment can cause sequential reactions involving the periodontal tissue and alveolar bone, resulting in the release of numerous substances from the dental tissues and surrounding structures. To better understand the biological processes involved in orthodontic treatment, improve treatment, and reduce adverse side effects, several of these substances have been proposed as biomarkers. Potential biological markers can be collected from different tissue samples, and suitable sampling is important to accurately reflect biological processes. This paper covers the tissue changes that are involved during orthodontic tooth movement such as at compression region (involving osteoblasts), tension region (involving osteoclasts), dental root, and pulp tissues. Besides, the involvement of stem cells and their development towards osteoblasts and osteoclasts during orthodontic treatment have also been explained. Several possible biomarkers representing these biological changes during specific phenomenon, that is, bone remodelling (formation and resorption), inflammation, and root resorption have also been proposed. The knowledge of these biomarkers could be used in accelerating orthodontic treatment. PMID:22125437

Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions

PubMed Central

Weber, Daniela; Davies, Michael J.; Grune, Tilman

2015-01-01

Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples. PMID:26141921

Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions.

PubMed

Weber, Daniela; Davies, Michael J; Grune, Tilman

2015-08-01

Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples. © 2015 Published by Elsevier Ltd.

Inhibitory effects of blockage of intermediate conductance Ca(2+)-activated K (+) channels on proliferation of hepatocellular carcinoma cells.

PubMed

Yang, Xiao-wei; Liu, Jin-wen; Zhang, Ru-chao; Yin, Qian; Shen, Wen-zhuang; Yi, Ji-lin

2013-02-01

The roles of intermediate conductance Ca(2+)-activated K(+) channel (IKCa1) in the pathogenesis of hepatocellular carcinoma (HCC) were investigated. Immunohistochemistry and Western blotting were used to detect the expression of IKCa1 protein in 50 HCC and 20 para-carcinoma tissue samples. Real-time PCR was used to detect the transcription level of IKCa1 mRNA in 13 HCC and 11 para-carcinoma tissue samples. The MTT assay was used to measure the function of IKCa1 in human HCC cell line HepG2 in vitro. TRAM-34, a specific blocker of IKCa1, was used to intervene with the function of IKCa1. As compared with para-carcinoma tissue, an over-expression of IKCa1 protein was detected in HCC tissue samples (P<0.05). The mRNA expression level of IKCa1 in HCC tissues was 2.17 times higher than that in para-carcinoma tissues. The proliferation of HepG2 cells was suppressed by TRAM-34 (0.5, 1.0, 2.0 and 4.0 μmol/L) in vitro (P<0.05). Our results suggested that IKCa1 may play a role in the proliferation of human HCC, and IKCa1 blockers may represent a potential therapeutic strategy for HCC.

Assessment of bioburden on human and animal tissues: part 2--results of testing of human tissue and qualification of a composite sample for routine bioburden determination.

PubMed

Kowalski, John B; Merritt, Karen; Gocke, David; Osborne, Joel

2012-08-01

A quantitative method was developed and validated to assess bioburden on tissue from human donors and to compare bioburden determination results to swab culture results from the same donor. An initial study with allograft tissue from 101 donors showed a wide range of bioburden levels; values from no colony-forming units (CFU) detected to >28,000 CFU were observed. Tissues from donors that had swab cultures negative for objectionable microorganisms generally had lower bioburden than tissues from donors where objectionable microorganisms were recovered by swab culturing. In a follow-up study with 1,445 donors, a wide range of bioburden levels was again observed on tissues from donors that were swab culture negative for objectionable microorganisms. Tissues from 885 (61%) of these donors had no recoverable bioburden (<2 CFU). Importantly, tissues from 560 (39%) of the donors had recoverable bioburden which ranged from 1 to >24,000 CFU. Identification of bioburden isolates showed a diversity of genera and species. In compliance with the recent revision of the American Association of Tissue Banks K2.210 Standard, the quantitative bioburden determination method was validated with a composite tissue sample that contains bone and soft tissue sections tested together in one extraction vessel. A recovery efficiency of 68% was validated and the composite sample was shown to be representative of all of the tissues recovered from a donor. The use of the composite sample in conjunction with the quantitative bioburden determination method will facilitate an accurate assessment of the numbers and types of contaminating microorganisms on allografts prior to disinfection/sterilization. This information will ensure that disinfection/sterilization processes are properly validated and the capability of the overall allograft process is understood on a donor by donor basis.

The use of FDTD in establishing in vitro experimentation conditions representative of lifelike cell phone radiation on the spermatozoa.

PubMed

Mouradi, Rand; Desai, Nisarg; Erdemir, Ahmet; Agarwal, Ashok

2012-01-01

Recent studies have shown that exposing human semen samples to cell phone radiation leads to a significant decline in sperm parameters. In daily living, a cell phone is usually kept in proximity to the groin, such as in a trouser pocket, separated from the testes by multiple layers of tissue. The aim of this study was to calculate the distance between cell phone and semen sample to set up an in vitro experiment that can mimic real life conditions (cell phone in trouser pocket separated by multiple tissue layers). For this reason, a computational model of scrotal tissues was designed by considering these separating layers, the results of which were used in a series of simulations using the Finite Difference Time Domain (FDTD) method. To provide an equivalent effect of multiple tissue layers, these results showed that the distance between a cell phone and semen sample should be 0.8 cm to 1.8 cm greater than the anticipated distance between a cell phone and the testes.

Lipidomic analysis of biological samples: Comparison of liquid chromatography, supercritical fluid chromatography and direct infusion mass spectrometry methods.

PubMed

Lísa, Miroslav; Cífková, Eva; Khalikova, Maria; Ovčačíková, Magdaléna; Holčapek, Michal

2017-11-24

Lipidomic analysis of biological samples in a clinical research represents challenging task for analytical methods given by the large number of samples and their extreme complexity. In this work, we compare direct infusion (DI) and chromatography - mass spectrometry (MS) lipidomic approaches represented by three analytical methods in terms of comprehensiveness, sample throughput, and validation results for the lipidomic analysis of biological samples represented by tumor tissue, surrounding normal tissue, plasma, and erythrocytes of kidney cancer patients. Methods are compared in one laboratory using the identical analytical protocol to ensure comparable conditions. Ultrahigh-performance liquid chromatography/MS (UHPLC/MS) method in hydrophilic interaction liquid chromatography mode and DI-MS method are used for this comparison as the most widely used methods for the lipidomic analysis together with ultrahigh-performance supercritical fluid chromatography/MS (UHPSFC/MS) method showing promising results in metabolomics analyses. The nontargeted analysis of pooled samples is performed using all tested methods and 610 lipid species within 23 lipid classes are identified. DI method provides the most comprehensive results due to identification of some polar lipid classes, which are not identified by UHPLC and UHPSFC methods. On the other hand, UHPSFC method provides an excellent sensitivity for less polar lipid classes and the highest sample throughput within 10min method time. The sample consumption of DI method is 125 times higher than for other methods, while only 40μL of organic solvent is used for one sample analysis compared to 3.5mL and 4.9mL in case of UHPLC and UHPSFC methods, respectively. Methods are validated for the quantitative lipidomic analysis of plasma samples with one internal standard for each lipid class. Results show applicability of all tested methods for the lipidomic analysis of biological samples depending on the analysis requirements. Copyright © 2017 Elsevier B.V. All rights reserved.

Reliable noninvasive measurement of blood gases

DOEpatents

Thomas, Edward V.; Robinson, Mark R.; Haaland, David M.; Alam, Mary K.

1994-01-01

Methods and apparatus for, preferably, determining noninvasively and in vivo at least two of the five blood gas parameters (i.e., pH, PCO.sub.2, [HCO.sub.3.sup.- ], PO.sub.2, and O.sub.2 sat.) in a human. The non-invasive method includes the steps of: generating light at three or more different wavelengths in the range of 500 nm to 2500 nm; irradiating blood containing tissue; measuring the intensities of the wavelengths emerging from the blood containing tissue to obtain a set of at least three spectral intensities v. wavelengths; and determining the unknown values of at least two of pH, [HCO.sub.3.sup.- ], PCO.sub.2 and a measure of oxygen concentration. The determined values are within the physiological ranges observed in blood containing tissue. The method also includes the steps of providing calibration samples, determining if the spectral intensities v. wavelengths from the tissue represents an outlier, and determining if any of the calibration samples represents an outlier. The determination of the unknown values is performed by at least one multivariate algorithm using two or more variables and at least one calibration model. Preferably, there is a separate calibration for each blood gas parameter being determined. The method can be utilized in a pulse mode and can also be used invasively. The apparatus includes a tissue positioning device, a source, at least one detector, electronics, a microprocessor, memory, and apparatus for indicating the determined values.

Optical coefficient measurements using bulk living tissue by an optical fiber puncture with FOV change

NASA Astrophysics Data System (ADS)

Nakazawa, Haruna; Doi, Marika; Ogawa, Emiyu; Arai, Tsunenori

2018-02-01

To avoid an instability of the optical coefficient measurement using sliced tissue preparation, we proposed the combination of light intensity measurement through an optical fiber puncturing into a bulk tissue varying field of view (FOV) and ray tracing calculation using Monte-Carlo method. The optical coefficients of myocardium such as absorption coefficient μa, scattering coefficient μs, and anisotropic parameter g are used in the myocardium optical propagation. Since optical coefficients obtained using thin sliced tissue could be instable because they are affected by dehydration and intracellular fluid effusion on the sample surface, variety of coefficients have been reported over individual optical differences of living samples. The proposed method which combined the experiment using the bulk tissue with ray tracing calculation were performed. In this method, a 200 μmΦ high-NA silica fiber installed in a 21G needle was punctured up to the bottom of the myocardial bulk tissue over 3 cm in thickness to measure light intensity changing the fiber-tip depth and FOV. We found that the measured attenuation coefficients decreased as the FOV increased. The ray trace calculation represented the same FOV dependence in above mentioned experimental result. We think our particular fiber punctured measurement using bulk tissue varying FOV with Inverse Monte-Carlo method might be useful to obtain the optical coefficients to avoid sample preparation instabilities.

MALDI (matrix assisted laser desorption ionization) Imaging Mass Spectrometry (IMS) of skin: Aspects of sample preparation.

PubMed

de Macedo, Cristiana Santos; Anderson, David M; Schey, Kevin L

2017-11-01

MALDI (matrix assisted laser desorption ionization) Imaging Mass Spectrometry (IMS) allows molecular analysis of biological materials making possible the identification and localization of molecules in tissues, and has been applied to address many questions on skin pathophysiology, as well as on studies about drug absorption and metabolism. Sample preparation for MALDI IMS is the most important part of the workflow, comprising specimen collection and preservation, tissue embedding, cryosectioning, washing, and matrix application. These steps must be carefully optimized for specific analytes of interest (lipids, proteins, drugs, etc.), representing a challenge for skin analysis. In this review, critical parameters for MALDI IMS sample preparation of skin samples will be described. In addition, specific applications of MALDI IMS of skin samples will be presented including wound healing, neoplasia, and infection. Copyright © 2017 Elsevier B.V. All rights reserved.

IUTA: a tool for effectively detecting differential isoform usage from RNA-Seq data.

PubMed

Niu, Liang; Huang, Weichun; Umbach, David M; Li, Leping

2014-10-06

Most genes in mammals generate several transcript isoforms that differ in stability and translational efficiency through alternative splicing. Such alternative splicing can be tissue- and developmental stage-specific, and such specificity is sometimes associated with disease. Thus, detecting differential isoform usage for a gene between tissues or cell lines/types (differences in the fraction of total expression of a gene represented by the expression of each of its isoforms) is potentially important for cell and developmental biology. We present a new method IUTA that is designed to test each gene in the genome for differential isoform usage between two groups of samples. IUTA also estimates isoform usage for each gene in each sample as well as averaged across samples within each group. IUTA is the first method to formulate the testing problem as testing for equal means of two probability distributions under the Aitchison geometry, which is widely recognized as the most appropriate geometry for compositional data (vectors that contain the relative amount of each component comprising the whole). Evaluation using simulated data showed that IUTA was able to provide test results for many more genes than was Cuffdiff2 (version 2.2.0, released in Mar. 2014), and IUTA performed better than Cuffdiff2 for the limited number of genes that Cuffdiff2 did analyze. When applied to actual mouse RNA-Seq datasets from six tissues, IUTA identified 2,073 significant genes with clear patterns of differential isoform usage between a pair of tissues. IUTA is implemented as an R package and is available at http://www.niehs.nih.gov/research/resources/software/biostatistics/iuta/index.cfm. Both simulation and real-data results suggest that IUTA accurately detects differential isoform usage. We believe that our analysis of RNA-seq data from six mouse tissues represents the first comprehensive characterization of isoform usage in these tissues. IUTA will be a valuable resource for those who study the roles of alternative transcripts in cell development and disease.

The value of banked samples for oncology drug discovery and development.

PubMed

Shaw, Peter M; Patterson, Scott D

2011-01-01

To gain insights into human biology and pathobiology, ready access to banked human tissue samples that encompass a representative cross section of the population is required. For optimal use, the banked human tissue needs to be appropriately consented, collected, annotated, and stored. If any of these elements are missing, the studies using these samples are compromised. These elements are critical whether the research is for academic or pharmaceutical industry purposes. An additional temporal element that adds enormous value to such banked samples is treatment and outcome information from the people who donated the tissue. To achieve these aims, many different groups have to work effectively together, not least of which are the individuals who donate their tissue with appropriate consent. Such research is unlikely to benefit the donors but others who succumb to the same disease. The development of a large accessible human tissue bank resource (National Cancer Institute's Cancer HUman Biobank [caHUB]) that provides an ongoing supply of human tissue for all working toward the common goal of understanding human health and disease has a number of advantages. These include, but are not limited to, access to a broad cross section of healthy and diseased populations beyond what individual collections may achieve for understanding disease pathobiology, therapeutic target discovery, as well as a source of material for diagnostic assay validation. Models will need to be developed to enable fair access to caHUB under terms that enable appropriate intellectual property protection and ultimate data sharing to ensure that the biobank successfully distributes samples to a broad range of researchers.

The developmental basis for germline mosaicism in mouse and Drosophila melanogaster.

PubMed

Drost, J B; Lee, W R

1998-01-01

Data involving germline mosaics in Drosophila melanogaster and mouse are reconciled with developmental observations. Mutations that become fixed in the early embryo before separation of soma from the germline may, by the sampling process of development, continue as part of germline and/or differentiate into any somatic tissue. The cuticle of adult D. melanogaster, because of segmental development, can be used to estimate the proportion of mutant nuclei in the early embryo, but most somatic tissues and the germlines of both species continue from samples too small to be representative of the early embryo. Because of the small sample of cells/nuclei that remain in the germline after separation of soma in both species, mosaic germlines have percentages of mutant cells that vary widely, with a mean of 50% and an unusual platykurtic, flat-topped distribution. While the sampling process leads to similar statistical results for both species, their patterns of development are very different. In D. melanogaster the first differentiation is the separation of soma from germline with the germline continuing from a sample of only two to four nuclei, whereas the adult cuticle is a representative sample of cleavage nuclei. The presence of mosaicism in D. melanogaster germline is independent of mosaicism in the eye, head, and thorax. This independence was used to determine that mutations can occur at any of the early embryonic cell divisions and still average 50% mutant germ cells when the germline is mosaic; however, the later the mutation occurs, the higher the proportion of completely nonmutant germlines. In contrast to D. melanogaster, the first differentiation in the mouse does not separate soma from germline but produces the inner cell mass that is representative of the cleavage nuclei. Following formation of the primitive streak, the primordial germ cells develop at the base of the allantois and among a clonally related sample of cells, providing the same statistical distribution in the mouse germlines as in D. melanogaster. The proportion of mutations that are fixed during early embryonic development is greatly underestimated. For example, a DNA lesion in a postmeiotic gamete that becomes fixed as a dominant mutation during early embryonic development of the F1 may produce an individual completely mutant in the germ line and relevant somatic tissue or, alternatively, the F1 germline may be completely mutant but with no relevant somatic tissues for detecting the mutation until the F2. In both cases the mutation would be classified as complete in the F1 and F2, respectively, and not recognized as embryonic in origin. Because germ cells differentiate later in mammalian development, there are more opportunities for correlation between germline and soma in the mammal than Drosophila. However, because the germ cells and any somatic tissue, like blood, are derived from small samples, there may be many individuals that test negative in blood but have germlines that are either mosaic or entirely mutant.

Comparison of prevalence estimation of Mycobacterium avium subsp. paratuberculosis infection by sampling slaughtered cattle with macroscopic lesions vs. systematic sampling.

PubMed

Elze, J; Liebler-Tenorio, E; Ziller, M; Köhler, H

2013-07-01

The objective of this study was to identify the most reliable approach for prevalence estimation of Mycobacterium avium ssp. paratuberculosis (MAP) infection in clinically healthy slaughtered cattle. Sampling of macroscopically suspect tissue was compared to systematic sampling. Specimens of ileum, jejunum, mesenteric and caecal lymph nodes were examined for MAP infection using bacterial microscopy, culture, histopathology and immunohistochemistry. MAP was found most frequently in caecal lymph nodes, but sampling more tissues optimized the detection rate. Examination by culture was most efficient while combination with histopathology increased the detection rate slightly. MAP was detected in 49/50 animals with macroscopic lesions representing 1.35% of the slaughtered cattle examined. Of 150 systematically sampled macroscopically non-suspect cows, 28.7% were infected with MAP. This indicates that the majority of MAP-positive cattle are slaughtered without evidence of macroscopic lesions and before clinical signs occur. For reliable prevalence estimation of MAP infection in slaughtered cattle, systematic random sampling is essential.

Understanding the effect of pulsed electric fields on thermostability of connective tissue isolated from beef pectoralis muscle using a model system.

PubMed

Alahakoon, A U; Oey, I; Silcock, P; Bremer, P

2017-10-01

Brisket is a low value/tough meat cut that contains a large amount of connective tissue. Conversion of collagen into gelatin during heating reduces the toughness of the connective tissue however this conversion is slow at low cooking temperatures (around 60°C). The objective of this project was to determine the ability of pulsed electric field (PEF) processing to reduce the thermal stability of connective tissue. To achieve this, a novel model system was designed in which connective tissue obtained from beef deep pectotalis muscle (brisket) was exposed to PEF at combinations of electric field strength (1.0 and 1.5kV/cm) and specific energy (50 and 100kJ/kg) within an agar matrix at electrical conductivities representing the electrical conductivity found in brisket. Differential scanning calorimetry showed that PEF treatment significantly (p<0.05) decreased the denaturation temperature of connective tissue compared to untreated samples. Increasing electric field strength and the specific energy increased the Ringer soluble collagen fraction. PEF treated samples showed higher solubilization compared to the untreated samples at both 60°C and 70°C in heat solubility test. SEM examination of PEF treated (at 1.5kV/cm and 100kJ/kg) and untreated samples revealed that PEF appeared to increase the porosity of the connective tissue structure. These finding suggest that PEF processing is a technology that could be used to improve the tenderness and decrease the cooking time of collagen rich, meat cuts. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proteomics Analysis of Tissue Samples Reveals Changes in Mitochondrial Protein Levels in Parathyroid Hyperplasia over Adenoma

PubMed Central

AKPINAR, GURLER; KASAP, MURAT; CANTURK, NUH ZAFER; ZULFIGAROVA, MEHIN; ISLEK, EYLÜL ECE; GULER, SERTAC ATA; SIMSEK, TURGAY; CANTURK, ZEYNEP

2017-01-01

Background/Aim: To unveil the pathophysiology of primary hyperparathyroidism, molecular details of parathyroid hyperplasia and adenoma have to be revealed. Such details will provide the tools necessary for differentiation of these two look-alike diseases. Therefore, in the present study, a comparative proteomic study using postoperative tissue samples from the parathyroid adenoma and parathyroid hyperplasia patients was performed. Materials and Methods: Protein extracts were prepared from tissue samples (n=8 per group). Protein pools were created for each group and subjected to DIGE and conventional 2DE. Following image analysis, spots representing the differentially regulated proteins were excised from the and used for identification via MALDI-TOF/TOF analysis. Results: The identities of 40 differentially-expressed proteins were revealed. Fourteen of these proteins were over-expressed in the hyperplasia while 26 of them were over-expressed in the adenoma. Conclusion: Most proteins found to be over-expressed in the hyperplasia samples were mitochondrial, underlying the importance of the mitochondrial activity as a potential biomarker for differentiation of parathyroid hyperplasia from adenoma. PMID:28446534

What are farmers really planting? Measuring the presence and effectiveness of Bt cotton in Pakistan.

PubMed

Spielman, David J; Zaidi, Fatima; Zambrano, Patricia; Khan, Asif Ali; Ali, Shaukat; Cheema, H Masooma Naseer; Nazli, Hina; Khan, Rao Sohail Ahmad; Iqbal, Arshad; Zia, Muhammad Amir; Ali, Ghulam Muhammad

2017-01-01

Genetically modified, insect-resistant Bacillus thuringiensis (Bt) cotton is cultivated extensively in Pakistan. Past studies, however, have raised concerns about the prevalence of Bt cotton varieties possessing weak or nonperforming insect-resistance traits conferred by the cry gene. We examine this issue using data drawn from a representative sample of cotton-growing households that were surveyed in six agroclimatic zones spanning 28 districts in Pakistan in 2013, as well as measurements of Cry protein levels in cotton tissue samples collected from the sampled households' main fields. The resultant dataset combines information from 593 sampled households with corresponding plant tissue diagnostics from 70 days after sowing, as well as information from 589 sampled households with corresponding diagnostics from 120 days after sowing. Our analysis indicates that 11 percent of farmers believed they were cultivating Bt cotton when, in fact, the Cry toxin was not present in the tested tissue at 70 days after sowing (i.e., a Type I error). The analysis further indicates that 5 percent of farmers believed they were cultivating non-Bt cotton when, in fact, the Cry toxin was present in the tested tissue (i.e., a Type II error). In addition, 17 percent of all sampled farmers were uncertain whether or not they were cultivating Bt cotton. Overall, 33 percent of farmers either did not know or were mistaken in their beliefs about the presence of the cry gene in the cotton they cultivated. Results also indicate that toxic protein levels in the plant tissue samples occurred below threshold levels for lethality in a significant percentage of cases, although these measurements may also be affected by factors related to tissue sample collection, handling, storage, and testing procedures. Nonetheless, results strongly suggest wide variability both in farmers' beliefs and in gene expression. Such variability has implications for policy and regulation in Pakistan's transgenic cotton seed market.

Comparison of inferred fractions of n-3 and n-6 polyunsaturated fatty acids in feral domestic cat diets with those in commercial feline extruded diets.

PubMed

Backus, Robert C; Thomas, David G; Fritsche, Kevin L

2013-04-01

To compare presumed fatty acid content in natural diets of feral domestic cats (inferred from body fat polyunsatrated fatty acids content) with polyunsaturated fatty acid content of commercial feline extruded diets. Subcutaneous and intra-abdominal adipose tissue samples (approx 1 g) from previously frozen cadavers of 7 adult feral domestic cats trapped in habitats remote from human activity and triplicate samples (200 g each) of 7 commercial extruded diets representing 68% of market share obtained from retail stores. Lipid, triacylglycerol, and phospholipid fractions in adipose tissue samples and ether extracts of diet samples were determined by gas chromatography of methyl esters. Triacylglycerol and phospholipid fractions in the adipose tissue were isolated by thin-layer chromatography. Diet samples were also analyzed for proximate contents. For the adipose tissue samples, with few exceptions, fatty acids fractions varied only moderately with lipid fraction and site from which tissue samples were obtained. Linoleic, α-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acid fractions were 15.0% to 28.2%, 4.5% to 18.7%, 0.9% to 5.0%, < 0.1% to 0.2%, and 0.6% to 1.7%, respectively. As inferred from the adipose findings, dietary fractions of docosahexaenoic and α-linolenic acid were significantly greater than those in the commercial feline diets, but those for linoleic and eicosapentaenoic acids were not significantly different. The fatty acid content of commercial extruded feline diets differed from the inferred content of natural feral cat diets, in which dietary n-3 and possibly n-6 polyunsaturated fatty acids were more abundant. The impact of this difference on the health of pet cats is not known.

Reconstruction of the forehead acoustic properties in an Indo-Pacific humpback dolphin (Sousa chinensis), with investigation on the responses of soft tissue sound velocity to temperature.

PubMed

Song, Zhongchang; Zhang, Yu; Berggren, Per; Wei, Chong

2017-02-01

Computed tomography (CT) imaging and ultrasound experimental measurements were combined to reconstruct the acoustic properties (density, velocity, and impedance) of the head from a deceased Indo-Pacific humpback dolphin (Sousa chinensis). The authors extracted 42 soft forehead tissue samples to estimate the sound velocity and density properties at room temperature, 25.0  °C. Hounsfield Units (HUs) of the samples were read from CT scans. Linear relationships between the tissues' HUs and velocity, and HUs and density were revealed through regression analyses. The distributions of the head acoustic properties at axial, coronal, and sagittal cross sections were reconstructed, suggesting that the forehead soft tissues were characterized by low-velocity in the melon, high-velocity in the muscle and connective tissues. Further, the sound velocities of melon, muscle, and connective tissue pieces were measured under different temperatures to investigate tissues' velocity response to temperature. The results demonstrated nonlinear relationships between tissues' sound velocity and temperature. This study represents a first attempt to provide general information on acoustic properties of this species. The results could provide meaningful information for understanding the species' bioacoustic characteristics and for further investigation on sound beam formation of the dolphin.

4D (x-y-z-t) imaging of thick biological samples by means of Two-Photon inverted Selective Plane Illumination Microscopy (2PE-iSPIM)

PubMed Central

Lavagnino, Zeno; Sancataldo, Giuseppe; d’Amora, Marta; Follert, Philipp; De Pietri Tonelli, Davide; Diaspro, Alberto; Cella Zanacchi, Francesca

2016-01-01

In the last decade light sheet fluorescence microscopy techniques, such as selective plane illumination microscopy (SPIM), has become a well established method for developmental biology. However, conventional SPIM architectures hardly permit imaging of certain tissues since the common sample mounting procedure, based on gel embedding, could interfere with the sample morphology. In this work we propose an inverted selective plane microscopy system (iSPIM), based on non-linear excitation, suitable for 3D tissue imaging. First, the iSPIM architecture provides flexibility on the sample mounting, getting rid of the gel-based mounting typical of conventional SPIM, permitting 3D imaging of hippocampal slices from mouse brain. Moreover, all the advantages brought by two photon excitation (2PE) in terms of reduction of scattering effects and contrast improvement are exploited, demonstrating an improved image quality and contrast compared to single photon excitation. The system proposed represents an optimal platform for tissue imaging and it smooths the way to the applicability of light sheet microscopy to a wider range of samples including those that have to be mounted on non-transparent surfaces. PMID:27033347

On the use of the Reciprocity Gap Functional in inverse scattering with near-field data: An application to mammography

NASA Astrophysics Data System (ADS)

Delbary, Fabrice; Aramini, Riccardo; Bozza, Giovanni; Brignone, Massimo; Piana, Michele

2008-11-01

Microwave tomography is a non-invasive approach to the early diagnosis of breast cancer. However the problem of visualizing tumors from diffracted microwaves is a difficult nonlinear ill-posed inverse scattering problem. We propose a qualitative approach to the solution of such a problem, whereby the shape and location of cancerous tissues can be detected by means of a combination of the Reciprocity Gap Functional method and the Linear Sampling method. We validate this approach to synthetic near-fields produced by a finite element method for boundary integral equations, where the breast is mimicked by the axial view of two nested cylinders, the external one representing the skin and the internal one representing the fat tissue.

Quantification of Toxoplasma gondii in tissue samples of experimentally infected goats by magnetic capture and real-time PCR.

PubMed

Juránková, Jana; Opsteegh, Marieke; Neumayerová, Helena; Kovařčík, Kamil; Frencová, Anita; Baláž, Vojtěch; Volf, Jiří; Koudela, Břetislav

2013-03-31

Undercooked meat containing tissue cysts is one of the most common sources of Toxoplasma gondii infection in humans. Goats are very susceptible to clinical toxoplasmosis, and especially kids are common food animals, thereby representing a risk for human infection. A sequence-specific magnetic capture method was used for isolation of T. gondii DNA from tissue samples from experimentally infected goat-kids and real-time PCR for the 529 bp repeat element allowed quantification of T. gondii DNA. The contamination level in different types of tissue and in two groups of goats euthanized 30 and 90 dpi was compared. The highest concentration of T. gondii DNA in both groups of goats was found in lung tissue, but only the higher parasite count in lung tissue compared to other organs in group A (euthanized 30 dpi) was statistically significant. T. gondii concentrations were higher in liver and dorsal muscle samples from goats euthanized 90 dpi than in goats euthanized at 30 dpi, while the T. gondii concentration in hearts decreased. This study describes for the first time distribution of T. gondii parasites in post-weaned goat kids. New information about T. gondii predilection sites in goats and about the progression of infection between 30 and 90 dpi was achieved. Copyright © 2012 Elsevier B.V. All rights reserved.

Tissues from the irradiated dog/mouse archive

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gayle Woloschak

The purpose of this project is to organize the databases/information and organize and move the tissues from the long-term dog (4,000 dogs) and mouse (over 30,000 mice) radiation experiments done at Argonne National Laboratory during the 1970's and 80's to Northwestern University. These studies were done with the intention of understanding the effects of exposure to radiation at a variety of different doses, dose-rates, and radiation qualities on end-points such as life-shortening, carcinogenesis, cause of death, shifts in disease incidence and other biological parameters. Organ and tissue samples from these animals including cancers, metastases and other significant degenerative and inflammatorymore » lesions and those in a regular protocol of normal tissues were preserved in paraffin blocks, tissue impressions and sections and represent a great resource for the radiation biology community. These collections are particularly significant since these experiments are not likely to be repeated because of the extreme cost of monies and time for such large-scale animal studies. The long-term goal is to make these tissues and databases available to the wider scientific community so that questions such as tissue sensitivity, early and late effects, low dose and protracted dose responses of normal and tumor tissues, etc. can be examined and defined. Recent advances in biology particularly at the subcellular and molecular level now permit microarray-based gene expression array analyses from paraffin-embedded tissues (where RNA samples are significantly degraded), synchrotron-based studies of metal and other elemental distribution patterns in tissues, PCR-based analyses for mutation detection, and other similar approaches that were not available when the long¬ term animal studies were designed and initiated. Understanding the basis and progression of radiation damage should also permit rational approaches to prevention and mitigation of those damages. Therefore, as stated earlier, these tissues and their related documentation, represent a significant resource for future studies. For this project, we propose to accomplish the following objectives: (1) inventory and organize the tissues, blood smears, wet-tissues and paper-¬based information that is available in the tissue bank at Argonne National Laboratory; (2) convert the existing Oracle database of the mouse studies to MS Access( the dog data is already in this format which is far more user friendly and widely used in business and research) , (3) move the remaining samples and documentation from dogs that had been transferred from ANL to New Mexico (in Dr. F. Hahn's care) to Northwestern University and add these to the inventory; (4) move the tissues and Access database at Argonne National Laboratory to Northwestern University.« less

Relationships of mercury concentrations across tissue types, muscle regions and fins for two shark species.

PubMed

O'Bryhim, Jason R; Adams, Douglas H; Spaet, Julia L Y; Mills, Gary; Lance, Stacey L

2017-04-01

Mercury (Hg) exposure poses a threat to both fish and human health. Sharks are known to bioaccumulate Hg, however, little is known regarding how Hg is distributed between different tissue groups (e.g. muscle regions, organs). Here we evaluated total mercury (THg) concentrations from eight muscle regions, four fins (first dorsal, left and right pectorals, caudal-from both the inner core and trailing margin of each fin), and five internal organs (liver, kidney, spleen, heart, epigonal organ) from two different shark species, bonnethead (Sphyrna tiburo) and silky shark (Carcharhinus falciformis) to determine the relationships of THg concentrations between and within tissue groups. Total Hg concentrations were highest in the eight muscle regions with no significant differences in THg concentrations between the different muscle regions and muscle types (red and white). Results from tissue collected from any muscle region would be representative of all muscle sample locations. Total Hg concentrations were lowest in samples taken from the fin inner core of the first dorsal, pectoral, and caudal (lower lobe) fins. Mercury concentrations for samples taken from the trailing margin of the dorsal, pectoral, and caudal fins (upper and lower lobe) were also not significantly different from each other for both species. Significant relationships were found between THg concentrations in dorsal axial muscle tissue and the fin inner core, liver, kidney, spleen and heart for both species as well as the THg concentrations between the dorsal fin trailing margin and the heart for the silky shark and all other sampled tissue types for the bonnethead shark. Our results suggest that biopsy sampling of dorsal muscle can provide data that can effectively estimate THg concentrations in specific organs without using more invasive, or lethal methods. Copyright © 2017 Elsevier Ltd. All rights reserved.

RNA Extraction from Animal and Human's Cancerous Tissues: Does Tissue Matter?

PubMed

Samadani, Ali Akbar; Nikbakhsh, Novin; Fattahi, Sadegh; Pourbagher, Roghayeh; Aghajanpour Mir, Seyyed Mohsen; Mousavi Kani, Narges; Abedian, Zeinab; Akhavan-Niaki, Haleh

2015-01-01

The reliability of gene expression profiling, based technologies and methods to find transcriptional differences representative of the original samples is influenced by the quality of the extracted RNA. Hence, RNA extraction is the first step to investigate the gene expression and its function. Consequently, the quality of extracted RNA is really significant. Correspondingly, this research was accomplished to optimize the RNA extraction methods and compare the amounts of tissue or quality of tissue. Relatively, the cancerous tissue of human stomach in fresh and frozen conditions and also the mouse fresh tissue were studied. Some factors like the amount of samples, efficacy differences of diverse extraction buffers (TriPure, Trizol) and also the efficacy of b-mercaptoethanol were compared and investigated. The results indicated that the less amount (1-2 mg) compared to other amounts (2-5 mg, 5-15 mg) yielded the best quality and the RNA bands (5S, 18S, 28S) were observed perfectly. Relatively, comparing and measuring some kinds of buffers (Trizol, TriPure) indicated no difference in RNA extraction quality. The last investigated factor was the effect of b- mercaptoethanol which was used along with TriPure to remove the RNAse. Conclusively, no effective impression was observed.

Trace element contamination in feather and tissue samples from Anna’s hummingbirds

USGS Publications Warehouse

Mikoni, Nicole A.; Poppenga, Robert H.; Ackerman, Joshua T.; Foley, Janet E.; Hazlehurst, Jenny; Purdin, Güthrum; Aston, Linda; Hargrave, Sabine; Jelks, Karen; Tell, Lisa A.

2017-01-01

Trace element contamination (17 elements; Be, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, Cd, Ba, Hg, Tl, and Pb) of live (feather samples only) and deceased (feather and tissue samples) Anna's hummingbirds (Calypte anna) was evaluated. Samples were analyzed using inductively coupled plasma-mass spectrometry (ICP-MS; 17 elements) and atomic absorption spectrophotometry (Hg only). Mean plus one standard deviation (SD) was considered the benchmark, and concentrations above the mean + 1 SD were considered elevated above normal. Contour feathers were sampled from live birds of varying age, sex, and California locations. In order to reduce thermal impacts, minimal feathers were taken from live birds, therefore a novel method was developed for preparation of low mass feather samples for ICP-MS analysis. The study found that the novel feather preparation method enabled small mass feather samples to be analyzed for trace elements using ICP-MS. For feather samples from live birds, all trace elements, with the exception of beryllium, had concentrations above the mean + 1 SD. Important risk factors for elevated trace element concentrations in feathers of live birds were age for iron, zinc, and arsenic, and location for iron, manganese, zinc, and selenium. For samples from deceased birds, ICP-MS results from body and tail feathers were correlated for Fe, Zn, and Pb, and feather concentrations were correlated with renal (Fe, Zn, Pb) or hepatic (Hg) tissue concentrations. Results for AA spectrophotometry analyzed samples from deceased birds further supported the ICP-MS findings where a strong correlation between mercury concentrations in feather and tissue (pectoral muscle) samples was found. These study results support that sampling feathers from live free-ranging hummingbirds might be a useful, non-lethal sampling method for evaluating trace element exposure and provides a sampling alternative since their small body size limits traditional sampling of blood and tissues. The results from this study provide a benchmark for the distribution of trace element concentrations in feather and tissue samples from hummingbirds and suggests a reference mark for exceeding normal. Lastly, pollinating avian species are minimally represented in the literature as bioindicators for environmental trace element contamination. Given that trace elements can move through food chains by a variety of routes, our study indicates that hummingbirds are possible bioindicators of environmental trace element contamination.

An MRI-Based Atlas for Correlation of Imaging and Pathologic Findings in Alzheimer's Disease.

PubMed

Raman, Mekala R; Schwarz, Christopher G; Murray, Melissa E; Lowe, Val J; Dickson, Dennis W; Jack, Clifford R; Kantarci, Kejal

2016-05-01

Pathologic diagnosis is the gold standard in evaluating imaging measures developed as biomarkers for pathologically defined disorders. A brain MRI atlas representing autopsy-sampled tissue can be used to directly compare imaging and pathology findings. Our objective was to develop a brain MRI atlas representing the cortical regions that are routinely sampled at autopsy for the diagnosis of Alzheimer's disease (AD). Subjects (n = 22; ages at death = 70-95) with a range of pathologies and antemortem 3T MRI were included. Histology slides from 8 cortical regions sampled from the left hemisphere at autopsy guided the localization of the atlas regions of interest (ROIs) on each subject's antemortem 3D T1 -weighted MRI. These ROIs were then registered to a common template and combined to form one ROI representing the volume of tissue that was sampled by the pathologists. A subset of the subjects (n = 4; ages at death = 79-95) had amyloid PET imaging. Density of β-amyloid immunostain was quantified from the autopsy-sampled regions in the 4 subjects using a custom-designed ImageScope algorithm. Median uptake values were calculated in each ROI on the amyloid-PET images. We found an association between β-amyloid plaque density in 8 ROIs of the 4 subjects (total ROI n = 32) and median PiB SUVR (r(2) = .64; P < .0001). In an atlas developed for imaging and pathologic correlation studies, we demonstrated that antemortem amyloid burden measured in the atlas ROIs on amyloid PET is strongly correlated with β-amyloid density measured on histology. This atlas can be used in imaging and pathologic correlation studies. © 2016 The Authors. Journal of Neuroimaging published by Wiley Periodicals, Inc. on behalf of American Society of Neuroimaging.

LIBS analysis of artificial calcified tissues matrices.

PubMed

Kasem, M A; Gonzalez, J J; Russo, R E; Harith, M A

2013-04-15

In most laser-based analytical methods, the reproducibility of quantitative measurements strongly depends on maintaining uniform and stable experimental conditions. For LIBS analysis this means that for accurate estimation of elemental concentration, using the calibration curves obtained from reference samples, the plasma parameters have to be kept as constant as possible. In addition, calcified tissues such as bone are normally less "tough" in their texture than many samples, especially metals. Thus, the ablation process could change the sample morphological features rapidly, and result in poor reproducibility statistics. In the present work, three artificial reference sample sets have been fabricated. These samples represent three different calcium based matrices, CaCO3 matrix, bone ash matrix and Ca hydroxyapatite matrix. A comparative study of UV (266 nm) and IR (1064 nm) LIBS for these three sets of samples has been performed under similar experimental conditions for the two systems (laser energy, spot size, repetition rate, irradiance, etc.) to examine the wavelength effect. The analytical results demonstrated that UV-LIBS has improved reproducibility, precision, stable plasma conditions, better linear fitting, and the reduction of matrix effects. Bone ash could be used as a suitable standard reference material for calcified tissue calibration using LIBS with a 266 nm excitation wavelength. Copyright © 2013 Elsevier B.V. All rights reserved.

Multimodal-3D imaging based on μMRI and μCT techniques bridges the gap with histology in visualization of the bone regeneration process.

PubMed

Sinibaldi, R; Conti, A; Sinjari, B; Spadone, S; Pecci, R; Palombo, M; Komlev, V S; Ortore, M G; Tromba, G; Capuani, S; Guidotti, R; De Luca, F; Caputi, S; Traini, T; Della Penna, S

2018-03-01

Bone repair/regeneration is usually investigated through X-ray computed microtomography (μCT) supported by histology of extracted samples, to analyse biomaterial structure and new bone formation processes. Magnetic resonance imaging (μMRI) shows a richer tissue contrast than μCT, despite at lower resolution, and could be combined with μCT in the perspective of conducting non-destructive 3D investigations of bone. A pipeline designed to combine μMRI and μCT images of bone samples is here described and applied on samples of extracted human jawbone core following bone graft. We optimized the coregistration procedure between μCT and μMRI images to avoid bias due to the different resolutions and contrasts. Furthermore, we used an Adaptive Multivariate Clustering, grouping homologous voxels in the coregistered images, to visualize different tissue types within a fused 3D metastructure. The tissue grouping matched the 2D histology applied only on 1 slice, thus extending the histology labelling in 3D. Specifically, in all samples, we could separate and map 2 types of regenerated bone, calcified tissue, soft tissues, and/or fat and marrow space. Remarkably, μMRI and μCT alone were not able to separate the 2 types of regenerated bone. Finally, we computed volumes of each tissue in the 3D metastructures, which might be exploited by quantitative simulation. The 3D metastructure obtained through our pipeline represents a first step to bridge the gap between the quality of information obtained from 2D optical microscopy and the 3D mapping of the bone tissue heterogeneity and could allow researchers and clinicians to non-destructively characterize and follow-up bone regeneration. Copyright © 2017 John Wiley & Sons, Ltd.

Soft-tissue imaging with C-arm cone-beam CT using statistical reconstruction

NASA Astrophysics Data System (ADS)

Wang, Adam S.; Webster Stayman, J.; Otake, Yoshito; Kleinszig, Gerhard; Vogt, Sebastian; Gallia, Gary L.; Khanna, A. Jay; Siewerdsen, Jeffrey H.

2014-02-01

The potential for statistical image reconstruction methods such as penalized-likelihood (PL) to improve C-arm cone-beam CT (CBCT) soft-tissue visualization for intraoperative imaging over conventional filtered backprojection (FBP) is assessed in this work by making a fair comparison in relation to soft-tissue performance. A prototype mobile C-arm was used to scan anthropomorphic head and abdomen phantoms as well as a cadaveric torso at doses substantially lower than typical values in diagnostic CT, and the effects of dose reduction via tube current reduction and sparse sampling were also compared. Matched spatial resolution between PL and FBP was determined by the edge spread function of low-contrast (˜40-80 HU) spheres in the phantoms, which were representative of soft-tissue imaging tasks. PL using the non-quadratic Huber penalty was found to substantially reduce noise relative to FBP, especially at lower spatial resolution where PL provides a contrast-to-noise ratio increase up to 1.4-2.2× over FBP at 50% dose reduction across all objects. Comparison of sampling strategies indicates that soft-tissue imaging benefits from fully sampled acquisitions at dose above ˜1.7 mGy and benefits from 50% sparsity at dose below ˜1.0 mGy. Therefore, an appropriate sampling strategy along with the improved low-contrast visualization offered by statistical reconstruction demonstrates the potential for extending intraoperative C-arm CBCT to applications in soft-tissue interventions in neurosurgery as well as thoracic and abdominal surgeries by overcoming conventional tradeoffs in noise, spatial resolution, and dose.

Glioma tissue obtained by modern ultrasonic aspiration with a simple sterile suction trap for primary cell culture and pathological evaluation.

PubMed

Schroeteler, Juliane; Reeker, Ralf; Suero Molina, Eric; Brokinkel, Benjamin; Holling, Markus; Grauer, Oliver M; Senner, Volker; Stummer, Walter; Ewelt, Christian

2014-01-01

Ultrasonic aspiration is widely used in the resection of brain tumors. Nevertheless, tumor tissue fragments obtained by ultrasonic aspiration are usually discarded. In this study, we demonstrate that these fragments are possible sources of material for histopathological study and tissue culture and compare their microscopic features and viability in tissue culture of cavitron ultrasonic surgical aspirator tissue fragments. Brain tumor tissue collected by ultrasonic aspiration (CUSA EXcel®; Integra Radionics Inc.) in a simple sterile suction trap during resection was processed for primary cell culture. Cell viability and immunohistological markers were measured by the WST-1 test, microscopy and immunofluorescent evaluation. Six gliomas are presented to demonstrate that these tissue fragments show good preservation of histological detail and tissue viability in culture. Utilization of this material may facilitate pathological interpretation by providing a more representative sample of tumor histology as well as an adequate and sterile biosource of material for tissue culture studies.

9 CFR 55.23 - Responsibilities of States and enrolled herd owners.

Code of Federal Regulations, 2010 CFR

2010-01-01

.... In cases where animals escape or disappear and thus are not available for tissue sampling and testing, an APHIS representative will investigate whether the unavailability of animals for testing... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Responsibilities of States and...

9 CFR 55.23 - Responsibilities of States and enrolled herd owners.

Code of Federal Regulations, 2011 CFR

2011-01-01

.... In cases where animals escape or disappear and thus are not available for tissue sampling and testing, an APHIS representative will investigate whether the unavailability of animals for testing... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Responsibilities of States and...

9 CFR 55.23 - Responsibilities of States and enrolled herd owners.

Code of Federal Regulations, 2012 CFR

2012-01-01

.... In cases where animals escape or disappear and thus are not available for tissue sampling and testing, an APHIS representative will investigate whether the unavailability of animals for testing... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Responsibilities of States and...

Fetal Tissues Tested for Microbial Sterility by Culture- and PCR-Based Methods Can be Safely Used in Clinics.

PubMed

Vitrenko, Yakov; Kostenko, Iryna; Kulebyakina, Kateryna; Duda, Alla; Klunnyk, Mariya; Sorochynska, Khrystyna

2017-02-16

Cell preparations to be used in clinical practice must be free of infectious agents. Safety concerns are especially elevated upon the use of human fetal tissues, which are otherwise highly advantageous in cell therapy. We demonstrate that treating fetal samples with antibiotic, extensive washing, and homogenization prior to cryoconservation efficiently removes microbes in general. Screening a large collection by an automatic culture system showed that 89.2% fetal tissue samples were sterile, while contamination was detected in 10.8% samples. Liver and chorion were contaminated more than the brain, kidney, lung, and soft tissues. Broad-range PCR from the bacterial 16s rRNA gene was adopted as a confirmatory assay; however, the concordance between the culture-based and PCR assays was weak. Taxonomic identification was done for contaminated samples by bacteriological methods and sequencing 16s rRNA PCR products. The two approaches revealed different spectra of taxonomic groups sharing only Lactobacillus, the most frequently found genus. In addition, other representatives of vaginal microbiota were detected by culture-based identification, while PCR product sequencing has also revealed a subset of nosocomial microorganisms. Importantly, species known to cause sepsis were identified by both techniques, arguing for their indispensability and mutual complementarity. We suggest that most contaminations are taken up during collection of fetal material rather than originating from an in utero infection. In conclusion, a rigorous microbiological control by culture and PCR is a prerequisite for safe clinical use of fetal tissue suspensions.

Transcriptome profiling of Kentucky bluegrass (Poa pratensis L.) accessions in response to salt stress.

PubMed

Bushman, B Shaun; Amundsen, Keenan L; Warnke, Scott E; Robins, Joseph G; Johnson, Paul G

2016-01-13

Kentucky bluegrass (Poa pratensis L.) is a prominent turfgrass in the cool-season regions, but it is sensitive to salt stress. Previously, a relatively salt tolerant Kentucky bluegrass accession was identified that maintained green colour under consistent salt applications. In this study, a transcriptome study between the tolerant (PI 372742) accession and a salt susceptible (PI 368233) accession was conducted, under control and salt treatments, and in shoot and root tissues. Sample replicates grouped tightly by tissue and treatment, and fewer differentially expressed transcripts were detected in the tolerant PI 372742 samples compared to the susceptible PI 368233 samples, and in root tissues compared to shoot tissues. A de novo assembly resulted in 388,764 transcripts, with 36,587 detected as differentially expressed. Approximately 75 % of transcripts had homology based annotations, with several differences in GO terms enriched between the PI 368233 and PI 372742 samples. Gene expression profiling identified salt-responsive gene families that were consistently down-regulated in PI 372742 and unlikely to contribute to salt tolerance in Kentucky bluegrass. Gene expression profiling also identified sets of transcripts relating to transcription factors, ion and water transport genes, and oxidation-reduction process genes with likely roles in salt tolerance. The transcript assembly represents the first such assembly in the highly polyploidy, facultative apomictic Kentucky bluegrass. The transcripts identified provide genetic information on how this plant responds to and tolerates salt stress in both shoot and root tissues, and can be used for further genetic testing and introgression.

Impact of Collection and Storage of Lung Tumor Tissue on Whole Genome Expression Profiling

PubMed Central

Freidin, Maxim B.; Bhudia, Neesa; Lim, Eric; Nicholson, Andrew G.; Cookson, William O.; Moffatt, Miriam F.

2012-01-01

Gene expression profiling could assist in revealing biomarkers of lung cancer prognosis and progression. The handling of biological samples may strongly influence global gene expression, a fact that has not been addressed in many studies. We sought to investigate the changes in gene expression that may occur as a result of sample processing time and conditions. Using Illumina Human WG-6 arrays, we quantified gene expression in lung carcinoma samples from six patients obtained at chest opening before and immediately after lung resection with storage in RNAlater [T1a(CO) and T1b(LR)], after receipt of the sample for histopathology, placed in RNAlater [T2a(HP)]; snap frozen [T2b(HP.SF)]; or snap frozen and stored for 1 week [T2c(HP.SFA)], as well as formalin-fixed, paraffin-embedded (FFPE) block samples. Sampling immediately after resection closely represented the tissue obtained in situ, with only 1% of genes differing more than twofold [T1a(CO) versus T1b(LR)]. Delaying tissue harvest for an average of 30 minutes from the operating theater had a significant impact on gene expression, with approximately 25% of genes differing between T1a(CO) and T2a(HP). Many genes previously identified as lung cancer biomarkers were altered during this period. Examination of FFPE specimens showed minimal correlation with fresh samples. This study shows that tissue collection immediately after lung resection with conservation in RNAlater is an optimal strategy for gene expression profiling. PMID:22240448

Core needle biopsy guidance based on EMOCT imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Iftimia, Nicusor V.; Park, Jesung; Maguluri, Gopi

2016-03-01

We present a novel method, based on encoder mapping OCT imaging, for real-time guidance of core biopsy procedures. This method provides real-time feedback to the interventional radiologist, such that he/she can reorient the needle during the biopsy and sample the most representative area of the suspicious mass that is being investigated. This aspect is very important for tailoring therapy to the specific cancer based on biomarker analysis, which will become one of the next big advances in our search for the optimal cancer therapy. To enable individualized treatment, the genetic constitution and the DNA repair status in the affected areas is needed for each patient. Thus, representative sampling of the tumor is needed for analyzing various biomarkers, which are used as a tool to personalize cancer therapy. The encoder-based OCT enables samping of large size masses and provides full control on the imaging probe, which is passed through the bore of the biopsy guidance needle. The OCT image is built gradually, based on the feedback of an optical encoder which senses the incremental movement of the needle with a few microns resolution. Tissue mapping is independent of the needle speed, while it is advanced through the tissue. The OCT frame is analyzed in real-time and tissue cellularity is reported in a very simple manner (pie chart). Our preliminary study on a rabbit model of cancer has demonstrated the capability of this technology for accurately differentiating between viable cancer and heterogeneous or necrotic tissue.

Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA)*

PubMed Central

Bader, Sabine; Zajac, Magdalena; Friess, Thomas; Ruge, Elisabeth; Rieder, Natascha; Gierke, Berthold; Heubach, Yvonne; Thomas, Marlene; Pawlak, Michael

2015-01-01

Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect using FFPE tissues. PMID:26106084

Microwave fixation versus formalin fixation of surgical and autopsy tissue.

PubMed

Login, G R

1978-05-01

Microwave irradiation of surgical and autopsy tissue penetrates, fixes, and hardens the tissue almost immediately (the fluid media used in the microwave consisted of saline, ten percent phosphate buffered formalin, and distilled water). Tissue sections from a representative sample of organs were tested. Comparable sections were simultaneously fixed in a phosphate buffered ten percent formalin bath in a vaccum oven as a control. Hematoxylin and eosin were used to stain the sections. Results equal to and superior to the control method were obtained. Saline microwave fixation was superior to formalin microwave fixation. Tissues placed in Zenker's solution and fixed in standard microwave oven (for approximately one minute) yielded results at least equal to conventional Zenker fixation (approximately two hours). No tissue hardening resulted from Zenker microwave fixation. A unique time versus temperature graph (microwave heating curve) reduces individual variation with this technique.

Contaminants in fish tissue from US lakes and reservoirs: A ...

EPA Pesticide Factsheets

An unequal probability design was used to develop national estimates for 268 persistent, bioaccumulative, and toxic chemicals in fish tissue from lakes and reservoirs of the conterminous United States (excluding the Laurentian Great Lakes and Great Salt Lake). Predator (fillet) and bottom-dweller (whole-body) composites were collected from 500 lakes selected randomly from the target population of 147,343 lakes in the lower 48 states. Each of these composite types comprised nationally representative samples whose results were extrapolated to the sampled population of an estimated 76,559 lakes for predators and 46,190 lakes for bottom dwellers. Mercury and PCBs were detected in all fish samples. Dioxins and furans were detected in 81% and 99% of predator and bottom-dweller samples, respectively. Cumulative frequency distributions showed that mercury concentrations exceeded the EPA 300 ppb mercury fish tissue criterion at nearly half of the lakes in the sampled population. Total PCB concentrations exceeded a 12 ppb human health risk-based consumption limit at nearly 17% of lakes, and dioxins and furans exceeded a 0.15 ppt (toxic equivalent or TEQ) risk-based threshold at nearly 8% of lakes in the sampled population. In contrast, 43 target chemicals were not detected in any samples. No detections were reported for nine organophosphate pesticides, one PCB congener, 16 polycyclic aromatic hydrocarbons, or 17 other semivolatile organic chemicals. An unequal prob

Contaminants in fish tissue from US lakes and reservoirs: a national probabilistic study.

PubMed

Stahl, Leanne L; Snyder, Blaine D; Olsen, Anthony R; Pitt, Jennifer L

2009-03-01

An unequal probability design was used to develop national estimates for 268 persistent, bioaccumulative, and toxic chemicals in fish tissue from lakes and reservoirs of the conterminous United States (excluding the Laurentian Great Lakes and Great Salt Lake). Predator (fillet) and bottom-dweller (whole body) composites were collected from 500 lakes selected randomly from the target population of 147,343 lakes in the lower 48 states. Each of these composite types comprised nationally representative samples whose results were extrapolated to the sampled population of an estimated 76,559 lakes for predators and 46,190 lakes for bottom dwellers. Mercury and PCBs were detected in all fish samples. Dioxins and furans were detected in 81% and 99% of predator and bottom-dweller samples, respectively. Cumulative frequency distributions showed that mercury concentrations exceeded the EPA 300 ppb mercury fish tissue criterion at nearly half of the lakes in the sampled population. Total PCB concentrations exceeded a 12 ppb human health risk-based consumption limit at nearly 17% of lakes, and dioxins and furans exceeded a 0.15 ppt (toxic equivalent or TEQ) risk-based threshold at nearly 8% of lakes in the sampled population. In contrast, 43 target chemicals were not detected in any samples. No detections were reported for nine organophosphate pesticides, one PCB congener, 16 polycyclic aromatic hydrocarbons, or 17 other semivolatile organic chemicals.

Monitoring of tissue heating with medium intensity focused ultrasound via four dimensional optoacoustic tomography

NASA Astrophysics Data System (ADS)

Oyaga Landa, Francisco Javier; Ronda Penacoba, Silvia; Deán-Ben, Xosé Luís.; Montero de Espinosa, Francisco; Razansky, Daniel

2018-02-01

Medium intensity focused ultrasound (MIFU) holds promise in important clinical applications. Generally, the aim in MIFU is to stimulate physiological mechanisms that reinforce healing responses, avoiding reaching temperatures that can cause permanent tissue damage. The outcome of interventions is then strongly affected by the temperature distribution in the treated region, and accurate monitoring represents a significant clinical need. In this work, we showcase the capacities of 4D optoacoustic imaging to monitor tissue heating during MIFU. The proposed method allows localizing the ultrasound focus, estimating the peak temperature and measuring the size of the heat-affected volume. Calibration experiments in a tissue-mimicking phantom demonstrate that the optoacoustically-estimated temperature accurately matches thermocouple readings. The good performance of the suggested approach in real tissues is further showcased in experiments with bovine muscle samples.

Inorganic elements in green sea turtles (Chelonia mydas): relationships among external and internal tissues

USGS Publications Warehouse

Faust, Derek R.; Hooper, Michael J.; Cobb, George P.; Barnes, Melanie; Shaver, Donna; Ertolacci, Shauna; Smith, Philip N.

2014-01-01

Inorganic elements from anthropogenic sources have entered marine environments worldwide and are detectable in marine organisms, including sea turtles. Threatened and endangered classifications of sea turtles have heretofore made assessments of contaminant concentrations difficult because of regulatory restrictions on obtaining samples using nonlethal techniques. In the present study, claw and skin biopsy samples were examined as potential indicators of internal tissue burdens in green sea turtles (Chelonia mydas). Significant relationships were observed between claw and liver, and claw and muscle concentrations of mercury, nickel, arsenic, and selenium (p < 0.05). Similarly, significant relationships were observed between skin biopsy concentrations and those in liver, kidney, and muscle tissues for mercury, arsenic, selenium, and vanadium (p < 0.05). Concentrations of arsenic, barium, chromium, nickel, strontium, vanadium, and zinc in claws and skin biopsies were substantially elevated when compared with all other tissues, indicating that these highly keratinized tissues may represent sequestration or excretion pathways. Correlations between standard carapace length and cobalt, lead, and manganese concentrations were observed (p < 0.05), indicating that tissue concentrations of these elements may be related to age and size. Results suggest that claws may indeed be useful indicators of mercury and nickel concentrations in liver and muscle tissues, whereas skin biopsy inorganic element concentrations may be better suited as indicators of mercury, selenium, and vanadium concentrations in liver, kidney, and muscle tissues of green sea turtles.

First-time detection of Mycobacterium bovis in livestock tissues and milk in the West Bank, Palestinian Territories.

PubMed

Ereqat, Suheir; Nasereddin, Abedelmajeed; Levine, Hagai; Azmi, Kifaya; Al-Jawabreh, Amer; Greenblatt, Charles L; Abdeen, Ziad; Bar-Gal, Gila Kahila

2013-01-01

Bovine tuberculosis, bTB, is classified by the WHO as one of the seven neglected zoonontic diseases that cause animal health problems and has high potential to infect humans. In the West Bank, bTB was not studied among animals and the prevalence of human tuberculosis caused by M. bovis is unknown. Therefore, the aim of this study was to estimate the prevalence of bTB among cattle and goats and identify the molecular characteristics of bTB in our area. A total of 208 tissue samples, representing 104 animals, and 150 raw milk samples, obtained from cows and goats were examined for the presence of mycobacteria. The tissue samples were collected during routine meat inspection from the Jericho abattoir. DNA was extracted from all samples, milk and tissue biopsies (n = 358), and screened for presence of TB DNA by amplifying a 123-bp segment of the insertion sequence IS6110. Eight out of 254 animals (3.1%) were found to be TB positive based on the IS6110-PCR. Identification of M. bovis among the positive TB samples was carried out via real time PCR followed by high resolution melt curve analysis, targeting the A/G transition along the oxyR gene. Spoligotyping analysis revealed a new genotype of M. bovis that was revealed from one tissue sample. Detection of M. bovis in tissue and milk of livestock suggests that apparently healthy cattle and goats are a potential source of infection of bTB and may pose a risk to public health. Hence, appropriate measures including meat inspection at abattoirs in the region are required together with promotion of a health campaign emphasizing the importance of drinking pasteurized milk. In addition, further studies are essential at the farm level to determine the exact prevalence of bTB in goats and cattle herds in the West Bank and Israel.

First-Time Detection of Mycobacterium bovis in Livestock Tissues and Milk in the West Bank, Palestinian Territories

PubMed Central

Ereqat, Suheir; Nasereddin, Abedelmajeed; Levine, Hagai; Azmi, Kifaya; Al-Jawabreh, Amer; Greenblatt, Charles L.; Abdeen, Ziad; Bar-Gal, Gila Kahila

2013-01-01

Background Bovine tuberculosis, bTB, is classified by the WHO as one of the seven neglected zoonontic diseases that cause animal health problems and has high potential to infect humans. In the West Bank, bTB was not studied among animals and the prevalence of human tuberculosis caused by M. bovis is unknown. Therefore, the aim of this study was to estimate the prevalence of bTB among cattle and goats and identify the molecular characteristics of bTB in our area. Methodology/principal findings A total of 208 tissue samples, representing 104 animals, and 150 raw milk samples, obtained from cows and goats were examined for the presence of mycobacteria. The tissue samples were collected during routine meat inspection from the Jericho abattoir. DNA was extracted from all samples, milk and tissue biopsies (n = 358), and screened for presence of TB DNA by amplifying a 123-bp segment of the insertion sequence IS6110. Eight out of 254 animals (3.1%) were found to be TB positive based on the IS6110-PCR. Identification of M. bovis among the positive TB samples was carried out via real time PCR followed by high resolution melt curve analysis, targeting the A/G transition along the oxyR gene. Spoligotyping analysis revealed a new genotype of M. bovis that was revealed from one tissue sample. Significance Detection of M. bovis in tissue and milk of livestock suggests that apparently healthy cattle and goats are a potential source of infection of bTB and may pose a risk to public health. Hence, appropriate measures including meat inspection at abattoirs in the region are required together with promotion of a health campaign emphasizing the importance of drinking pasteurized milk. In addition, further studies are essential at the farm level to determine the exact prevalence of bTB in goats and cattle herds in the West Bank and Israel. PMID:24069475

Efficacy of Alkaline Hydrolysis as an Alternative Method for Treatment and Disposal of Infectious Animal Waste.

PubMed

Kaye, Gordon; Weber, Peter; Evans, Ann; Venezia, Richard

1998-05-01

The efficacy of alkaline hydrolysis as an alternative for incineration or autoclaving during treatment and disposal of infectious waste was evaluated by testing for the destruction of samples of pure cultures of selected infectious microorganisms during digestion of 114 to 136-kg loads of animal carcasses in an animal tissue digestor at the Albany Medical College. Ten milliliter samples of pure cultures of each microorganism were divided among 3 dialysis bags made from narrow diameter dialysis tubing, and each of these bags was placed inside another dialysis bag made from larger diameter dialysis tubing. Each double-bagged sample was suspended from the cover of the carcass basket of the tissue digestor so that it was completely covered by hot alkaline digestion solution during the carcass digestion process. The following organisms were required by the New York State Department of Health as representative pathogens for testing sterilization capabilities of the procedure: Staphylococcus aureus, Mycobacterium fortuitum, Candida albicans, Bacillus subtilis, Pseudomonas aeruginosa, Aspergillus fumigatus, Mycobacterium bovis BCG, MS-2 bacteriophage, and Giardia muris. Animal carcasses included pigs, sheep, rabbits, dogs, rats, mice, and guinea pigs. The tissue digestor was operated at 110 to 120 C and approximately 15 lb/in2 (gauge) for 18 h before the system was allowed to cool to 50 C and dialysis bags were retrieved and submitted for microbial culture. None of the samples obtained from the dialysis bags after the digestion process yielded indicator bacteria or yeast. Giardia cysts were completely destroyed; only small fragments of what appeared to be cyst wall could be recognized with light microscopic examination. No plaque-forming units were detected with MS-2 bacteriophage after digestion. Samples of the hydrolyzate also did not yield growth on culture media. Animal carcasses were completely solubilized and digested, with only the inorganic components of the bones and teeth remaining after draining and rinsing of the digestion vessel. Alkaline hydrolysis, as performed in this tissue digestor, completely destroyed all representative classes of potentially infectious agents as well as disposing of animal carcasses by solubilization and digestion.

Evidence of presence of Mycobacterium tuberculosis in bovine tissue samples by multiplex PCR: possible relevance to reverse zoonosis.

PubMed

Mittal, M; Chakravarti, S; Sharma, V; Sanjeeth, B S; Churamani, C P; Kanwar, N S

2014-04-01

Bovine tuberculosis, caused by Mycobacterium bovis, remains one of the most important zoonotic health concerns worldwide. The transmission of Mycobacterium tuberculosis from humans to animals also occurs especially in countries where there is close interaction of humans with the animals. In the present study, thirty bovine lung tissue autopsy samples from an organized dairy farm located in North India were screened for the presence of Mycobacterium tuberculosis complex by smear microscopy, histopathological findings and PCR. Differential diagnosis of M. tuberculosis and M. bovis was made based on the deletion of mce-3 operon in M. bovis. The present study found eight of these samples positive for M. tuberculosis by multiplex PCR. Sequencing was performed on two PCR-positive representative samples and on annotation, and BLAST analysis confirmed the presence of gene fragment specific to Mycobacterium tuberculosis. The presence of M. tuberculosis in all the positive samples raises the possibility of human-to-cattle transmission and possible adaptation of this organism in bovine tissues. This study accentuates the importance of screening and differential diagnosis of Mycobacterium tuberculosis complex in humans and livestock for adopting effective TB control and eradication programmes. © 2014 Blackwell Verlag GmbH.

Veterinary Medicine and Multi-Omics Research for Future Nutrition Targets: Metabolomics and Transcriptomics of the Common Degenerative Mitral Valve Disease in Dogs.

PubMed

Li, Qinghong; Freeman, Lisa M; Rush, John E; Huggins, Gordon S; Kennedy, Adam D; Labuda, Jeffrey A; Laflamme, Dorothy P; Hannah, Steven S

2015-08-01

Canine degenerative mitral valve disease (DMVD) is the most common form of heart disease in dogs. The objective of this study was to identify cellular and metabolic pathways that play a role in DMVD by performing metabolomics and transcriptomics analyses on serum and tissue (mitral valve and left ventricle) samples previously collected from dogs with DMVD or healthy hearts. Gas or liquid chromatography followed by mass spectrophotometry were used to identify metabolites in serum. Transcriptomics analysis of tissue samples was completed using RNA-seq, and selected targets were confirmed by RT-qPCR. Random Forest analysis was used to classify the metabolites that best predicted the presence of DMVD. Results identified 41 known and 13 unknown serum metabolites that were significantly different between healthy and DMVD dogs, representing alterations in fat and glucose energy metabolism, oxidative stress, and other pathways. The three metabolites with the greatest single effect in the Random Forest analysis were γ-glutamylmethionine, oxidized glutathione, and asymmetric dimethylarginine. Transcriptomics analysis identified 812 differentially expressed transcripts in left ventricle samples and 263 in mitral valve samples, representing changes in energy metabolism, antioxidant function, nitric oxide signaling, and extracellular matrix homeostasis pathways. Many of the identified alterations may benefit from nutritional or medical management. Our study provides evidence of the growing importance of integrative approaches in multi-omics research in veterinary and nutritional sciences.

Deep learning for tissue microarray image-based outcome prediction in patients with colorectal cancer

NASA Astrophysics Data System (ADS)

Bychkov, Dmitrii; Turkki, Riku; Haglund, Caj; Linder, Nina; Lundin, Johan

2016-03-01

Recent advances in computer vision enable increasingly accurate automated pattern classification. In the current study we evaluate whether a convolutional neural network (CNN) can be trained to predict disease outcome in patients with colorectal cancer based on images of tumor tissue microarray samples. We compare the prognostic accuracy of CNN features extracted from the whole, unsegmented tissue microarray spot image, with that of CNN features extracted from the epithelial and non-epithelial compartments, respectively. The prognostic accuracy of visually assessed histologic grade is used as a reference. The image data set consists of digitized hematoxylin-eosin (H and E) stained tissue microarray samples obtained from 180 patients with colorectal cancer. The patient samples represent a variety of histological grades, have data available on a series of clinicopathological variables including long-term outcome and ground truth annotations performed by experts. The CNN features extracted from images of the epithelial tissue compartment significantly predicted outcome (hazard ratio (HR) 2.08; CI95% 1.04-4.16; area under the curve (AUC) 0.66) in a test set of 60 patients, as compared to the CNN features extracted from unsegmented images (HR 1.67; CI95% 0.84-3.31, AUC 0.57) and visually assessed histologic grade (HR 1.96; CI95% 0.99-3.88, AUC 0.61). As a conclusion, a deep-learning classifier can be trained to predict outcome of colorectal cancer based on images of H and E stained tissue microarray samples and the CNN features extracted from the epithelial compartment only resulted in a prognostic discrimination comparable to that of visually determined histologic grade.

Biodynamic imaging for phenotypic profiling of three-dimensional tissue culture

PubMed Central

Sun, Hao; Merrill, Daniel; An, Ran; Turek, John; Matei, Daniela; Nolte, David D.

2017-01-01

Abstract. Three-dimensional (3-D) tissue culture represents a more biologically relevant environment for testing new drugs compared to conventional two-dimensional cancer cell culture models. Biodynamic imaging is a high-content 3-D optical imaging technology based on low-coherence interferometry and digital holography that uses dynamic speckle as high-content image contrast to probe deep inside 3-D tissue. Speckle contrast is shown to be a scaling function of the acquisition time relative to the persistence time of intracellular transport and hence provides a measure of cellular activity. Cellular responses of 3-D multicellular spheroids to paclitaxel are compared among three different growth techniques: rotating bioreactor (BR), hanging-drop (HD), and nonadherent (U-bottom, UB) plate spheroids, compared with ex vivo living tissues. HD spheroids have the most homogeneous tissue, whereas BR spheroids display large sample-to-sample variability as well as spatial heterogeneity. The responses of BR-grown tumor spheroids to paclitaxel are more similar to those of ex vivo biopsies than the responses of spheroids grown using HD or plate methods. The rate of mitosis inhibition by application of taxol is measured through tissue dynamics spectroscopic imaging, demonstrating the ability to monitor antimitotic chemotherapy. These results illustrate the potential use of low-coherence digital holography for 3-D pharmaceutical screening applications. PMID:28301634

Biodynamic imaging for phenotypic profiling of three-dimensional tissue culture

NASA Astrophysics Data System (ADS)

Sun, Hao; Merrill, Daniel; An, Ran; Turek, John; Matei, Daniela; Nolte, David D.

2017-01-01

Three-dimensional (3-D) tissue culture represents a more biologically relevant environment for testing new drugs compared to conventional two-dimensional cancer cell culture models. Biodynamic imaging is a high-content 3-D optical imaging technology based on low-coherence interferometry and digital holography that uses dynamic speckle as high-content image contrast to probe deep inside 3-D tissue. Speckle contrast is shown to be a scaling function of the acquisition time relative to the persistence time of intracellular transport and hence provides a measure of cellular activity. Cellular responses of 3-D multicellular spheroids to paclitaxel are compared among three different growth techniques: rotating bioreactor (BR), hanging-drop (HD), and nonadherent (U-bottom, UB) plate spheroids, compared with ex vivo living tissues. HD spheroids have the most homogeneous tissue, whereas BR spheroids display large sample-to-sample variability as well as spatial heterogeneity. The responses of BR-grown tumor spheroids to paclitaxel are more similar to those of ex vivo biopsies than the responses of spheroids grown using HD or plate methods. The rate of mitosis inhibition by application of taxol is measured through tissue dynamics spectroscopic imaging, demonstrating the ability to monitor antimitotic chemotherapy. These results illustrate the potential use of low-coherence digital holography for 3-D pharmaceutical screening applications.

Preliminary assessment of facial soft tissue thickness utilizing three-dimensional computed tomography models of living individuals.

PubMed

Parks, Connie L; Richard, Adam H; Monson, Keith L

2014-04-01

Facial approximation is the technique of developing a representation of the face from the skull of an unknown individual. Facial approximation relies heavily on average craniofacial soft tissue depths. For more than a century, researchers have employed a broad array of tissue depth collection methodologies, a practice which has resulted in a lack of standardization in craniofacial soft tissue depth research. To combat such methodological inconsistencies, Stephan and Simpson 2008 [15] examined and synthesized a large number of previously published soft tissue depth studies. Their comprehensive meta-analysis produced a pooled dataset of averaged tissue depths and a simplified methodology, which the researchers suggest be utilized as a minimum standard protocol for future craniofacial soft tissue depth research. The authors of the present paper collected craniofacial soft tissue depths using three-dimensional models generated from computed tomography scans of living males and females of four self-identified ancestry groups from the United States ranging in age from 18 to 62 years. This paper assesses the differences between: (i) the pooled mean tissue depth values from the sample utilized in this paper and those published by Stephan 2012 [21] and (ii) the mean tissue depth values of two demographically similar subsets of the sample utilized in this paper and those published by Rhine and Moore 1984 [16]. Statistical test results indicate that the tissue depths collected from the sample evaluated in this paper are significantly and consistently larger than those published by Stephan 2012 [21]. Although a lack of published variance data by Rhine and Moore 1984 [16] precluded a direct statistical assessment, a substantive difference was also concluded. Further, the dataset presented in this study is representative of modern American adults and is, therefore, appropriate for use in constructing contemporary facial approximations. Published by Elsevier Ireland Ltd.

Defects formation and spiral waves in a network of neurons in presence of electromagnetic induction.

PubMed

Rostami, Zahra; Jafari, Sajad

2018-04-01

Complex anatomical and physiological structure of an excitable tissue (e.g., cardiac tissue) in the body can represent different electrical activities through normal or abnormal behavior. Abnormalities of the excitable tissue coming from different biological reasons can lead to formation of some defects. Such defects can cause some successive waves that may end up to some additional reorganizing beating behaviors like spiral waves or target waves. In this study, formation of defects and the resulting emitted waves in an excitable tissue are investigated. We have considered a square array network of neurons with nearest-neighbor connections to describe the excitable tissue. Fundamentally, electrophysiological properties of ion currents in the body are responsible for exhibition of electrical spatiotemporal patterns. More precisely, fluctuation of accumulated ions inside and outside of cell causes variable electrical and magnetic field. Considering undeniable mutual effects of electrical field and magnetic field, we have proposed the new Hindmarsh-Rose (HR) neuronal model for the local dynamics of each individual neuron in the network. In this new neuronal model, the influence of magnetic flow on membrane potential is defined. This improved model holds more bifurcation parameters. Moreover, the dynamical behavior of the tissue is investigated in different states of quiescent, spiking, bursting and even chaotic state. The resulting spatiotemporal patterns are represented and the time series of some sampled neurons are displayed, as well.

Mesenchymal Stem Cell Levels of Human Spinal Tissues.

PubMed

Harris, Liam; Vangsness, C Thomas

2018-05-01

Systematic review. The aim of this study was to investigate, quantify, compare, and compile the various mesenchymal stem cell (MSC) tissue sources within human spinal tissues to act as a compendium for clinical and research application. Recent years have seen a dramatic increase in academic and clinical understanding of human MSCs. Previously limited to cells isolated from bone marrow, the past decade has illicited the characterization and isolation of human MSCs from adipose, bone marrow, synovium, muscle, periosteum, peripheral blood, umbilical cord, placenta, and numerous other tissues. As researchers explore practical applications of cells in these tissues, the absolute levels of MSCs in specific spinal tissue will be critical to guide future research. The PubMED, MEDLINE, EMBASE, and Cochrane databases were searched for articles relating to the harvest, characterization, isolation, and quantification of human MSCs from spinal tissues. Selected articles were examined for relevant data, categorized according to type of spinal tissue, and when possible, standardized to facilitate comparisons between sites. Human MSC levels varied widely between spinal tissues. Yields for intervertebral disc demonstrated roughly 5% of viable cells to be positive for MSC surface markers. Cartilage endplate cells yielded 18,500 to 61,875 cells/0.8 mm thick sample of cartilage end plate. Ligamentum flavum yielded 250,000 to 500,000 cells/g of tissue. Annulus fibrosus fluorescence activated cell sorting treatment found 29% of cells positive for MSC marker Stro-1. Nucleus pulposus yielded mean tissue samples of 40,584 to 234,137 MSCs per gram of tissue. Numerous tissues within and surrounding the spine represent a consistent and reliable source for the harvest and isolation of human MSCs. Among the tissues of the spine, the annulus fibrosus and ligamentum flavum each offer considerable levels of MSCs, and may prove comparable to that of bone marrow. 5.

Image analysis of representative food structures: application of the bootstrap method.

PubMed

Ramírez, Cristian; Germain, Juan C; Aguilera, José M

2009-08-01

Images (for example, photomicrographs) are routinely used as qualitative evidence of the microstructure of foods. In quantitative image analysis it is important to estimate the area (or volume) to be sampled, the field of view, and the resolution. The bootstrap method is proposed to estimate the size of the sampling area as a function of the coefficient of variation (CV(Bn)) and standard error (SE(Bn)) of the bootstrap taking sub-areas of different sizes. The bootstrap method was applied to simulated and real structures (apple tissue). For simulated structures, 10 computer-generated images were constructed containing 225 black circles (elements) and different coefficient of variation (CV(image)). For apple tissue, 8 images of apple tissue containing cellular cavities with different CV(image) were analyzed. Results confirmed that for simulated and real structures, increasing the size of the sampling area decreased the CV(Bn) and SE(Bn). Furthermore, there was a linear relationship between the CV(image) and CV(Bn) (.) For example, to obtain a CV(Bn) = 0.10 in an image with CV(image) = 0.60, a sampling area of 400 x 400 pixels (11% of whole image) was required, whereas if CV(image) = 1.46, a sampling area of 1000 x 100 pixels (69% of whole image) became necessary. This suggests that a large-size dispersion of element sizes in an image requires increasingly larger sampling areas or a larger number of images.

A national statistical survey assessment of mercury concentrations in fillets of fish collected in the U.S. EPA national rivers and streams assessment of the continental USA.

PubMed

Wathen, John B; Lazorchak, James M; Olsen, Anthony R; Batt, Angela

2015-03-01

The U.S. EPA conducted a national statistical survey of fish fillet tissue with a sample size of 541 sites on boatable rivers =>5th order in 2008-2009. This is the first such study of mercury (Hg) in fish tissue from river sites focused on potential impacts to human health from fish consumption to also address wildlife impacts. Sample sites were identified as being urban or non-urban. All sample mercury concentrations were above the 3.33ugkg(-1) (ppb) quantitation limit, and an estimated 25.4% (±4.4%) of the 51663 river miles assessed exceeded the U.S. EPA 300ugkg(-1) fish-tissue based water quality criterion for mercury, representing 13144±181.8 river miles. Estimates of river miles exceeding comparable aquatic life thresholds (translated from fillet concentrations to whole fish equivalents) in avian species were similar to the number of river miles exceeding the human health threshold, whereas some mammalian species were more at risk than human from lower mercury concentrations. A comparison of means from the non-urban and urban data and among three ecoregions did not indicate a statistically significant difference in fish tissue Hg concentrations at p<0.05. Published by Elsevier Ltd.

Biodistribution Analysis of Oncolytic Adenoviruses in Patient Autopsy Samples Reveals Vascular Transduction of Noninjected Tumors and Tissues.

PubMed

Koski, Anniina; Bramante, Simona; Kipar, Anja; Oksanen, Minna; Juhila, Juuso; Vassilev, Lotta; Joensuu, Timo; Kanerva, Anna; Hemminki, Akseli

2015-10-01

In clinical trials with oncolytic adenoviruses, there has been no mortality associated with treatment vectors. Likewise, in the Advanced Therapy Access Program (ATAP), where 290 patients were treated with 10 different viruses, no vector-related mortality was observed. However, as the patient population who received adenovirus treatments in ATAP represented heavily pretreated patients, often with very advanced disease, some patients died relatively soon after receiving their virus treatment mandating autopsy to investigate cause of death. Eleven such autopsies were performed and confirmed disease progression as the cause of death in each case. The regulatory requirement for investigating the safety of advanced therapy medical products presented a unique opportunity to study tissue samples collected as a routine part of the autopsies. Oncolytic adenoviral DNA was recovered in a wide range of tissues, including injected and noninjected tumors and various normal tissues, demonstrating the ability of the vector to disseminate through the vascular route. Furthermore, we recovered and cultured viable virus from samples of noninjected brain metastases of an intravenously treated patient, confirming that oncolytic adenovirus can reach tumors through the intravascular route. Data presented here give mechanistic insight into mode of action and biodistribution of oncolytic adenoviruses in cancer patients.

A comparison study of different excitation wavelengths to determine the relative content of key biomolecules in breast cancer and breast normal tissue

NASA Astrophysics Data System (ADS)

Sordillo, Laura A.; Sordillo, Peter P.; Budansky, Yury; Pu, Yang; Alfano, R. R.

2015-03-01

Fluorescence profiles from breast cancer and breast normal tissue samples with excitation wavelengths at 280 nm and 340 nm were obtained using the conventional LS-50 Perkin-Elmer spectrometer. Fluorescence ratios from these tissue samples, demonstrated by emission peaks at 340 nm, 440 nm and 460 nm and likely representing tryptophan and NADH, show increased relative content of tryptophan in malignant samples. Double ratio (DR) techniques were used to measure the severity of disease. The single excitation double ratio (Single-DR) method utilizes the emission intensity peaks from the spectrum acquired using a single excitation of 280 nm; while the dual excitation double ratio (dual-DR) method utilizes the emission intensity peaks from the spectra acquired using an excitation of 280 nm and 340 nm. Single-DR and dual-DR from 13 patients with breast carcinoma were compared in terms of their efficiency to distinguish high from low/intermediate tumors. Similar results were found with both methods. Results suggest that dual excitation wavelengths may be as effective as single excitation wavelength in calculating the relative content of biomolecules in breast cancer tissue, as well as for the assessment of the malignant potential of these tumors.

DALMATIAN: An Algorithm for Automatic Cell Detection and Counting in 3D.

PubMed

Shuvaev, Sergey A; Lazutkin, Alexander A; Kedrov, Alexander V; Anokhin, Konstantin V; Enikolopov, Grigori N; Koulakov, Alexei A

2017-01-01

Current 3D imaging methods, including optical projection tomography, light-sheet microscopy, block-face imaging, and serial two photon tomography enable visualization of large samples of biological tissue. Large volumes of data obtained at high resolution require development of automatic image processing techniques, such as algorithms for automatic cell detection or, more generally, point-like object detection. Current approaches to automated cell detection suffer from difficulties originating from detection of particular cell types, cell populations of different brightness, non-uniformly stained, and overlapping cells. In this study, we present a set of algorithms for robust automatic cell detection in 3D. Our algorithms are suitable for, but not limited to, whole brain regions and individual brain sections. We used watershed procedure to split regional maxima representing overlapping cells. We developed a bootstrap Gaussian fit procedure to evaluate the statistical significance of detected cells. We compared cell detection quality of our algorithm and other software using 42 samples, representing 6 staining and imaging techniques. The results provided by our algorithm matched manual expert quantification with signal-to-noise dependent confidence, including samples with cells of different brightness, non-uniformly stained, and overlapping cells for whole brain regions and individual tissue sections. Our algorithm provided the best cell detection quality among tested free and commercial software.

Quantification of tumor morphology via 3D histology: application to oral cavity cancers

NASA Astrophysics Data System (ADS)

Doyle, Scott; Brandwein-Gensler, Margaret; Tomaszewski, John

2016-03-01

Traditional histopathology quantifies disease through the study of glass slides, i.e. two-dimensional samples that are representative of the overall process. We hypothesize that 3D reconstruction can enhance our understanding of histopathologic interpretations. To test this hypothesis, we perform a pilot study of the risk model for oral cavity cancer (OCC), which stratifies patients into low-, intermediate-, and high-risk for locoregional disease-free survival. Classification is based on study of hematoxylin and eosin (H and E) stained tissues sampled from the resection specimens. In this model, the Worst Pattern of Invasion (WPOI) is assessed, representing specific architectural features at the interface between cancer and non-cancer tissue. Currently, assessment of WPOI is based on 2D sections of tissue, representing complex 3D structures of tumor growth. We believe that by reconstructing a 3D model of tumor growth and quantifying the tumor-host interface, we can obtain important diagnostic information that is difficult to assess in 2D. Therefore, we introduce a pilot study framework for visualizing tissue architecture and morphology in 3D from serial sections of histopathology. This framework can be used to enhance predictive models for diseases where severity is determined by 3D biological structure. In this work we utilize serial H and E-stained OCC resections obtained from 7 patients exhibiting WPOI-3 (low risk of recurrence) through WPOI-5 (high risk of recurrence). A supervised classifier automatically generates a map of tumor regions on each slide, which are then co-registered using an elastic deformation algorithm. A smooth 3D model of the tumor region is generated from the registered maps, which is suitable for quantitative tumor interface morphology feature extraction. We report our preliminary models created with this system and suggest further enhancements to traditional histology scoring mechanisms that take spatial architecture into consideration.

Mercury correlations among blood, muscle, and hair of northern elephant seals during the breeding and molting fasts

USGS Publications Warehouse

Peterson, Sarah; Ackerman, Joshua T.; Costa, Daniel P.

2016-01-01

Mercury (Hg) biomonitoring and toxicological risk assessments for marine mammals commonly sample different tissues, making comparisons to toxicity benchmarks and among species and regions difficult. Few studies have examined how life history events, such as fasting, influence the relationship between total Hg (THg) concentrations in different tissues. We evaluated the relationships between THg concentrations in blood, muscle, and hair of female and male northern elephant seals (Mirounga angustirostris) at the start and end of the breeding and molting fasts. The relationships between tissues varied among tissue pairs and differed by sampling period and sex. Blood and muscle were generally related at all time periods; however, hair, an inert tissue, did not strongly represent the metabolically active tissues (blood and muscle) at all times of year. The strongest relationships between THg concentrations in hair and those in blood or muscle were observed during periods of active hair growth (end of the molting period) or during time periods when internal body conditions were similar to those when the hair was grown (end of the breeding fast). Our results indicate that THg concentrations in blood or muscle can be translated to the other tissue type using the equations we developed, but that THg concentrations in hair were generally a poor index of internal THg concentrations except during the end of fasting periods.

Increased variability in ApcMin/+ intestinal tissue can be measured with microultrasound

NASA Astrophysics Data System (ADS)

Fatehullah, A.; Sharma, S.; Newton, I. P.; Langlands, A. J.; Lay, H.; Nelson, S. A.; McMahon, R. K.; McIlvenny, N.; Appleton, P. L.; Cochran, S.; Näthke, I. S.

2016-07-01

Altered tissue structure is a feature of many disease states and is usually measured by microscopic methods, limiting analysis to small areas. Means to rapidly and quantitatively measure the structure and organisation of large tissue areas would represent a major advance not just for research but also in the clinic. Here, changes in tissue organisation that result from heterozygosity in Apc, a precancerous situation, are comprehensively measured using microultrasound and three-dimensional high-resolution microscopy. Despite its normal appearance in conventionally examined cross-sections, both approaches revealed a significant increase in the variability of tissue organisation in Apc heterozygous tissue. These changes preceded the formation of aberrant crypt foci or adenoma. Measuring these premalignant changes using microultrasound provides a potential means to detect microscopically abnormal regions in large tissue samples, independent of visual examination or biopsies. Not only does this provide a powerful tool for studying tissue structure in experimental settings, the ability to detect and monitor tissue changes by microultrasound could be developed into a powerful adjunct to screening endoscopy in the clinic.

How Representative Are Research Tissue Biobanks of the Local Populations? Experience of the Infectious Diseases Biobank at King's College, London, UK.

PubMed

Kozlakidis, Zisis; Mant, Christine; Peters, Barry; Post, Frank; Fox, Julie; Philpott-Howard, John; Tong, William C-Y; Edgeworth, Jonathan; Peakman, Mark; Malim, Michael; Cason, John

2011-09-01

Biobanks have a primary responsibility to collect tissues that are a true reflection of their local population and thereby promote translational research, which is applicable to the community. The Infectious Diseases BioBank (IDB) at King's College London is located in the southeast of the city, an area that is ethnically diverse. Transplantation programs have frequently reported a low rate of donation among some ethnic minorities. To determine whether patients who volunteered peripheral venous blood samples to the IDB were representative of the local community, we compared local government demographic data to characteristics of patients who have donated to the IDB. There was a good match between these statistics, indicating that the IDB's volunteer population of human immunodeficiency virus patients was similar to local demographics.

How Representative Are Research Tissue Biobanks of the Local Populations? Experience of the Infectious Diseases Biobank at King's College, London, UK

PubMed Central

Kozlakidis, Zisis; Mant, Christine; Peters, Barry; Post, Frank; Fox, Julie; Philpott-Howard, John; Tong, William C.-Y.; Edgeworth, Jonathan; Peakman, Mark; Malim, Michael

2011-01-01

Biobanks have a primary responsibility to collect tissues that are a true reflection of their local population and thereby promote translational research, which is applicable to the community. The Infectious Diseases BioBank (IDB) at King's College London is located in the southeast of the city, an area that is ethnically diverse. Transplantation programs have frequently reported a low rate of donation among some ethnic minorities. To determine whether patients who volunteered peripheral venous blood samples to the IDB were representative of the local community, we compared local government demographic data to characteristics of patients who have donated to the IDB. There was a good match between these statistics, indicating that the IDB's volunteer population of human immunodeficiency virus patients was similar to local demographics. PMID:21977243

Analysis of the Macaca mulatta transcriptome and the sequence divergence between Macaca and human.

PubMed

Magness, Charles L; Fellin, P Campion; Thomas, Matthew J; Korth, Marcus J; Agy, Michael B; Proll, Sean C; Fitzgibbon, Matthew; Scherer, Christina A; Miner, Douglas G; Katze, Michael G; Iadonato, Shawn P

2005-01-01

We report the initial sequencing and comparative analysis of the Macaca mulatta transcriptome. Cloned sequences from 11 tissues, nine animals, and three species (M. mulatta, M. fascicularis, and M. nemestrina) were sampled, resulting in the generation of 48,642 sequence reads. These data represent an initial sampling of the putative rhesus orthologs for 6,216 human genes. Mean nucleotide diversity within M. mulatta and sequence divergence among M. fascicularis, M. nemestrina, and M. mulatta are also reported.

An unsupervised MVA method to compare specific regions in human breast tumor tissue samples using ToF-SIMS.

PubMed

Bluestein, Blake M; Morrish, Fionnuala; Graham, Daniel J; Guenthoer, Jamie; Hockenbery, David; Porter, Peggy L; Gamble, Lara J

2016-03-21

Imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) and principal component analysis (PCA) were used to investigate two sets of pre- and post-chemotherapy human breast tumor tissue sections to characterize lipids associated with tumor metabolic flexibility and response to treatment. The micron spatial resolution imaging capability of ToF-SIMS provides a powerful approach to attain spatially-resolved molecular and cellular data from cancerous tissues not available with conventional imaging techniques. Three ca. 1 mm(2) areas per tissue section were analyzed by stitching together 200 μm × 200 μm raster area scans. A method to isolate and analyze specific tissue regions of interest by utilizing PCA of ToF-SIMS images is presented, which allowed separation of cellularized areas from stromal areas. These PCA-generated regions of interest were then used as masks to reconstruct representative spectra from specifically stromal or cellular regions. The advantage of this unsupervised selection method is a reduction in scatter in the spectral PCA results when compared to analyzing all tissue areas or analyzing areas highlighted by a pathologist. Utilizing this method, stromal and cellular regions of breast tissue biopsies taken pre- versus post-chemotherapy demonstrate chemical separation using negatively-charged ion species. In this sample set, the cellular regions were predominantly all cancer cells. Fatty acids (i.e. palmitic, oleic, and stearic), monoacylglycerols, diacylglycerols and vitamin E profiles were distinctively different between the pre- and post-therapy tissues. These results validate a new unsupervised method to isolate and interpret biochemically distinct regions in cancer tissues using imaging ToF-SIMS data. In addition, the method developed here can provide a framework to compare a variety of tissue samples using imaging ToF-SIMS, especially where there is section-to-section variability that makes it difficult to use a serial hematoxylin and eosin (H&E) stained section to direct the SIMS analysis.

Analysis of iron, zinc, selenium and cadmium in paraffin-embedded prostate tissue specimens using inductively coupled plasma mass-spectrometry

USGS Publications Warehouse

Sarafanov, A.G.; Todorov, T.I.; Kajdacsy-Balla, A.; Gray, Michael A.; MacIas, V.; Centeno, J.A.

2008-01-01

Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a valuable and abundant resource of pathologic material for various biomedical studies. In the present study, we report the application of high-resolution inductively coupled mass-spectrometry (ICP-MS) for quantification of Fe, Zn, Se and Cd in FFPE prostate tissue. These elements have a possible role in the development of prostate diseases: while Zn and Se are needed for a healthy prostate, Cd shows multiple toxic and carcinogenic effects. Excessive accumulation of Fe induces the production of highly reactive hydroxyl radical species, which may play a role in cancer etiopathogenesis. To assess whether the levels of these metals in the FFPE prostate tissue represent their original content, we compared their levels with those in the fresh tissue (on dry weight basis) in samples obtained from 15 patients. We found that in FFPE tissue, the recoveries of Se, Fe, Cd and Zn were progressively decreased, 97??11% (r=0.88), 82??22% (r=0.86), 59??23% (r=0.69) and 24??11% (r=0.38), respectively. Thus, the use of correction factors, determined as k=0.16 for Se, k=0.20 for Fe, k=0.27 for Cd and k=0.67 for Zn, is required to estimate the retrospective levels of these elements in the parental non-processed fresh (wet) prostate tissue. The technique used in this study enables the analysis of archival FFPE prostate tissue for the concentrations of Fe, Zn, Se and Cd to study association between the levels of these metals and prostate disease. ?? 2008.

Molecular weight profiles of proanthocyanidin polymers

Treesearch

Vincent M. Williams; Lawrence J. Porter; Richard W. Hemingway

1983-01-01

The MW profiles of proanthocyanidin polymers (condensed tannins) from 32 samples representing a wide range of plant tissues of many different species have been obtained by gel permeation chromatography of the peracetate derivatives. The tannins vary widely in MW, with M values for the peracetates in the range 1600-5500. The MW profiles vary greatly from those with...

Statistical Survey of Persistent Organic Pollutants: Risk Estimations to Humans and Wildlife through Consumption of Fish from U.S. Rivers

EPA Science Inventory

U.S. EPA conducted a national statistical survey of fish tissue contamination at 540 river sites (representing 82 954 river km) in 2008–2009, and analyzed samples for 50 persistent organic pollutants (POPs), including 21 PCB congeners, 8 PBDE congeners, and 21 organoc...

Ptychographic X-ray nanotomography quantifies mineral distributions in human dentine

NASA Astrophysics Data System (ADS)

Zanette, I.; Enders, B.; Dierolf, M.; Thibault, P.; Gradl, R.; Diaz, A.; Guizar-Sicairos, M.; Menzel, A.; Pfeiffer, F.; Zaslansky, P.

2015-03-01

Bones are bio-composites with biologically tunable mechanical properties, where a polymer matrix of nanofibrillar collagen is reinforced by apatite mineral crystals. Some bones, such as antler, form and change rapidly, while other bone tissues, such as human tooth dentine, develop slowly and maintain constant composition and architecture for entire lifetimes. When studying apatite mineral microarchitecture, mineral distributions or mineralization activity of bone-forming cells, representative samples of tissue are best studied at submicrometre resolution while minimizing sample-preparation damage. Here, we demonstrate the power of ptychographic X-ray tomography to map variations in the mineral content distribution in three dimensions and at the nanometre scale. Using this non-destructive method, we observe nanostructures surrounding hollow tracts that exist in human dentine forming dentinal tubules. We reveal unprecedented quantitative details of the ultrastructure clearly revealing the spatially varying mineralization density. Such information is essential for understanding a variety of natural and therapeutic effects for example in bone tissue healing and ageing.

Boron concentration measurements by alpha spectrometry and quantitative neutron autoradiography in cells and tissues treated with different boronated formulations and administration protocols.

PubMed

Bortolussi, Silva; Ciani, Laura; Postuma, Ian; Protti, Nicoletta; Luca Reversi; Bruschi, Piero; Ferrari, Cinzia; Cansolino, Laura; Panza, Luigi; Ristori, Sandra; Altieri, Saverio

2014-06-01

The possibility to measure boron concentration with high precision in tissues that will be irradiated represents a fundamental step for a safe and effective BNCT treatment. In Pavia, two techniques have been used for this purpose, a quantitative method based on charged particles spectrometry and a boron biodistribution imaging based on neutron autoradiography. A quantitative method to determine boron concentration by neutron autoradiography has been recently set-up and calibrated for the measurement of biological samples, both solid and liquid, in the frame of the feasibility study of BNCT. This technique was calibrated and the obtained results were cross checked with those of α spectrometry, in order to validate them. The comparisons were performed using tissues taken form animals treated with different boron administration protocols. Subsequently the quantitative neutron autoradiography was employed to measure osteosarcoma cell samples treated with BPA and with new boronated formulations. © 2013 Published by Elsevier Ltd.

Inorganic elements in green sea turtles (Chelonia mydas): relationships among external and internal tissues.

PubMed

Faust, Derek R; Hooper, Michael J; Cobb, George P; Barnes, Melanie; Shaver, Donna; Ertolacci, Shauna; Smith, Philip N

2014-09-01

Inorganic elements from anthropogenic sources have entered marine environments worldwide and are detectable in marine organisms, including sea turtles. Threatened and endangered classifications of sea turtles have heretofore made assessments of contaminant concentrations difficult because of regulatory restrictions on obtaining samples using nonlethal techniques. In the present study, claw and skin biopsy samples were examined as potential indicators of internal tissue burdens in green sea turtles (Chelonia mydas). Significant relationships were observed between claw and liver, and claw and muscle concentrations of mercury, nickel, arsenic, and selenium (p < 0.05). Similarly, significant relationships were observed between skin biopsy concentrations and those in liver, kidney, and muscle tissues for mercury, arsenic, selenium, and vanadium (p < 0.05). Concentrations of arsenic, barium, chromium, nickel, strontium, vanadium, and zinc in claws and skin biopsies were substantially elevated when compared with all other tissues, indicating that these highly keratinized tissues may represent sequestration or excretion pathways. Correlations between standard carapace length and cobalt, lead, and manganese concentrations were observed (p < 0.05), indicating that tissue concentrations of these elements may be related to age and size. Results suggest that claws may indeed be useful indicators of mercury and nickel concentrations in liver and muscle tissues, whereas skin biopsy inorganic element concentrations may be better suited as indicators of mercury, selenium, and vanadium concentrations in liver, kidney, and muscle tissues of green sea turtles. © 2014 SETAC.

Hog Charm II tetracycline test screening results compared with a liquid chromatography tandem mass spectrometry 10-μg/kg method.

PubMed

Salter, Robert; Holmes, Steven; Legg, David; Coble, Joel; George, Bruce

2012-02-01

Pork tissue samples that tested positive and negative by the Charm II tetracycline test screening method in the slaughter plant laboratory were tested with the modified AOAC International liquid chromatography tandem mass spectrometry (LC-MS-MS) method 995.09 to determine the predictive value of the screening method at detecting total tetracyclines at 10 μg/kg of tissue, in compliance with Russian import regulations. There were 218 presumptive-positive tetracycline samples of 4,195 randomly tested hogs. Of these screening test positive samples, 83% (182) were positive, >10 μg/kg by LC-MS-MS; 12.8% (28) were false violative, greater than limit of detection (LOD) but <10 μg/kg; and 4.2% (8) were not detected at the LC-MS-MS LOD. The 36 false-violative and not-detected samples represent 1% of the total samples screened. Twenty-seven of 30 randomly selected tetracycline screening negative samples tested below the LC-MS-MS LOD, and 3 samples tested <3 μg/kg chlortetracycline. Results indicate that the Charm II tetracycline test is effective at predicting hogs containing >10 μg/kg total tetracyclines in compliance with Russian import regulations.

Structural and molecular interrogation of intact biological systems

PubMed Central

Chung, Kwanghun; Wallace, Jenelle; Kim, Sung-Yon; Kalyanasundaram, Sandhiya; Andalman, Aaron S.; Davidson, Thomas J.; Mirzabekov, Julie J.; Zalocusky, Kelly A.; Mattis, Joanna; Denisin, Aleksandra K.; Pak, Sally; Bernstein, Hannah; Ramakrishnan, Charu; Grosenick, Logan; Gradinaru, Viviana; Deisseroth, Karl

2014-01-01

Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease. PMID:23575631

Quantitative diagnosis of tongue cancer from histological images in an animal model

NASA Astrophysics Data System (ADS)

Lu, Guolan; Qin, Xulei; Wang, Dongsheng; Muller, Susan; Zhang, Hongzheng; Chen, Amy; Chen, Zhuo G.; Fei, Baowei

2016-03-01

We developed a chemically-induced oral cancer animal model and a computer aided method for tongue cancer diagnosis. The animal model allows us to monitor the progress of the lesions over time. Tongue tissue dissected from mice was sent for histological processing. Representative areas of hematoxylin and eosin stained tissue from tongue sections were captured for classifying tumor and non-tumor tissue. The image set used in this paper consisted of 214 color images (114 tumor and 100 normal tissue samples). A total of 738 color, texture, morphometry and topology features were extracted from the histological images. The combination of image features from epithelium tissue and its constituent nuclei and cytoplasm has been demonstrated to improve the classification results. With ten iteration nested cross validation, the method achieved an average sensitivity of 96.5% and a specificity of 99% for tongue cancer detection. The next step of this research is to apply this approach to human tissue for computer aided diagnosis of tongue cancer.

MicroRNA Expression in Laser Micro-dissected Breast Cancer Tissue Samples - a Pilot Study.

PubMed

Seclaman, Edward; Narita, Diana; Anghel, Andrei; Cireap, Natalia; Ilina, Razvan; Sirbu, Ioan Ovidiu; Marian, Catalin

2017-10-28

Breast cancer continues to represent a significant public health burden despite outstanding research advances regarding the molecular mechanisms of cancer biology, biomarkers for diagnostics and prognostic and therapeutic management of this disease. The studies of micro RNAs in breast cancer have underlined their potential as biomarkers and therapeutic targets; however most of these studies are still done on largely heterogeneous whole breast tissue samples. In this pilot study we have investigated the expression of four micro RNAs (miR-21, 145, 155, 92) known to be involved in breast cancer, in homogenous cell populations collected by laser capture microdissection from breast tissue section slides. Micro RNA expression was assessed by real time PCR, and associations with clinical and pathological characteristics were also explored. Our results have confirmed previous associations of miR-21 expression with poor prognosis characteristics of breast cancers such as high stage, large and highly proliferative tumors. No statistically significant associations were found with the other micro RNAs investigated, possibly due to the small sample size of our study. Our results also suggest that miR-484 could be a suitable endogenous control for data normalization in breast tissues, these results needing further confirmation by future studies. In summary, our pilot study showed the feasibility of detecting micro RNAs expression in homogenous laser captured microdissected invasive breast cancer samples, and confirmed some of the previously reported associations with poor prognostic characteristics of breast tumors.

Extraction of high-quality DNA from ethanol-preserved tropical plant tissues.

PubMed

Bressan, Eduardo A; Rossi, Mônica L; Gerald, Lee T S; Figueira, Antonio

2014-04-24

Proper conservation of plant samples, especially during remote field collection, is essential to assure quality of extracted DNA. Tropical plant species contain considerable amounts of secondary compounds, such as polysaccharides, phenols, and latex, which affect DNA quality during extraction. The suitability of ethanol (96% v/v) as a preservative solution prior to DNA extraction was evaluated using leaves of Jatropha curcas and other tropical species. Total DNA extracted from leaf samples stored in liquid nitrogen or ethanol from J. curcas and other tropical species (Theobroma cacao, Coffea arabica, Ricinus communis, Saccharum spp., and Solanum lycopersicon) was similar in quality, with high-molecular-weight DNA visualized by gel electrophoresis. DNA quality was confirmed by digestion with EcoRI or HindIII and by amplification of the ribosomal gene internal transcribed spacer region. Leaf tissue of J. curcas was analyzed by light and transmission electron microscopy before and after exposure to ethanol. Our results indicate that leaf samples can be successfully preserved in ethanol for long periods (30 days) as a viable method for fixation and conservation of DNA from leaves. The success of this technique is likely due to reduction or inactivation of secondary metabolites that could contaminate or degrade genomic DNA. Tissue conservation in 96% ethanol represents an attractive low-cost alternative to commonly used methods for preservation of samples for DNA extraction. This technique yields DNA of equivalent quality to that obtained from fresh or frozen tissue.

Extraction of high-quality DNA from ethanol-preserved tropical plant tissues

PubMed Central

2014-01-01

Background Proper conservation of plant samples, especially during remote field collection, is essential to assure quality of extracted DNA. Tropical plant species contain considerable amounts of secondary compounds, such as polysaccharides, phenols, and latex, which affect DNA quality during extraction. The suitability of ethanol (96% v/v) as a preservative solution prior to DNA extraction was evaluated using leaves of Jatropha curcas and other tropical species. Results Total DNA extracted from leaf samples stored in liquid nitrogen or ethanol from J. curcas and other tropical species (Theobroma cacao, Coffea arabica, Ricinus communis, Saccharum spp., and Solanum lycopersicon) was similar in quality, with high-molecular-weight DNA visualized by gel electrophoresis. DNA quality was confirmed by digestion with EcoRI or HindIII and by amplification of the ribosomal gene internal transcribed spacer region. Leaf tissue of J. curcas was analyzed by light and transmission electron microscopy before and after exposure to ethanol. Our results indicate that leaf samples can be successfully preserved in ethanol for long periods (30 days) as a viable method for fixation and conservation of DNA from leaves. The success of this technique is likely due to reduction or inactivation of secondary metabolites that could contaminate or degrade genomic DNA. Conclusions Tissue conservation in 96% ethanol represents an attractive low-cost alternative to commonly used methods for preservation of samples for DNA extraction. This technique yields DNA of equivalent quality to that obtained from fresh or frozen tissue. PMID:24761774

MicroRNAs are suitable for assessment as biomarkers from formalin-fixed paraffin-embedded tissue, and miR-24 represents an appropriate reference microRNA for diffuse large B-cell lymphoma studies.

PubMed

Culpin, Rachel Emily; Sieniawski, Michal; Proctor, Stephen John; Menon, Geetha; Mainou-Fowler, Tryfonia

2013-03-01

Tissue biopsy specimens in the form of formalin-fixed paraffin-embedded tissue (FFPET) represent a valuable resource for biomarker identification and validation. However, to date, they remain an underused asset due to uncertainty regarding RNA extraction and the reliability of downstream techniques, including quantitative RT-PCR. Recently, much interest has emerged in the study of microRNAs; small single-stranded RNAs with a role in transcriptional regulation, that are thought to be well preserved in FFPET. In this study, we show that microRNA expression is comparable between FFPET and matched fresh-frozen samples (miR-17-5p: p=0.01, miR-92: p=0.003), and demonstrate that no significant deterioration in expression occurs over prolonged FFPET storage (p=0.06). Furthermore, microRNA expression is equivalent dependant on RNA extraction method (p<0.001) or DNAse treatment of total RNA (p<0.001). Finally, we validate miR-24 as a suitable reference microRNA for diffuse large B-cell lymphoma (DLBCL) FFPET studies.

Suppression of Botrytis cinerea on necrotic grapevine tissues by early-season applications of natural products and biological control agents.

PubMed

Calvo-Garrido, Carlos; Viñas, Inmaculada; Elmer, Philip A G; Usall, Josep; Teixidó, Neus

2014-04-01

Necrotic tissues within grape (Vitis vinifera) bunches represent an important source of Botrytis cinerea inoculum for Botrytis bunch rot (BBR) at harvest in vineyards. This research quantified the incidence of B. cinerea on necrotic floral and fruit tissues and the efficacy of biologically based treatments for suppression of B. cinerea secondary inoculum within developing bunches. At veraison (2009 and 2010), samples of aborted flowers, aborted fruits and calyptras were collected, and the incidence and sporulation of B. cinerea were determined. Aborted fruits presented significantly higher incidence in untreated samples. Early-season applications of Candida sake plus Fungicover®, Fungicover alone or Ulocladium oudemansii significantly reduced B. cinerea incidence on aborted flowers and calyptras by 46-85%. Chitosan treatment significantly reduced B. cinerea incidence on calyptras. None of the treatments reduced B. cinerea incidence on aborted fruits. Treatments significantly reduced sporulation severity by 48% or more. Treatments were effective at reducing B. cinerea secondary inoculum on necrotic tissues, in spite of the variable control on aborted fruits. This is the first report to quantify B. cinerea on several tissues of bunch trash and to describe the effective suppression of saprophytic B. cinerea inoculum by biologically based treatments. © 2013 Society of Chemical Industry.

Pathobiological investigation of naturally infected canine rabies cases from Sri Lanka.

PubMed

Beck, S; Gunawardena, P; Horton, D L; Hicks, D J; Marston, D A; Ortiz-Pelaez, A; Fooks, A R; Núñez, A

2017-04-12

The recommended screening of rabies in 'suspect' animal cases involves testing fresh brain tissue. The preservation of fresh tissue however can be difficult under field conditions and formalin fixation provides a simple alternative that may allow a confirmatory diagnosis. The occurrence and location of histopathological changes and immunohistochemical (IHC) labelling for rabies in formalin fixed paraffin embedded (FFPE) canine brain is described in samples from 57 rabies suspect cases from Sri-Lanka. The presence of Negri bodies and immunohistochemical detection of rabies virus antigen were evaluated in the cortex, hippocampus, cerebellum and brainstem. The effect of autolysis and artefactual degeneration of the tissue was also assessed. Rabies was confirmed in 53 of 57 (93%) cases by IHC. IHC labelling was statistically more abundant in the brainstem. Negri bodies were observed in 32 of 53 (60.4%) of the positive cases. Although tissue degradation had no effect on IHC diagnosis, it was associated with an inability to detect Negri bodies. In 13 cases, a confirmatory Polymerase chain reaction (PCR) testing for rabies virus RNA was undertaken by extracting RNA from fresh frozen tissue, and also attempted using FFPE samples. PCR detection using fresh frozen samples was in agreement with the IHC results. The PCR method from FFPE tissues was suitable for control material but unsuccessful in our field cases. Histopathological examination of the brain is essential to define the differential diagnoses of behaviour modifying conditions in rabies virus negative cases, but it is unreliable as the sole method for rabies diagnosis, particularly where artefactual change has occurred. Formalin fixation and paraffin embedding does not prevent detection of rabies virus via IHC labelling even where artefactual degeneration has occurred. This could represent a pragmatic secondary assay for rabies diagnosis in the field because formalin fixation can prevent sample degeneration. The brain stem was shown to be the site with most viral immunoreactivity; supporting recommended sampling protocols in favour of improved necropsy safety in the field. PCR testing of formalin fixed tissue may be successful in certain circumstances as an alternative test.

Human iPSC-derived neurons and lymphoblastoid cells for personalized medicine research in neuropsychiatric disorders.

PubMed

Gurwitz, David

2016-09-01

The development and clinical implementation of personalized medicine crucially depends on the availability of high-quality human biosamples; animal models, although capable of modeling complex human diseases, cannot reflect the large variation in the human genome, epigenome, transcriptome, proteome, and metabolome. Although the biosamples available from public biobanks that store human tissues and cells may represent the large human diversity for most diseases, these samples are not always sufficient for developing biomarkers for patient-tailored therapies for neuropsychiatric disorders. Postmortem human tissues are available from many biobanks; nevertheless, collections of neuronal human cells from large patient cohorts representing the human diversity remain scarce. Two tools are gaining popularity for personalized medicine research on neuropsychiatric disorders: human induced pluripotent stem cell-derived neurons and human lymphoblastoid cell lines. This review examines and contrasts the advantages and limitations of each tool for personalized medicine research.

PIXE analysis of mineral matter in thin sections of human lung

NASA Astrophysics Data System (ADS)

Annegarn, H. J.; Pillay, A. E.; Da Vies, J. C. A.; Faure, D.; Sellschop, J. P. F.

1988-12-01

It is postulated that insoluble mineral residues in the lungs of deceased miners may provide a quantitative measure of the integrated lifetime dust exposure. For epidemiological surveys rapid instrumental techniques are required to analyse representative samples of lung tissue. Particle-induced X-ray emission (PIXE) has been evaluated for analysis of microtomed slices of wax-embedded lung and lymph node (Hilar gland) tissue from deceased miners. The 50 μm slices, mounted on Mylar backings and placed in a He atmosphere, were irradiated using 3.2 MeV protons. PIXE analysis provided adequate sensitivity for key mineral elements including Si, Cr and Ti. The porous, nonuniform nature of lung tissue made it impossible to measure the tissue mass in the irradiated area, preventing the calculation of mass concentrations. Instead, biological sulphur was used as an internal standard, assuming that the fraction of S in soft, fat-free tissue is constant. Results are presented for lung and lymph node tissue from gold, chrome, copper, platinum and asbestos miners. Si mineral residues in lymph node tissue were found to be concentrated by a factor 50 relative to lung. Cr residues were clearly observed in the chrome miner's lung, but no excess of Cu was present in the copper miner's lung. There is evidence of preferential Si removal relative to Ti. Results warrant further development of PIXE for scanning of large numbers of lung samples prepared in the above manner.

X-ray phase contrast imaging of the breast: Analysis of tissue simulating materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vedantham, Srinivasan; Karellas, Andrew

Purpose: Phase contrast imaging, particularly of the breast, is being actively investigated. The purpose of this work is to investigate the x-ray phase contrast properties of breast tissues and commonly used breast tissue substitutes or phantom materials with an aim of determining the phantom materials best representative of breast tissues. Methods: Elemental compositions of breast tissues including adipose, fibroglandular, and skin were used to determine the refractive index, n= 1 -{delta}+i {beta}. The real part of the refractive index, specifically the refractive index decrement ({delta}), over the energy range of 5-50 keV were determined using XOP software (version 2.3, Europeanmore » Synchrotron Radiation Facility, France). Calcium oxalate and calcium hydroxyapatite were considered to represent the material compositions of microcalcifications in vivo. Nineteen tissue substitutes were considered as possible candidates to represent adipose tissue, fibroglandular tissue and skin, and four phantom materials were considered as possible candidates to represent microcalcifications. For each material, either the molecular formula, if available, or the elemental composition based on weight fraction, was used to determine {delta}. At each x-ray photon energy, the absolute percent difference in {delta} between the breast tissue and the substitute material was determined, from which three candidates were selected. From these candidate tissue substitutes, the material that minimized the absolute percent difference in linear attenuation coefficient {mu}, and hence {beta}, was considered to be best representative of that breast tissue. Results: Over the energy range of 5-50 keV, while the {delta} of CB3 and fibroglandular tissue-equivalent material were within 1% of that of fibroglandular tissue, the {mu} of fibroglandular tissue-equivalent material better approximated the fibroglandular tissue. While the {delta} of BR10 and adipose tissue-equivalent material were within 1% of that of adipose tissue, the tissue-equivalent material better approximated the adipose tissue in terms of {mu}. Polymethyl methacrylate, a commonly used tissue substitute, exhibited {delta} greater than fibroglandular tissue by {approx}12%. The A-150 plastic closely approximated the skin. Several materials exhibited {delta} between that of adipose and fibroglandular tissue. However, there was an energy-dependent mismatch in terms of equivalent fibroglandular weight fraction between {delta} and {mu} for these materials. For microcalcifications, aluminum and calcium carbonate were observed to straddle the {delta} and {mu} of calcium oxalate and calcium hydroxyapatite. Aluminum oxide, commonly used to represent microcalcifications in the American College of Radiology recommended phantoms for accreditation exhibited {delta} greater than calcium hydroxyapatite by {approx}23%. Conclusions: A breast phantom comprising A-150 plastic to represent the skin, commercially available adipose and fibroglandular tissue-equivalent formulations to represent adipose and fibroglandular tissue, respectively, was found to be best suited for x-ray phase-sensitive imaging of the breast. Calcium carbonate or aluminum can be used to represent microcalcifications.« less

X-ray phase contrast imaging of the breast: Analysis of tissue simulating materials1

PubMed Central

Vedantham, Srinivasan; Karellas, Andrew

2013-01-01

Purpose: Phase contrast imaging, particularly of the breast, is being actively investigated. The purpose of this work is to investigate the x-ray phase contrast properties of breast tissues and commonly used breast tissue substitutes or phantom materials with an aim of determining the phantom materials best representative of breast tissues. Methods: Elemental compositions of breast tissues including adipose, fibroglandular, and skin were used to determine the refractive index, n = 1 − δ + i β. The real part of the refractive index, specifically the refractive index decrement (δ), over the energy range of 5–50 keV were determined using XOP software (version 2.3, European Synchrotron Radiation Facility, France). Calcium oxalate and calcium hydroxyapatite were considered to represent the material compositions of microcalcifications in vivo. Nineteen tissue substitutes were considered as possible candidates to represent adipose tissue, fibroglandular tissue and skin, and four phantom materials were considered as possible candidates to represent microcalcifications. For each material, either the molecular formula, if available, or the elemental composition based on weight fraction, was used to determine δ. At each x-ray photon energy, the absolute percent difference in δ between the breast tissue and the substitute material was determined, from which three candidates were selected. From these candidate tissue substitutes, the material that minimized the absolute percent difference in linear attenuation coefficient μ, and hence β, was considered to be best representative of that breast tissue. Results: Over the energy range of 5–50 keV, while the δ of CB3 and fibroglandular tissue-equivalent material were within 1% of that of fibroglandular tissue, the μ of fibroglandular tissue-equivalent material better approximated the fibroglandular tissue. While the δ of BR10 and adipose tissue-equivalent material were within 1% of that of adipose tissue, the tissue-equivalent material better approximated the adipose tissue in terms of μ. Polymethyl methacrylate, a commonly used tissue substitute, exhibited δ greater than fibroglandular tissue by ∼12%. The A-150 plastic closely approximated the skin. Several materials exhibited δ between that of adipose and fibroglandular tissue. However, there was an energy-dependent mismatch in terms of equivalent fibroglandular weight fraction between δ and μ for these materials. For microcalcifications, aluminum and calcium carbonate were observed to straddle the δ and μ of calcium oxalate and calcium hydroxyapatite. Aluminum oxide, commonly used to represent microcalcifications in the American College of Radiology recommended phantoms for accreditation exhibited δ greater than calcium hydroxyapatite by ∼23%. Conclusions: A breast phantom comprising A-150 plastic to represent the skin, commercially available adipose and fibroglandular tissue-equivalent formulations to represent adipose and fibroglandular tissue, respectively, was found to be best suited for x-ray phase-sensitive imaging of the breast. Calcium carbonate or aluminum can be used to represent microcalcifications. PMID:23556900

Bomb-curve radiocarbon measurement of recent biologic tissues and applications to wildlife forensics and stable isotope (paleo)ecology

NASA Astrophysics Data System (ADS)

Uno, Kevin T.; Quade, Jay; Fisher, Daniel C.; Wittemyer, George; Douglas-Hamilton, Iain; Andanje, Samuel; Omondi, Patrick; Litoroh, Moses; Cerling, Thure E.

2013-07-01

Above-ground thermonuclear weapons testing from 1952 through 1962 nearly doubled the concentration of radiocarbon (14C) in the atmosphere. As a result, organic material formed during or after this period may be radiocarbon-dated using the abrupt rise and steady fall of the atmospheric 14C concentration known as the bomb-curve. We test the accuracy of accelerator mass spectrometry radiocarbon dating of 29 herbivore and plant tissues collected on known dates between 1905 and 2008 in East Africa. Herbivore samples include teeth, tusks, soft tissue, hair, and horn. Tissues formed after 1955 are dated to within 0.3-1.3 y of formation, depending on the tissue type, whereas tissues older than ca. 1955 have high age uncertainties (>17 y) due to the Suess effect. 14C dating of tissues has applications to stable isotope (paleo)ecology and wildlife forensics. We use data from 41 additional samples to determine growth rates of tusks, molars, and hair, which improve interpretations of serial stable isotope data for (paleo)ecological studies. 14C dating can also be used to calculate the time interval represented in periodic histological structures in dental tissues (i.e., perikymata), which in turn may be used as chronometers in fossil teeth. Bomb-curve 14C dating of confiscated animal tissues (e.g., ivory statues) can be used to determine whether trade of the item is legal, because many Convention of International Trade of Endangered Species restrictions are based on the age of the tissue, and thus can serve as a powerful forensic tool to combat illegal trade in animal parts.

Bomb-curve radiocarbon measurement of recent biologic tissues and applications to wildlife forensics and stable isotope (paleo)ecology

PubMed Central

Uno, Kevin T.; Quade, Jay; Fisher, Daniel C.; Wittemyer, George; Douglas-Hamilton, Iain; Andanje, Samuel; Omondi, Patrick; Litoroh, Moses; Cerling, Thure E.

2013-01-01

Above-ground thermonuclear weapons testing from 1952 through 1962 nearly doubled the concentration of radiocarbon (14C) in the atmosphere. As a result, organic material formed during or after this period may be radiocarbon-dated using the abrupt rise and steady fall of the atmospheric 14C concentration known as the bomb-curve. We test the accuracy of accelerator mass spectrometry radiocarbon dating of 29 herbivore and plant tissues collected on known dates between 1905 and 2008 in East Africa. Herbivore samples include teeth, tusks, soft tissue, hair, and horn. Tissues formed after 1955 are dated to within 0.3–1.3 y of formation, depending on the tissue type, whereas tissues older than ca. 1955 have high age uncertainties (>17 y) due to the Suess effect. 14C dating of tissues has applications to stable isotope (paleo)ecology and wildlife forensics. We use data from 41 additional samples to determine growth rates of tusks, molars, and hair, which improve interpretations of serial stable isotope data for (paleo)ecological studies. 14C dating can also be used to calculate the time interval represented in periodic histological structures in dental tissues (i.e., perikymata), which in turn may be used as chronometers in fossil teeth. Bomb-curve 14C dating of confiscated animal tissues (e.g., ivory statues) can be used to determine whether trade of the item is legal, because many Convention of International Trade of Endangered Species restrictions are based on the age of the tissue, and thus can serve as a powerful forensic tool to combat illegal trade in animal parts. PMID:23818577

Bomb-curve radiocarbon measurement of recent biologic tissues and applications to wildlife forensics and stable isotope (paleo)ecology.

PubMed

Uno, Kevin T; Quade, Jay; Fisher, Daniel C; Wittemyer, George; Douglas-Hamilton, Iain; Andanje, Samuel; Omondi, Patrick; Litoroh, Moses; Cerling, Thure E

2013-07-16

Above-ground thermonuclear weapons testing from 1952 through 1962 nearly doubled the concentration of radiocarbon ((14)C) in the atmosphere. As a result, organic material formed during or after this period may be radiocarbon-dated using the abrupt rise and steady fall of the atmospheric (14)C concentration known as the bomb-curve. We test the accuracy of accelerator mass spectrometry radiocarbon dating of 29 herbivore and plant tissues collected on known dates between 1905 and 2008 in East Africa. Herbivore samples include teeth, tusks, soft tissue, hair, and horn. Tissues formed after 1955 are dated to within 0.3-1.3 y of formation, depending on the tissue type, whereas tissues older than ca. 1955 have high age uncertainties (>17 y) due to the Suess effect. (14)C dating of tissues has applications to stable isotope (paleo)ecology and wildlife forensics. We use data from 41 additional samples to determine growth rates of tusks, molars, and hair, which improve interpretations of serial stable isotope data for (paleo)ecological studies. (14)C dating can also be used to calculate the time interval represented in periodic histological structures in dental tissues (i.e., perikymata), which in turn may be used as chronometers in fossil teeth. Bomb-curve (14)C dating of confiscated animal tissues (e.g., ivory statues) can be used to determine whether trade of the item is legal, because many Convention of International Trade of Endangered Species restrictions are based on the age of the tissue, and thus can serve as a powerful forensic tool to combat illegal trade in animal parts.

Positive cell-free fetal DNA testing for trisomy 13 reveals confined placental mosaicism.

PubMed

Hall, April L; Drendel, Holli M; Verbrugge, Jennifer L; Reese, Angela M; Schumacher, Katherine L; Griffith, Christopher B; Weaver, David D; Abernathy, Mary P; Litton, Christian G; Vance, Gail H

2013-09-01

We report on a case in which cell-free fetal DNA was positive for trisomy 13 most likely due to confined placental mosaicism. Cell-free fetal DNA testing analyzes DNA derived from placental trophoblast cells and can lead to incorrect results that are not representative of the fetus. We sought to confirm commercial cell-free fetal DNA testing results by chorionic villus sampling and amniocentesis. These results were followed up by postnatal chromosome analysis of cord blood and placental tissue. First-trimester cell-free fetal DNA test results were positive for trisomy 13. Cytogenetic analysis of chorionic villus sampling yielded a mosaic karyotype of 47,XY,+13[10]/46,XY[12]. G-banded analysis of amniotic fluid was normal, 46,XY. Postnatal cytogenetic analysis of cord blood was normal. Karyotyping of tissues from four quadrants of the placenta demonstrated mosaicism for trisomy 13 in two of the quadrants and a normal karyotype in the other two. Our case illustrates several important aspects of this new testing methodology: that cell-free fetal DNA may not be representative of the fetal karyotype; that follow-up with diagnostic testing of chorionic villus sampling and/or amniotic fluid for abnormal test results should be performed; and that pretest counseling regarding the full benefits, limitations, and possible testing outcomes of cell-free fetal DNA screening is important.

Isolation and characterization of canine perivascular stem/stromal cells for bone tissue engineering.

PubMed

James, Aaron W; Zhang, Xinli; Crisan, Mihaela; Hardy, Winters R; Liang, Pei; Meyers, Carolyn A; Lobo, Sonja; Lagishetty, Venu; Childers, Martin K; Asatrian, Greg; Ding, Catherine; Yen, Yu-Hsin; Zou, Erin; Ting, Kang; Peault, Bruno; Soo, Chia

2017-01-01

For over 15 years, human subcutaneous adipose tissue has been recognized as a rich source of tissue resident mesenchymal stem/stromal cells (MSC). The isolation of perivascular progenitor cells from human adipose tissue by a cell sorting strategy was first published in 2008. Since this time, the interest in using pericytes and related perivascular stem/stromal cell (PSC) populations for tissue engineering has significantly increased. Here, we describe a set of experiments identifying, isolating and characterizing PSC from canine tissue (N = 12 canine adipose tissue samples). Results showed that the same antibodies used for human PSC identification and isolation are cross-reactive with canine tissue (CD45, CD146, CD34). Like their human correlate, canine PSC demonstrate characteristics of MSC including cell surface marker expression, colony forming unit-fibroblast (CFU-F) inclusion, and osteogenic differentiation potential. As well, canine PSC respond to osteoinductive signals in a similar fashion as do human PSC, such as the secreted differentiation factor NEL-Like Molecule-1 (NELL-1). Nevertheless, important differences exist between human and canine PSC, including differences in baseline osteogenic potential. In summary, canine PSC represent a multipotent mesenchymogenic cell source for future translational efforts in tissue engineering.

RNA-Seq-based toxicogenomic assessment of fresh frozen and formalin-fixed tissues yields similar mechanistic insights.

PubMed

Auerbach, Scott S; Phadke, Dhiral P; Mav, Deepak; Holmgren, Stephanie; Gao, Yuan; Xie, Bin; Shin, Joo Heon; Shah, Ruchir R; Merrick, B Alex; Tice, Raymond R

2015-07-01

Formalin-fixed, paraffin-embedded (FFPE) pathology specimens represent a potentially vast resource for transcriptomic-based biomarker discovery. We present here a comparison of results from a whole transcriptome RNA-Seq analysis of RNA extracted from fresh frozen and FFPE livers. The samples were derived from rats exposed to aflatoxin B1 (AFB1 ) and a corresponding set of control animals. Principal components analysis indicated that samples were separated in the two groups representing presence or absence of chemical exposure, both in fresh frozen and FFPE sample types. Sixty-five percent of the differentially expressed transcripts (AFB1 vs. controls) in fresh frozen samples were also differentially expressed in FFPE samples (overlap significance: P < 0.0001). Genomic signature and gene set analysis of AFB1 differentially expressed transcript lists indicated highly similar results between fresh frozen and FFPE at the level of chemogenomic signatures (i.e., single chemical/dose/duration elicited transcriptomic signatures), mechanistic and pathology signatures, biological processes, canonical pathways and transcription factor networks. Overall, our results suggest that similar hypotheses about the biological mechanism of toxicity would be formulated from fresh frozen and FFPE samples. These results indicate that phenotypically anchored archival specimens represent a potentially informative resource for signature-based biomarker discovery and mechanistic characterization of toxicity. Copyright © 2014 John Wiley & Sons, Ltd.

Feasibility of contrast-enhanced ultrasound-guided biopsy of sentinel lymph nodes in dogs.

PubMed

Gelb, Hylton R; Freeman, Lynetta J; Rohleder, Jacob J; Snyder, Paul W

2010-01-01

Our goal was to develop and validate a technique to identify the sentinel lymph nodes of the mammary glands of healthy dogs with contrast-enhanced ultrasound, and evaluate the feasibility of obtaining representative samples of a sentinel lymph node under ultrasound guidance using a new biopsy device. Three healthy intact female adult hounds were anesthetized and each received an injection of octafluoropropane-filled lipid microspheres and a separate subcutaneous injection of methylene blue dye around a mammary gland. Ultrasound was then used to follow the contrast agent through the lymphatic channel to the sentinel lymph node. Lymph node biopsy was performed under ultrasound guidance, followed by an excisional biopsy of the lymph nodes and a regional mastectomy procedure. Excised tissues were submitted for histopathologic examination and evaluated as to whether they were representative of the node. The ultrasound contrast agent was easily visualized with ultrasound leading up to the sentinel lymph nodes. Eight normal lymph nodes (two inguinal, one axillary in two dogs; two inguinal in one dog) were identified and biopsied. Lymphoid tissue was obtained from all biopsy specimens. Samples from four of eight lymph nodes contained both cortical and medullary lymphoid tissue. Contrast-enhanced ultrasound can be successfully used to image and guide minimally invasive biopsy of the normal sentinel lymph nodes draining the mammary glands in healthy dogs. Further work is needed to evaluate whether this technique may be applicable in patients with breast cancer or other conditions warranting evaluation of sentinel lymph nodes in animals.

Photoacoustic image-guided navigation system for surgery (Conference Presentation)

NASA Astrophysics Data System (ADS)

Park, Sara; Jang, Jongseong; Kim, Jeesu; Kim, Young Soo; Kim, Chulhong

2017-03-01

Identifying and delineating invisible anatomical and pathological details during surgery guides surgical procedures in real time. Various intraoperative imaging modalities have been increasingly employed to minimize such surgical risks as anatomical changes, damage to normal tissues, and human error. However, current methods provide only structural information, which cannot identify critical structures such as blood vessels. The logical next step is an intraoperative imaging modality that can provide functional information. Here, we have successfully developed a photoacoustic (PA) image-guided navigation system for surgery by integrating a position tracking system and a real-time clinical photoacoustic/ultrasound (PA/US) imaging system. PA/US images were acquired in real time and overlaid on pre-acquired cross-sectional magnetic resonance (MR) images. In the overlaid images, PA images represent the optical absorption characteristics of the surgical field, while US and MR images represent the morphological structure of surrounding tissues. To test the feasibility of the system, we prepared a tissue mimicking phantom which contained two samples, methylene blue as a contrast agent and water as a control. We acquired real-time overlaid PA/US/MR images of the phantom, which were well-matched with the optical and morphological properties of the samples. The developed system is the first approach to a novel intraoperative imaging technology based on PA imaging, and we believe that the system can be utilized in various surgical environments in the near future, improving the efficacy of surgical guidance.

The pituitary gland under infrared light - in search of a representative spectrum for homogeneous regions.

PubMed

Banas, A; Banas, K; Furgal-Borzych, A; Kwiatek, W M; Pawlicki, B; Breese, M B H

2015-04-07

The pituitary gland is a small but vital organ in the human body. It is located at the base of the brain and is often described as the master gland due to its multiple functions. The pituitary gland secretes and stores hormones, such as the thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), growth hormone (hGH), prolactin, gonadotropins, and luteinizing hormones, as well as the antidiuretic hormone (ADH). A proper diagnosis of pituitary disorders is of utmost importance as this organ participates in regulating a variety of body functions. Typical histopathological analysis provides much valuable information, but it gives no insight into the biochemical background of the changes that occur within the gland. One approach that could be used to evaluate the biochemistry of tissue sections obtained from pituitary disorders is Fourier Transform Infra-Red (FTIR) spectromicroscopy. In order to collect diagnostically valuable information large areas of tissue must be investigated. This work focuses on obtaining a unique and representative FTIR spectrum characteristic of one type of cell architecture within a sample. The idea presented is based on using hierarchical cluster analysis (HCA) for data evaluation to search for uniform patterns within samples from the perspective of FTIR spectra. The results obtained demonstrate that FTIR spectromicroscopy, combined with proper statistical evaluation, can be treated as a complementary method for histopathological analysis and ipso facto can increase the sensitivity and specificity for detecting various disorders not only for the pituitary gland, but also for other human tissues.

Raman spectroscopy and imaging: applications in human breast cancer diagnosis.

PubMed

Brozek-Pluska, Beata; Musial, Jacek; Kordek, Radzislaw; Bailo, Elena; Dieing, Thomas; Abramczyk, Halina

2012-08-21

The applications of spectroscopic methods in cancer detection open new possibilities in early stage diagnostics. Raman spectroscopy and Raman imaging represent novel and rapidly developing tools in cancer diagnosis. In the study described in this paper Raman spectroscopy has been employed to examine noncancerous and cancerous human breast tissues of the same patient. The most significant differences between noncancerous and cancerous tissues were found in regions characteristic for the vibrations of carotenoids, lipids and proteins. Particular attention was paid to the role played by unsaturated fatty acids in the differentiation between the noncancerous and the cancerous tissues. Comparison of Raman spectra of the noncancerous and the cancerous tissues with the spectra of oleic, linoleic, α-linolenic, γ-linolenic, docosahexaenoic and eicosapentaenoic acids has been presented. The role of sample preparation in the determination of cancer markers is also discussed in this study.

Transmission geometry laserspray ionization vacuum using an atmospheric pressure inlet.

PubMed

Lutomski, Corinne A; El-Baba, Tarick J; Inutan, Ellen D; Manly, Cory D; Wager-Miller, James; Mackie, Ken; Trimpin, Sarah

2014-07-01

This represents the first report of laserspray ionization vacuum (LSIV) with operation directly from atmospheric pressure for use in mass spectrometry. Two different types of electrospray ionization source inlets were converted to LSIV sources by equipping the entrance of the atmospheric pressure inlet aperture with a customized cone that is sealed with a removable glass plate holding the matrix/analyte sample. A laser aligned in transmission geometry (at 180° relative to the inlet) ablates the matrix/analyte sample deposited on the vacuum side of the glass slide. Laser ablation from vacuum requires lower inlet temperature relative to laser ablation at atmospheric pressure. However, higher inlet temperature is required for high-mass analytes, for example, α-chymotrypsinogen (25.6 kDa). Labile compounds such as gangliosides and cardiolipins are detected in the negative ion mode directly from mouse brain tissue as intact doubly deprotonated ions. Multiple charging enhances the ion mobility spectrometry separation of ions derived from complex tissue samples.

Transmission Geometry Laserspray Ionization Vacuum Using an Atmospheric Pressure Inlet

PubMed Central

2015-01-01

This represents the first report of laserspray ionization vacuum (LSIV) with operation directly from atmospheric pressure for use in mass spectrometry. Two different types of electrospray ionization source inlets were converted to LSIV sources by equipping the entrance of the atmospheric pressure inlet aperture with a customized cone that is sealed with a removable glass plate holding the matrix/analyte sample. A laser aligned in transmission geometry (at 180° relative to the inlet) ablates the matrix/analyte sample deposited on the vacuum side of the glass slide. Laser ablation from vacuum requires lower inlet temperature relative to laser ablation at atmospheric pressure. However, higher inlet temperature is required for high-mass analytes, for example, α-chymotrypsinogen (25.6 kDa). Labile compounds such as gangliosides and cardiolipins are detected in the negative ion mode directly from mouse brain tissue as intact doubly deprotonated ions. Multiple charging enhances the ion mobility spectrometry separation of ions derived from complex tissue samples. PMID:24896880

Transplantation of human fetal tissue for neurodegenerative diseases: validation of a new protocol for microbiological analysis and bacterial decontamination.

PubMed

Piroth, Tobias; Pauly, Marie-Christin; Schneider, Christian; Wittmer, Annette; Möllers, Sven; Döbrössy, Máté; Winkler, Christian; Nikkhah, Guido

2014-01-01

Restorative cell therapy concepts in neurodegenerative diseases are aimed at replacing lost neurons. Despite advances in research on pluripotent stem cells, fetal tissue from routine elective abortions is still regarded as the only safe cell source. Progenitor cells isolated from distinct first-trimester fetal CNS regions have already been used in clinical trials and will be used again in a new multicenter trial funded by the European Union (TRANSEURO). Bacterial contamination of human fetal tissue poses a potential risk of causing infections in the brain of the recipient. Thus, effective methods of microbial decontamination and validation of these methods are required prior to approval of a neurorestorative cell therapy trial. We have developed a protocol consisting of subsequent washing steps at different stages of tissue processing. Efficacy of microbial decontamination was assessed on rat embryonic tissue incubated with high concentrations of defined microbe solutions including representative bacterial and fungal species. Experimental microbial contamination was reduced by several log ranks. Subsequently, we have analyzed the spectrum of microbial contamination and the effect of subsequent washing steps on aborted human fetal tissue; 47.7% of the samples taken during human fetal tissue processing were positive for a microbial contamination, but after washing, no sample exhibited bacterial growth. Our data suggest that human fetal tissue for neural repair can carry microbes of various species, highlighting the need for decontamination procedures. The decontamination protocol described in this report has been shown to be effective as no microbes could be detected at the end of the procedure.

Evidence for plant-derived xenomiRs based on a large-scale analysis of public small RNA sequencing data from human samples.

PubMed

Zhao, Qi; Liu, Yuanning; Zhang, Ning; Hu, Menghan; Zhang, Hao; Joshi, Trupti; Xu, Dong

2018-01-01

In recent years, an increasing number of studies have reported the presence of plant miRNAs in human samples, which resulted in a hypothesis asserting the existence of plant-derived exogenous microRNA (xenomiR). However, this hypothesis is not widely accepted in the scientific community due to possible sample contamination and the small sample size with lack of rigorous statistical analysis. This study provides a systematic statistical test that can validate (or invalidate) the plant-derived xenomiR hypothesis by analyzing 388 small RNA sequencing data from human samples in 11 types of body fluids/tissues. A total of 166 types of plant miRNAs were found in at least one human sample, of which 14 plant miRNAs represented more than 80% of the total plant miRNAs abundance in human samples. Plant miRNA profiles were characterized to be tissue-specific in different human samples. Meanwhile, the plant miRNAs identified from microbiome have an insignificant abundance compared to those from humans, while plant miRNA profiles in human samples were significantly different from those in plants, suggesting that sample contamination is an unlikely reason for all the plant miRNAs detected in human samples. This study also provides a set of testable synthetic miRNAs with isotopes that can be detected in situ after being fed to animals.

Fiber Angle and Aspect Ratio Influence the Shear Mechanics of Oriented Electrospun Nanofibrous Scaffolds

PubMed Central

Driscoll, Tristan P.; Nerurkar, Nandan L.; Jacobs, Nathan T.; Elliott, Dawn M.; Mauck, Robert L.

2011-01-01

Fibrocartilages, including the knee meniscus and the annulus fibrosus (AF) of the intervertebral disc, play critical mechanical roles in load transmission across joints and their function is dependent upon well-defined structural hierarchies, organization, and composition. All, however, are compromised in the pathologic transformations associated with tissue degeneration. Tissue engineering strategies that address these key features, for example, aligned nanofibrous scaffolds seeded with mesenchymal stem cells (MSCs), represent a promising approach for the regeneration of these fibrous structures. While such engineered constructs can replicate native tissue structure and uniaxial tensile properties, the multidirectional loading encountered by these tissues in vivo necessitates that they function adequately in other loading modalities as well, including shear. As previous findings have shown that native tissue tensile and shear properties are dependent on fiber angle and sample aspect ratio, respectively, the objective of the present study was to evaluate the effects of a changing fiber angle and sample aspect ratio on the shear properties of aligned electrospun poly(ε-caprolactone) (PCL) scaffolds, and to determine how extracellular matrix deposition by resident MSCs modulates the measured shear response. Results show that fiber orientation and sample aspect ratio significantly influence the response of scaffolds in shear, and that measured shear strains can be predicted by finite element models. Furthermore, acellular PCL scaffolds possessed a relatively high shear modulus, 2–4 fold greater than native tissue, independent of fiber angle and aspect ratio. It was further noted that under testing conditions that engendered significant fiber stretch, the aggregate resistance to shear was higher, indicating a role for fiber stretch in the overall shear response. Finally, with time in culture, the shear modulus of MSC laden constructs increased, suggesting that deposited ECM contributes to the construct shear properties. Collectively, these findings show that aligned electrospun PCL scaffolds are a promising tool for engineering fibrocartilage tissues, and that the shear properties of both acellular and cell-seeded formulations can match or exceed native tissue benchmarks. PMID:22098865

Structural and functional studies of bioobjects prepared from femoral heads

NASA Astrophysics Data System (ADS)

Kirilova, I. A.; Sharkeev, Yu. P.; Podorozhnaya, V. T.; Popova, K. S.; Uvarkin, P. V.

2015-11-01

Results of examination of physicomechanical characteristics of samples of medial femoral head cuts are presented. The samples of medial femoral head cuts resected in 6 patients with coxarthrosis in primary endoprosthetic replacement of a coxofemoral joint have been tested for micro- and nanohardness. Young's modulus and elemental composition of bone tissue have been investigated. To estimate the architectonics of cancellous tissue of the femoral head, adjacent cuts of the same patient have been analyzed. The porosity of bone tissue was estimated from macroscopic images obtained using macrophotography. The total porosity is calculated as the ratio of the total length of straight line segments overlapping pores to the total length of secants. A three-point bending test of the samples has shown that their strength changed from 0.187 to 1.650 MPa and their elasticity modulus changes from 1.69 to 8.15 MPa. The microhardness of the samples changes in the range 220-265 MPa and the average microhardness of medial femoral head cuts is 240 MPa. The elemental composition of medial femoral head cuts is represented by basic Ca, P, O, Na and Mg elements as well as by Sn, S, Fe, Cr, and C in microamounts. The atomic Ca to P ratio for bone tissue is 1.55. It is revealed that pores of the upper part of the femoral head have a more regular shape and in the lower part they are more elongated along the cut and occupy a larger volume. The lower part of the femoral head has a higher porosity (39 and 33%) than the upper part (34 and 30%). The total porosity of all samples does not exceed 37%.

Long-term room temperature preservation of corpse soft tissue: an approach for tissue sample storage

PubMed Central

2011-01-01

Background Disaster victim identification (DVI) represents one of the most difficult challenges in forensic sciences, and subsequent DNA typing is essential. Collected samples for DNA-based human identification are usually stored at low temperature to halt the degradation processes of human remains. We have developed a simple and reliable procedure for soft tissue storage and preservation for DNA extraction. It ensures high quality DNA suitable for PCR-based DNA typing after at least 1 year of room temperature storage. Methods Fragments of human psoas muscle were exposed to three different environmental conditions for diverse time periods at room temperature. Storage conditions included: (a) a preserving medium consisting of solid sodium chloride (salt), (b) no additional substances and (c) garden soil. DNA was extracted with proteinase K/SDS followed by organic solvent treatment and concentration by centrifugal filter devices. Quantification was carried out by real-time PCR using commercial kits. Short tandem repeat (STR) typing profiles were analysed with 'expert software'. Results DNA quantities recovered from samples stored in salt were similar up to the complete storage time and underscored the effectiveness of the preservation method. It was possible to reliably and accurately type different genetic systems including autosomal STRs and mitochondrial and Y-chromosome haplogroups. Autosomal STR typing quality was evaluated by expert software, denoting high quality profiles from DNA samples obtained from corpse tissue stored in salt for up to 365 days. Conclusions The procedure proposed herein is a cost efficient alternative for storage of human remains in challenging environmental areas, such as mass disaster locations, mass graves and exhumations. This technique should be considered as an additional method for sample storage when preservation of DNA integrity is required for PCR-based DNA typing. PMID:21846338

Structural and functional studies of bioobjects prepared from femoral heads

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirilova, I. A., E-mail: IKirilova@niito.ru; Podorozhnaya, V. T., E-mail: VPodorognaya@niito.ru; Sharkeev, Yu. P., E-mail: sharkeev@ispms.tsc.ru

2015-11-17

Results of examination of physicomechanical characteristics of samples of medial femoral head cuts are presented. The samples of medial femoral head cuts resected in 6 patients with coxarthrosis in primary endoprosthetic replacement of a coxofemoral joint have been tested for micro- and nanohardness. Young’s modulus and elemental composition of bone tissue have been investigated. To estimate the architectonics of cancellous tissue of the femoral head, adjacent cuts of the same patient have been analyzed. The porosity of bone tissue was estimated from macroscopic images obtained using macrophotography. The total porosity is calculated as the ratio of the total length ofmore » straight line segments overlapping pores to the total length of secants. A three-point bending test of the samples has shown that their strength changed from 0.187 to 1.650 MPa and their elasticity modulus changes from 1.69 to 8.15 MPa. The microhardness of the samples changes in the range 220–265 MPa and the average microhardness of medial femoral head cuts is 240 MPa. The elemental composition of medial femoral head cuts is represented by basic Ca, P, O, Na and Mg elements as well as by Sn, S, Fe, Cr, and C in microamounts. The atomic Ca to P ratio for bone tissue is 1.55. It is revealed that pores of the upper part of the femoral head have a more regular shape and in the lower part they are more elongated along the cut and occupy a larger volume. The lower part of the femoral head has a higher porosity (39 and 33%) than the upper part (34 and 30%). The total porosity of all samples does not exceed 37%.« less

Potential interactions among disease, pesticides, water quality and adjacent land cover in amphibian habitats in the United States.

PubMed

Battaglin, W A; Smalling, K L; Anderson, C; Calhoun, D; Chestnut, T; Muths, E

2016-10-01

To investigate interactions among disease, pesticides, water quality, and adjacent land cover, we collected samples of water, sediment, and frog tissue from 21 sites in 7 States in the United States (US) representing a variety of amphibian habitats. All samples were analyzed for >90 pesticides and pesticide degradates, and water and frogs were screened for the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) using molecular methods. Pesticides and pesticide degradates were detected frequently in frog breeding habitats (water and sediment) as well as in frog tissue. Fungicides occurred more frequently in water, sediment, and tissue than was expected based upon their limited use relative to herbicides or insecticides. Pesticide occurrence in water or sediment was not a strong predictor of occurrence in tissue, but pesticide concentrations in tissue were correlated positively to agricultural and urban land, and negatively to forested land in 2-km buffers around the sites. Bd was detected in water at 45% of sites, and on 34% of swabbed frogs. Bd detections in water were not associated with differences in land use around sites, but sites with detections had colder water. Frogs that tested positive for Bd were associated with sites that had higher total fungicide concentrations in water and sediment, but lower insecticide concentrations in sediments relative to frogs that were Bd negative. Bd concentrations on frog swabs were positively correlated to dissolved organic carbon, and total nitrogen and phosphorus, and negatively correlated to pH and water temperature. Data were collected from a range of locations and amphibian habitats and represent some of the first field-collected information aimed at understanding the interactions between pesticides, land use, and amphibian disease. These interactions are of particular interest to conservation efforts as many amphibians live in altered habitats and may depend on wetlands embedded in these landscapes to survive. Published by Elsevier B.V.

Measuring tissue back-pressure--in vivo injection forces during subcutaneous injection.

PubMed

Allmendinger, Andrea; Mueller, Robert; Schwarb, Edward; Chipperfield, Mark; Huwyler, Joerg; Mahler, Hanns-Christian; Fischer, Stefan

2015-07-01

Limited information is available on injection forces of parenterals representing the in vivo situation. Scope of the present study was to investigate the contribution of the subcutaneous (sc) tissue layer to injection forces during in vivo injection. Göttingen minipigs received injections of isotonic dextran solutions (1-100 mPas) into the plica inguinalis using different injection rates and volumes (0.025-0.2 mL/s and 2.5 vs. 4.5 mL). The contribution of the sc back-pressure to injection forces was found to increase linearly with viscosity and injection rate ranging from 0.6 ± 0.5 N to 1.0 ± 0.4 N (1 mPas), 0.7 ± 0.2 N to 2.4 ± 1.9 N (10 mPas), and 1.8 ± 0.6 N to 4.7 ± 3.3 N (20 mPas) for injection rates of 0.025 to 0.2 mL/s, respectively. Variability increased with viscosity and injection rate. Values are average values from 10 randomized injections. A maximum of 12.9 N was reached for 20 mPas at 0.2 mL/s; 6.9 ± 0.3 N was determined for 100 mPas at 0.025 mL/s. No difference was found between injection volumes of 2.5 and 4.5 mL. The contribution of the tissue was differentiated from the contribution of the injection device and a local temperature effect. This effect was leading to warming of the (equilibrated) sample in the needle, therefore smaller injection forces than expected compensating tissue resistance to some parts. When estimating injection forces representative for the in vivo situation, the contribution of the tissue has to be considered as well as local warming of the sample in the needle during injection.

Potential interactions among disease, pesticides, water quality and adjacent land cover in amphibian habitats in the United States

USGS Publications Warehouse

Battaglin, William A.; Smalling, Kelly L.; Anderson, Chauncey; Calhoun, Daniel L.; Chestnut, Tara E.; Muths, Erin L.

2016-01-01

To investigate interactions among disease, pesticides, water quality, and adjacent land cover, we collected samples of water, sediment, and frog tissue from 21 sites in 7 States in the United States (US) representing a variety of amphibian habitats. All samples were analyzed for > 90 pesticides and pesticide degradates, and water and frogs were screened for the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) using molecular methods. Pesticides and pesticide degradates were detected frequently in frog breeding habitats (water and sediment) as well as in frog tissue. Fungicides occurred more frequently in water, sediment, and tissue than was expected based upon their limited use relative to herbicides or insecticides. Pesticide occurrence in water or sediment was not a strong predictor of occurrence in tissue, but pesticide concentrations in tissue were correlated positively to agricultural and urban land, and negatively to forested land in 2-km buffers around the sites. Bd was detected in water at 45% of sites, and on 34% of swabbed frogs. Bd detections in water were not associated with differences in land use around sites, but sites with detections had colder water. Frogs that tested positive for Bd were associated with sites that had higher total fungicide concentrations in water and sediment, but lower insecticide concentrations in sediments relative to frogs that were Bd negative. Bd concentrations on frog swabs were positively correlated to dissolved organic carbon, and total nitrogen and phosphorus, and negatively correlated to pH and water temperature.Data were collected from a range of locations and amphibian habitats and represent some of the first field-collected information aimed at understanding the interactions between pesticides, land use, and amphibian disease. These interactions are of particular interest to conservation efforts as many amphibians live in altered habitats and may depend on wetlands embedded in these landscapes to survive.

Histology and Biaxial Mechanical Behavior of Abdominal Aortic Aneurysm Tissue Samples.

PubMed

Pancheri, Francesco Q; Peattie, Robert A; Reddy, Nithin D; Ahamed, Touhid; Lin, Wenjian; Ouellette, Timothy D; Iafrati, Mark D; Luis Dorfmann, A

2017-03-01

Abdominal aortic aneurysms (AAAs) represent permanent, localized dilations of the abdominal aorta that can be life-threatening if progressing to rupture. Evaluation of risk of rupture depends on understanding the mechanical behavior of patient AAA walls. In this project, a series of patient AAA wall tissue samples have been evaluated through a combined anamnestic, mechanical, and histopathologic approach. Mechanical properties of the samples have been characterized using a novel, strain-controlled, planar biaxial testing protocol emulating the in vivo deformation of the aorta. Histologically, the tissue ultrastructure was highly disrupted. All samples showed pronounced mechanical stiffening with stretch and were notably anisotropic, with greater stiffness in the circumferential than the axial direction. However, there were significant intrapatient variations in wall stiffness and stress. In biaxial tests in which the longitudinal stretch was held constant at 1.1 as the circumferential stretch was extended to 1.1, the maximum average circumferential stress was 330 ± 70 kPa, while the maximum average axial stress was 190 ± 30 kPa. A constitutive model considering the wall as anisotropic with two preferred directions fit the measured data well. No statistically significant differences in tissue mechanical properties were found based on patient gender, age, maximum bulge diameter, height, weight, body mass index, or smoking history. Although a larger patient cohort is merited to confirm these conclusions, the project provides new insight into the relationships between patient natural history, histopathology, and mechanical behavior that may be useful in the development of accurate methods for rupture risk evaluation.

A screening of persistent organohalogenated contaminants in hair of East Greenland polar bears.

PubMed

Jaspers, Veerle L B; Dietz, Rune; Sonne, Christian; Letcher, Robert J; Eens, Marcel; Neels, Hugo; Born, Erik W; Covaci, Adrian

2010-10-15

In this pilot study, we report on levels of persistent organohalogenated contaminants (OHCs) in hair of polar bears (Ursus maritimus) from East Greenland sampled between 1999 and 2001. To our knowledge, this is the first study on the validation of polar bear hair as a non-invasive matrix representative of concentrations and profiles in internal organs and blood plasma. Because of low sample weights (13-140mg), only major bioaccumulative OHCs were detected above the limit of quantification: five polychlorinated biphenyl (PCB) congeners (CB 99, 138, 153, 170 and 180), one polybrominated diphenyl ether (PBDE) congener (BDE 47), oxychlordane, trans-nonachlor and β-hexachlorocyclohexane. The PCB profile in hair was similar to that of internal tissues (i.e. adipose, liver, brain and blood), with CB 153 and 180 as the major congeners in all matrices. A gender difference was found for concentrations in hair relative to concentrations in internal tissues. Females (n=6) were found to display negative correlations, while males (n=5) showed positive correlations, although p-values were not found significant. These negative correlations in females may reflect seasonal OHC mobilisation from periphery adipose tissue due to, for example, lactation and fasting. The lack of significance in most correlations may be due to small sample sizes and seasonal variability of concentrations in soft tissues. Further research with larger sample weights and sizes is therefore necessary to draw more definitive conclusions on the usefulness of hair for biomonitoring OHCs in polar bears and other fur mammals. Copyright © 2010 Elsevier B.V. All rights reserved.

Metal-clad waveguide characterization for contact-based light transmission into tissue

NASA Astrophysics Data System (ADS)

Chininis, Jeffrey; Whiteside, Paul; Hunt, Heather K.

2016-02-01

As contemporary laser dermatology procedures, like tattoo removal and skin resurfacing, become more popular, the complications of their operation are also becoming more prevalent. Frequent incidences of over-exposure, ocular injury, and excessive thermal damage represent mounting concerns for those seeking such procedures; moreover, each of these problems is a direct consequence of the standard, free-space method of laser transmission predominantly used in clinical settings. Therefore, an alternative method of light transmission is needed to minimize these problems. Here, we demonstrate and characterize an alternative method that uses planar waveguides to deliver light into sample tissue via direct contact. To do this, slab substrates made from glass were clad in layers of titanium and silver, constraining the light within the waveguide along the waveguide's length. By creating active areas on the waveguide surface, the propagating light could then optically tunnel into the tissue sample, when the waveguide was brought into contact with the tissue. SEM and EDS were used to characterize the metal film thickness and deposition rates onto the glass substrates. Laser light from a Q-switched Nd:YAG source operating at 532nm was coupled into the waveguide and transmitted into samples of pig skin. The amount of light transmitted was measured using photoacoustics techniques, in conjunction with a photodiode and integrating sphere. Transmitting light into tissue in this manner effectively resolves or circumvents the complications caused by free-space propagation methods as it reduces the operating distance to 0, which prevents hazardous back-reflections and allows for the ready incorporation of contact cooling technologies.

Comparison of oral microbiota in tumor and non-tumor tissues of patients with oral squamous cell carcinoma

PubMed Central

2012-01-01

Background Bacterial infections have been linked to malignancies due to their ability to induce chronic inflammation. We investigated the association of oral bacteria in oral squamous cell carcinoma (OSCC/tumor) tissues and compared with adjacent non-tumor mucosa sampled 5 cm distant from the same patient (n = 10). By using culture-independent 16S rRNA approaches, denaturing gradient gel electrophoresis (DGGE) and cloning and sequencing, we assessed the total bacterial diversity in these clinical samples. Results DGGE fingerprints showed variations in the band intensity profiles within non-tumor and tumor tissues of the same patient and among the two groups. The clonal analysis indicated that from a total of 1200 sequences characterized, 80 bacterial species/phylotypes were detected representing six phyla, Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Actinobacteria and uncultivated TM7 in non-tumor and tumor libraries. In combined library, 12 classes, 16 order, 26 families and 40 genera were observed. Bacterial species, Streptococcus sp. oral taxon 058, Peptostreptococcus stomatis, Streptococcus salivarius, Streptococcus gordonii, Gemella haemolysans, Gemella morbillorum, Johnsonella ignava and Streptococcus parasanguinis I were highly associated with tumor site where as Granulicatella adiacens was prevalent at non-tumor site. Streptococcus intermedius was present in 70% of both non-tumor and tumor sites. Conclusions The underlying changes in the bacterial diversity in the oral mucosal tissues from non-tumor and tumor sites of OSCC subjects indicated a shift in bacterial colonization. These most prevalent or unique bacterial species/phylotypes present in tumor tissues may be associated with OSCC and needs to be further investigated with a larger sample size. PMID:22817758

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome

PubMed Central

Camargo, Anamaria A.; Samaia, Helena P. B.; Dias-Neto, Emmanuel; Simão, Daniel F.; Migotto, Italo A.; Briones, Marcelo R. S.; Costa, Fernando F.; Aparecida Nagai, Maria; Verjovski-Almeida, Sergio; Zago, Marco A.; Andrade, Luis Eduardo C.; Carrer, Helaine; El-Dorry, Hamza F. A.; Espreafico, Enilza M.; Habr-Gama, Angelita; Giannella-Neto, Daniel; Goldman, Gustavo H.; Gruber, Arthur; Hackel, Christine; Kimura, Edna T.; Maciel, Rui M. B.; Marie, Suely K. N.; Martins, Elizabeth A. L.; Nóbrega, Marina P.; Paçó-Larson, Maria Luisa; Pardini, Maria Inês M. C.; Pereira, Gonçalo G.; Pesquero, João Bosco; Rodrigues, Vanderlei; Rogatto, Silvia R.; da Silva, Ismael D. C. G.; Sogayar, Mari C.; Sonati, Maria de Fátima; Tajara, Eloiza H.; Valentini, Sandro R.; Alberto, Fernando L.; Amaral, Maria Elisabete J.; Aneas, Ivy; Arnaldi, Liliane A. T.; de Assis, Angela M.; Bengtson, Mário Henrique; Bergamo, Nadia Aparecida; Bombonato, Vanessa; de Camargo, Maria E. R.; Canevari, Renata A.; Carraro, Dirce M.; Cerutti, Janete M.; Corrêa, Maria Lucia C.; Corrêa, Rosana F. R.; Costa, Maria Cristina R.; Curcio, Cyntia; Hokama, Paula O. M.; Ferreira, Ari J. S.; Furuzawa, Gilberto K.; Gushiken, Tsieko; Ho, Paulo L.; Kimura, Elza; Krieger, José E.; Leite, Luciana C. C.; Majumder, Paromita; Marins, Mozart; Marques, Everaldo R.; Melo, Analy S. A.; Melo, Monica; Mestriner, Carlos Alberto; Miracca, Elisabete C.; Miranda, Daniela C.; Nascimento, Ana Lucia T. O.; Nóbrega, Francisco G.; Ojopi, Élida P. B.; Pandolfi, José Rodrigo C.; Pessoa, Luciana G.; Prevedel, Aline C.; Rahal, Paula; Rainho, Claudia A.; Reis, Eduardo M. R.; Ribeiro, Marcelo L.; da Rós, Nancy; de Sá, Renata G.; Sales, Magaly M.; Sant'anna, Simone Cristina; dos Santos, Mariana L.; da Silva, Aline M.; da Silva, Neusa P.; Silva, Wilson A.; da Silveira, Rosana A.; Sousa, Josane F.; Stecconi, Daniella; Tsukumo, Fernando; Valente, Valéria; Soares, Fernando; Moreira, Eloisa S.; Nunes, Diana N.; Correa, Ricardo G.; Zalcberg, Heloisa; Carvalho, Alex F.; Reis, Luis F. L.; Brentani, Ricardo R.; Simpson, Andrew J. G.; de Souza, Sandro J.

2001-01-01

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning. PMID:11593022

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome.

PubMed

Camargo, A A; Samaia, H P; Dias-Neto, E; Simão, D F; Migotto, I A; Briones, M R; Costa, F F; Nagai, M A; Verjovski-Almeida, S; Zago, M A; Andrade, L E; Carrer, H; El-Dorry, H F; Espreafico, E M; Habr-Gama, A; Giannella-Neto, D; Goldman, G H; Gruber, A; Hackel, C; Kimura, E T; Maciel, R M; Marie, S K; Martins, E A; Nobrega, M P; Paco-Larson, M L; Pardini, M I; Pereira, G G; Pesquero, J B; Rodrigues, V; Rogatto, S R; da Silva, I D; Sogayar, M C; Sonati, M F; Tajara, E H; Valentini, S R; Alberto, F L; Amaral, M E; Aneas, I; Arnaldi, L A; de Assis, A M; Bengtson, M H; Bergamo, N A; Bombonato, V; de Camargo, M E; Canevari, R A; Carraro, D M; Cerutti, J M; Correa, M L; Correa, R F; Costa, M C; Curcio, C; Hokama, P O; Ferreira, A J; Furuzawa, G K; Gushiken, T; Ho, P L; Kimura, E; Krieger, J E; Leite, L C; Majumder, P; Marins, M; Marques, E R; Melo, A S; Melo, M B; Mestriner, C A; Miracca, E C; Miranda, D C; Nascimento, A L; Nobrega, F G; Ojopi, E P; Pandolfi, J R; Pessoa, L G; Prevedel, A C; Rahal, P; Rainho, C A; Reis, E M; Ribeiro, M L; da Ros, N; de Sa, R G; Sales, M M; Sant'anna, S C; dos Santos, M L; da Silva, A M; da Silva, N P; Silva, W A; da Silveira, R A; Sousa, J F; Stecconi, D; Tsukumo, F; Valente, V; Soares, F; Moreira, E S; Nunes, D N; Correa, R G; Zalcberg, H; Carvalho, A F; Reis, L F; Brentani, R R; Simpson, A J; de Souza, S J; Melo, M

2001-10-09

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription-PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Effects of vitrification cryopreservation on follicular morphology and stress relaxation behaviors of human ovarian tissues: sucrose versus trehalose as the non-permeable protective agent.

PubMed

Tian, Ting; Zhao, Gang; Han, Dan; Zhu, Kaixuan; Chen, Dawei; Zhang, Zhiguo; Wei, Zhaolian; Cao, Yunxia; Zhou, Ping

2015-04-01

Is sucrose more effective than trehalose in human ovarian tissue cryopreservation? The effect of sucrose as a cryoprotective agent (CPA) was not significantly different from that of trehalose in human ovarian tissue cryopreservation. Sugars have the ability to keep the cell membrane intact and can decrease the toxicity of CPAs. Sucrose is the most commonly used non-permeable CPA, while trehalose is rarely used in human ovarian tissue cryopreservation. Although various methods are utilized to evaluate the efficiency of human ovarian tissue cryopreservation, few studies have evaluated the effect of cryopreservation from the viewpoint of biomechanics. A total of 15 ovarian tissue samples were collected from 15 patients (20-41 years old) with benign ovarian tumors or malignancies, and each was dissected into six slices. Two slices were taken as the fresh control group. The remaining four slices were vitrified using different vitrification protocols. After warming, samples in each group were either fixed for histological evaluation or destined for stress relaxation test. The CPA solutions for the control and vitrified groups were composed of EDS and EDT (E, ethylene glycol; D, dimethylsulphoxide; S, sucrose; T, trehalose). The stress relaxation experiments were carried out at room temperature using a dynamic mechanical analyzer. Ovarian tissue samples were assessed for both their morphology and viscoelasticity. Stress relaxation data (SRD) were calculated as a percentage, representing the ability to maintain the initial stress after stretching. The percentage of morphologically normal follicles was compared between groups, which was represented by morphologic preservation ratio. The morphologic preservation ratio of the primordial follicles in the fresh control group (87.58%) was higher than that in group S (72.33%) (P = 0.000) and group T (79.56%) (P = 0.002). Although not statistically significant, compared with the S group, vitrification with T suggested a trend toward a higher morphologic preservation ratio of the primordial follicles. The SRD in the fresh control group (0.6433 ± 0.7233) was significantly different from that in group S (0.5200 ± 0.8331, P = 0.000) or in group T (0.5667 ± 0.6415, P = 0.000). However, no significant difference was found between groups S and T. Experimental samples were directly exposed to the air, which will result in a discrepancy in the viscoelastic properties between experimental tissues and in vivo tissues. Our study suggested a trend toward a higher morphologic preservation ratio of the primordial follicles after vitrification in trehalose compared with sucrose, which may provide a basis for further optimizing human ovarian tissue vitrification. In addition, it was possible to evaluate the effect of ovarian tissue cryopreservation from a biomechanics perspective. This study was supported by the grants from the Medical Scientific Research Subject, Health Ministry of Anhui Province (2010B014) and National Basic Research Program of China (973 Program) (2012CB944704), and the National Natural Science Foundation of China (Nos. 51276179 and 51476160). The authors declare that there is no conflict of interests regarding the publication of this original paper. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Epidermal growth factor receptor (EGFR) is overexpressed in high-grade dysplasia and adenocarcinoma of the esophagus and may represent a biomarker of histological progression in Barrett's esophagus (BE).

PubMed

Cronin, James; McAdam, Elizabeth; Danikas, Antonios; Tselepis, Chris; Griffiths, Paul; Baxter, John; Thomas, Linzi; Manson, James; Jenkins, Gareth

2011-01-01

The assessment of cancer risk in patients with Barrett's esophagus (BE) is currently fraught with difficulty. The current gold standard method of assessing cancer risk is histological assessment, with the appearance of high-grade dysplasia (HGD) as the key event monitored. Sampling error during endoscopy limits the usefulness of this approach, and there has been much recent interest in supplementing histological assessment with molecular markers, which may aid in patient stratification. No molecular marker has been yet validated to accurately correlate with esophageal histological progression. Here, we assessed the suitability of several membranous proteins as biomarkers by correlating their abundance with histological progression. In all, 107 patient samples, from 100 patients, were arranged on a tissue microarray (TMA) and represented the various stages of histological progression in BE. This TMA was probed with antibodies for eight receptor proteins (mostly membranous). Epidermal growth factor receptor (EGFR) staining was found to be the most promising biomarker identified with clear increases in staining accompanying histological progression. Further, immunohistochemistry was performed using the full-tissue sections from BE, HGD, and adenocarcinoma tissues, which confirmed the stepwise increase in EGFR abundance. Using a robust H-score analysis, EGFR abundance was shown to increase 13-fold in the adenocarcinoma tissues compared to the BE tissues. EGFR was "overexpressed" in 35% of HGD specimens and 80% of adenocarcinoma specimens when using the H-score of the BE patients (plus 3 s.d.) as the threshold to define overexpression. EGFR staining was also noted to be higher in BE tissues adjacent to HGD/adenocarcinoma. Western blotting, although showing more EGFR protein in the adenocarcinomas compared to the BE tissue, was highly variable. EGFR overexpression was accompanied by aneuploidy (gain) of chromosome 7, plus amplification of the EGFR locus. Finally, the bile acid deoxycholic acid (DCA) (at neutral and acidic pH) and acid alone was capable of upregulating EGFR mRNA in vitro, and in the case of neutral pH DCA, this was NF-κB dependent. EGFR is overexpressed during the histological progression in BE tissues and hence may be useful as a biomarker of histological progression. Furthermore, as EGFR is a membranous protein expressed on the luminal surface of the esophageal mucosa, it may also be a useful target for biopsy guidance during endoscopy.

A Microfluidic Interface for the Culture and Sampling of Adiponectin from Primary Adipocytes

PubMed Central

Godwin, Leah A.; Brooks, Jessica C.; Hoepfner, Lauren D.; Wanders, Desiree; Judd, Robert L.; Easley, Christopher J.

2014-01-01

Secreted from adipose tissue, adiponectin is a vital endocrine hormone that acts in glucose metabolism, thereby establishing its crucial role in diabetes, obesity, and other metabolic disease states. Insulin exposure to primary adipocytes cultured in static conditions has been shown to stimulate adiponectin secretion. However, conventional, static methodology for culturing and stimulating adipocytes falls short of truly mimicking physiological environments. Along with decreases in experimental costs and sample volume, and increased temporal resolution, microfluidic platforms permit small-volume flowing cell culture systems, which more accurately represent the constant flow conditions through vasculature in vivo. Here, we have integrated a customized primary tissue culture reservoir into a passively operated microfluidic device made of polydimethylsiloxane (PDMS). Fabrication of the reservoir was accomplished through unique PDMS “landscaping” above sampling channels, with a design strategy targeted to primary adipocytes to overcome issues of positive cell buoyancy. This reservoir allowed three-dimensional culture of primary murine adipocytes, accurate control over stimulants via constant perfusion, and sampling of adipokine secretion during various treatments. As the first report of primary adipocyte culture and sampling within microfluidic systems, this work sets the stage for future studies in adipokine secretion dynamics. PMID:25423362

Real-time optical coherence tomography observation of retinal tissue damage during laser photocoagulation therapy on ex-vivo porcine samples

NASA Astrophysics Data System (ADS)

Steiner, P.; Považay, B.; Stoller, M.; Morgenthaler, P.; Inniger, D.; Arnold, P.; Sznitman, R.; Meier, Ch.

2015-07-01

Retinal laser photocoagulation represents a widely used treatment for retinal pathologies such as diabetic chorioretinopathy or diabetic edema. For effective treatment, an appropriate choice of the treatment energy dose is crucial to prevent excessive tissue damage caused by over-irradiation of the retina. In this manuscript we investigate simultaneous and time-resolved optical coherence tomography for its applicability to provide feedback to the ophthalmologist about the introduced retinal damage during laser photocoagulation. Time-resolved and volumetric optical coherence tomography data of 96 lesions on ex-vivo porcine samples, set with a 577 nm laser prototype and irradiance of between 300 and 8800 W=cm2 were analyzed. Time-resolved scans were compared to volumetric scans of the lesion and correlated with ophthalmoscopic visibility. Lastly, image parameters extracted from optical coherence tomography Mscans, suitable for lesion classification were identified. Results presented in this work support the hypothesis that simultaneous optical coherence tomography provides valuable information about the extent of retinal tissue damage and may be used to guide retinal laser photocoagulation in the future.

Classification of prostate cancer grade using temporal ultrasound: in vivo feasibility study

NASA Astrophysics Data System (ADS)

Ghavidel, Sahar; Imani, Farhad; Khallaghi, Siavash; Gibson, Eli; Khojaste, Amir; Gaed, Mena; Moussa, Madeleine; Gomez, Jose A.; Siemens, D. Robert; Leveridge, Michael; Chang, Silvia; Fenster, Aaron; Ward, Aaron D.; Abolmaesumi, Purang; Mousavi, Parvin

2016-03-01

Temporal ultrasound has been shown to have high classification accuracy in differentiating cancer from benign tissue. In this paper, we extend the temporal ultrasound method to classify lower grade Prostate Cancer (PCa) from all other grades. We use a group of nine patients with mostly lower grade PCa, where cancerous regions are also limited. A critical challenge is to train a classifier with limited aggressive cancerous tissue compared to low grade cancerous tissue. To resolve the problem of imbalanced data, we use Synthetic Minority Oversampling Technique (SMOTE) to generate synthetic samples for the minority class. We calculate spectral features of temporal ultrasound data and perform feature selection using Random Forests. In leave-one-patient-out cross-validation strategy, an area under receiver operating characteristic curve (AUC) of 0.74 is achieved with overall sensitivity and specificity of 70%. Using an unsupervised learning approach prior to proposed method improves sensitivity and AUC to 80% and 0.79. This work represents promising results to classify lower and higher grade PCa with limited cancerous training samples, using temporal ultrasound.

Three dimensional mapping of strontium in bone by dual energy K-edge subtraction imaging

NASA Astrophysics Data System (ADS)

Cooper, D. M. L.; Chapman, L. D.; Carter, Y.; Wu, Y.; Panahifar, A.; Britz, H. M.; Bewer, B.; Zhouping, W.; Duke, M. J. M.; Doschak, M.

2012-09-01

The bones of many terrestrial vertebrates, including humans, are continually altered through an internal process of turnover known as remodeling. This process plays a central role in bone adaptation and disease. The uptake of fluorescent tetracyclines within bone mineral is widely exploited as a means of tracking new tissue formation. While investigation of bone microarchitecture has undergone a dimensional shift from 2D to 3D in recent years, we lack a 3D equivalent to fluorescent labeling. In the current study we demonstrate the ability of synchrotron radiation dual energy K-edge subtraction (KES) imaging to map the 3D distribution of elemental strontium within rat vertebral samples. This approach has great potential for ex vivo analysis of preclinical models and human tissue samples. KES also represents a powerful tool for investigating the pharmokinetics of strontium-based drugs recently approved in many countries around the globe for the treatment of osteoporosis.

Heavy metals in red crabs, Chaceon quinquedens, from the Gulf of Mexico.

PubMed

Perry, Harriet; Isphording, Wayne; Trigg, Christine; Riedel, Ralf

2015-12-30

The red crab, Chaceon quinquedens, is distributed in deep waters of the Gulf of Mexico (GOM) and is most abundant in an area associated with sediment deposition from the Mississippi River. Sediment geochemistry and biological and ecological traits of red crabs favor accumulation of contaminants. Red crabs, sediment, and bottom water samples were taken from three distinct geographic locations representing areas with differing exposure to contaminant laden effluents from the Mississippi River. Inductively coupled plasma spectrophotometry and atomic absorption spectrophotometry were employed to determine levels of heavy metals in red crab muscle tissue. Ion site partitioning was used to determine metal speciation in sediments. Red crabs showed evidence of heavy metal bioaccumulation in all sample areas with high variability in contaminant levels in individual crabs for some metals. Bioavailability of metals in sediment did not always result in accumulation in muscle tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

Plant Tissues in 3D via X-Ray Tomography: Simple Contrasting Methods Allow High Resolution Imaging

PubMed Central

Staedler, Yannick M.; Masson, David; Schönenberger, Jürg

2013-01-01

Computed tomography remains strongly underused in plant sciences despite its high potential in delivering detailed 3D phenotypical information because of the low X-ray absorption of most plant tissues. Existing protocols to study soft tissues display poor performance, especially when compared to those used on animals. More efficient protocols to study plant material are therefore needed. Flowers of Arabidopsis thaliana and Marcgravia caudata were immersed in a selection of contrasting agents used to treat samples for transmission electron microscopy. Grayscale values for floral tissues and background were measured as a function of time. Contrast was quantified via a contrast index. The thick buds of Marcgravia were scanned to determine which contrasting agents best penetrate thick tissues. The highest contrast increase with cytoplasm-rich tissues was obtained with phosphotungstate, whereas osmium tetroxide and bismuth tatrate displayed the highest contrast increase with vacuolated tissues. Phosphotungstate also displayed the best sample penetration. Furthermore, infiltration with phosphotungstate allowed imaging of all plants parts at a high resolution of 3 µm, which approaches the maximum resolution of our equipment: 1.5 µm. The high affinity of phosphotungstate for vasculature, cytoplasm-rich tissue, and pollen causes these tissues to absorb more X-rays than the surrounding tissues, which, in turn, makes these tissues appear brighter on the scan data. Tissues with different brightness can then be virtually dissected from each other by selecting the bracket of grayscale to be visualized. Promising directions for the future include in silico phenotyping and developmental studies of plant inner parts (e.g., ovules, vasculature, pollen, and cell nuclei) via virtual dissection as well as correlations of quantitative phenotypes with omics datasets. Therefore, this work represents a crucial improvement of previous methods, allowing new directions of research to be undertaken in areas ranging from morphology to systems biology. PMID:24086499

Microcystin accumulation in liver and muscle of tilapia in two large Brazilian hydroelectric reservoirs.

PubMed

Deblois, Charles P; Aranda-Rodriguez, Rocio; Giani, Alessandra; Bird, David F

2008-03-01

The objective of this study was to measure levels of the toxin microcystin in different tissues of fish known to feed on cyanobacteria during toxic bloom events. Wild Nile and redbreast tilapia (Oreochromis niloticus and Tilapia rendalli) were sampled from the catch of artisanal fishermen at eutrophic stations of Funil and Furnas reservoirs in southeastern Brazil. Phytoplankton communities in the two reservoirs were quite different taxonomically, but not dissimilar in microcystin content (200 microg g dry weight (DW) seston(-1) at Funil, 800 microg gDW seston(-1) at Furnas). All of the 27 fish sampled contained microcystin, ranging from 0.8 to 32.1 microg g liver(-1) and from 0.9 to 12.0 ng g muscle(-1). Most microcystin variants found in seston were also found in fish liver. T. rendalli had the lowest concentration in both tissues when compared to O. niloticus. In both reservoirs, one of every four fish sampled, always O. niloticus, had a level of microcystins beyond the World Health Organization tolerable daily intake (8 ng g tissue(-1)) and represented a risk for consumers. It is possible that closer study of inter-species variability in toxin burden in cyanobacteria-impacted water bodies will permit the development of guidelines for fish consumption that will better protect public health.

The effects of sample preparation on measured concentrations of eight elements in edible tissues of fish from streams contaminated by lead mining

USGS Publications Warehouse

Schmitt, Christopher J.; Finger, Susan E.

1987-01-01

The influence of sample preparation on measured concentrations of eight elements in the edible tissues of two black basses (Centrarchidae), two catfishes (Ictaluridae), and the black redhorse,Moxostoma duquesnei (Catostomidae) from two rivers in southeastern Missouri contaminated by mining and related activities was investigated. Concentrations of Pb, Cd, Cu, Zn, Fe, Mn, Ba, and Ca were measured in two skinless, boneless samples of axial muscle from individual fish prepared in a clean room. One sample (normally-processed) was removed from each fish with a knife in a manner typically used by investigators to process fish for elemental analysis and presumedly representative of methods employed by anglers when preparing fish for home consumption. A second sample (clean-processed) was then prepared from each normally-processed sample by cutting away all surface material with acid-cleaned instruments under ultraclean conditions. The samples were analyzed as a single group by atomic absorption spectrophotometry. Of the elements studied, only Pb regularly exceeded current guidelines for elemental contaminants in foods. Concentrations were high in black redhorse from contaminated sites, regardless of preparation method; for the other fishes, whether or not Pb guidelines were exceeded depended on preparation technique. Except for Mn and Ca, concentrations of all elements measured were significantly lower in cleanthan in normally-processed tissue samples. Absolute differences in measured concentrations between clean- and normally-processed samples were most evident for Pb and Ba in bass and catfish and for Cd and Zn in redhorse. Regardless of preparation method, concentrations of Pb, Ca, Mn, and Ba in individual fish were closely correlated; samples that were high or low in one of these four elements were correspondingly high or low in the other three. In contrast, correlations between Zn, Fe, and Cd occurred only in normallyprocessed samples, suggesting that these correlations resulted from high concentrations on the surfaces of some samples. Concentrations of Pb and Ba in edible tissues of fish from contaminated sites were highly correlated with Ca content, which was probably determined largely by the amount of tissue other than muscle in the sample because fish muscle contains relatively little Ca. Accordingly, variation within a group of similar samples can be reduced by normalizing Pb and Ba concentrations to a standard Ca concentration. When sample size (N) is large, this can be accomplished statistically by analysis of covariance; whenN is small, molar ratios of [Pb]/[Ca] and [Ba]/[Ca] can be computed. Without such adjustments, unrealistically large Ns are required to yield statistically reliable estimates of Pb concentrations in edible tissues. Investigators should acknowledge that reported concentrations of certain elements are only estimates, and that regardless of the care exercised during the collection, preparation, and analysis of samples, results should be interpreted with the awareness that contamination from external sources may have occurred.

The first investigation of Wilms' tumour atomic structure-nitrogen and carbon isotopic composition as a novel biomarker for the most individual approach in cancer disease

PubMed Central

Taran, Katarzyna; Frączek, Tomasz; Sikora-Szubert, Anita; Sitkiewicz, Anna; Młynarski, Wojciech; Kobos, Józef; Paneth, Piotr

2016-01-01

The paper describes a novel approach to investigating Wilms' tumour (nephroblastoma) biology at the atomic level. Isotope Ratio Mass Spectrometry (IRMS) was used to directly assess the isotope ratios of nitrogen and carbon in 84 Wilms' tumour tissue samples from 28 cases representing the histological spectrum of nephroblastoma. Marked differences in nitrogen and carbon isotope ratios were found between nephroblastoma histological types and along the course of cancer disease, with a breakout in isotope ratio of the examined elements in tumour tissue found between stages 2 and 3. Different isotopic compositions with regard to nitrogen and carbon content were observed in blastemal Wilms' tumour, with and without focal anaplasia, and in poorly- and well-differentiated epithelial nephroblastoma. This first assessment of nitrogen and carbon isotope ratio reveals the previously unknown part of Wilms' tumour biology and represents a potential novel biomarker, allowing for a highly individual approach to treating cancer. Furthermore, this method of estimating isotopic composition appears to be the most sensitive tool yet for cancer tissue evaluation, and a valuable complement to established cancer study methods with prospective clinical impact. PMID:27732932

Multiplexed color-coded probe-based gene expression assessment for clinical molecular diagnostics in formalin-fixed paraffin-embedded human renal allograft tissue.

PubMed

Adam, Benjamin; Afzali, Bahman; Dominy, Katherine M; Chapman, Erin; Gill, Reeda; Hidalgo, Luis G; Roufosse, Candice; Sis, Banu; Mengel, Michael

2016-03-01

Histopathologic diagnoses in transplantation can be improved with molecular testing. Preferably, molecular diagnostics should fit into standard-of-care workflows for transplant biopsies, that is, formalin-fixed paraffin-embedded (FFPE) processing. The NanoString(®) gene expression platform has recently been shown to work with FFPE samples. We aimed to evaluate its methodological robustness and feasibility for gene expression studies in human FFPE renal allograft samples. A literature-derived antibody-mediated rejection (ABMR) 34-gene set, comprised of endothelial, NK cell, and inflammation transcripts, was analyzed in different retrospective biopsy cohorts and showed potential to molecularly discriminate ABMR cases, including FFPE samples. NanoString(®) results were reproducible across a range of RNA input quantities (r = 0.998), with different operators (r = 0.998), and between different reagent lots (r = 0.983). There was moderate correlation between NanoString(®) with FFPE tissue and quantitative reverse transcription polymerase chain reaction (qRT-PCR) with corresponding dedicated fresh-stabilized tissue (r = 0.487). Better overall correlation with histology was observed with NanoString(®) (r = 0.354) than with qRT-PCR (r = 0.146). Our results demonstrate the feasibility of multiplexed gene expression quantification from FFPE renal allograft tissue. This represents a method for prospective and retrospective validation of molecular diagnostics and its adoption in clinical transplantation pathology. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Climatic signals registered as Carbon isotopic values in Metasequoia leaf tissues: A statistical analysis

NASA Astrophysics Data System (ADS)

Yang, H.; Blais, B.; Perez, G.; Pagani, M.

2006-12-01

To examine climatic signals registered as carbon isotopic values in leaf tissues of C3 plants, we collected mature leaf tissues from sun and shade leaves of Metasequoia trees germinated from the 1947 batch of seeds from China and planted along a latitudinal gradient of the United States. Samples from 40 individual trees, along with fossilized material from the early Tertiary of the Canadian Arctic, were analyzed for C and concentration and isotopic values using EA-IRMS after the removal of free lipids. The generated datasets were then merged with climate data compiled from each tree site recorded as average values over the past thirty years (1971-2002, NOAA database). When the isotope data were cross plotted against each geographic and climatic indicator, Latitude, Mean Annual Temperature (MAT), Average Summer Mean Temperature (ASMT)(June-August), Mean Annual Precipitation (MAP), and Average Summer Mean Precipitation (ASMP) respectively correlation patterns were revealed. The best correlating trend was obtained between temperature parameters and C isotopic values, and this correlation is stronger in the northern leaf samples than the southern samples. We discovered a strong positive correlation between latitude and the offset of C isotopic values between shade and sun leaves. This investigation represents a comprehensive examination on climatic signals registered as C isotopic values on a single species that is marked by single genetic source. The results bear implications on paleoclimatic interpretations of C isotopic signals obtained from fossil plant tissues.

Morphological, ultrastructural and functional imaging of frozen/thawed and vitrified/warmed human ovarian tissue retrieved from oncological patients.

PubMed

Fabbri, R; Vicenti, R; Macciocca, M; Martino, N A; Dell'Aquila, M E; Pasquinelli, G; Morselli-Labate, A M; Seracchioli, R; Paradisi, R

2016-08-01

Which is the best method for human ovarian tissue cryopreservation: slow freezing/rapid thawing (SF/RT) or vitrification/warming (V/W)? The conventional SF/RT protocol used in this study seems to better preserve the morpho-functional status of human cryopreserved ovarian tissue than the used open carrier V/W protocol. Cryopreservation of human ovarian tissue is generally performed using the SF/RT method. However, reduction in the follicular pool and stroma damage are often observed. An emerging alternative procedure is represented by V/W which seems to allow the maintenance of the morphological integrity of the stroma. This is a retrospective cohort study including six patients affected by oncological diseases and enrolled from January to December 2014. Ovarian tissue was laparoscopically harvested from the right and left ovaries and was cryopreserved using a routinary SF/RT protocol or a V/W method, involving tissue incubation in two solutions (containing propylene glycol, ethylene glycol and sucrose at different concentrations) and vitrification in an open system. For each patient, three pieces from each ovary were collected at the time of laparoscopy (fresh tissue) and after storage (SF/RT or V/W) and processed for light microscopy (LM) and transmission electron microscopy (TEM), to assess the morphological and ultrastructural features of follicles and stroma, and for laser scanning confocal microscopy (LSCM), to determine the functional energetic/redox stroma status. The preservation status of SF/RT and V/W ovarian tissues was compared with that of fresh ones, as well as between them. By LM and TEM, SF/RT and V/W samples showed cryodamage of small entity. Interstitial oedema and increased stromal cell vacuolization and chromatin clumping were observed in SF/RT samples; in contrast, V/W samples showed oocyte nuclei with slightly thickened chromatin and irregular shapes. The functional imaging analysis by LSCM revealed that the mitochondrial activity and intracellular reactive oxygen species levels were reduced both in SF/RT and in V/W samples compared with fresh samples. The study also showed progressive dysfunction of the mitochondrial activity going from the outer to the inner serial section of the ovarian cortex. The reduction of mitochondrial activity of V/W samples compared with fresh samples was significantly higher in the inner section than in the outer section. The results report the bioenergetic and oxidative status assessment of fresh and cryopreserved human ovarian tissue by LSCM, a technique recently applied to tissue samples. The use of LSCM on human ovarian tissues after SF/RT or V/W is a new application that requires validation. The procedures for mitochondrial staining with functional probes and fixing are not yet standardized. Xenografting of the cryopreserved ovarian tissue in severe combined immunodeficient mice and in vitro culture have not yet been performed. The identification of a cryopreservation method able to maintain the morpho-functional integrity of the ovarian tissue and a number of follicles comparable with those observed in fresh tissue might optimize results in clinical practice, in terms of recovery, duration of ovarian function and increased delivery outcomes after replanting. The SF/RT protocol allowed better morpho-functional tissue integrity than the V/W procedure. Funding was provided by Fondazione del Monte di Bologna e Ravenna, Italy. Dr N.A.M. was granted by the project ONEV MIUR PONa3 00134-n.254/R&C 18 5 2011 and the project GR-2011-02351396 (Ministry of Health, Young Researchers Grant 2011/2012). There are no competing interests. Clinical trial 74/2001/0 (approved:13 2 2002): 'Pilot study on cryopreservation of human ovarian tissue: morphological and immunohistochemical analysis before and after cryopreservation'. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Orthodontic treatment effects on inflammatory marker profiles in saliva before and after 2 archwire changes

NASA Astrophysics Data System (ADS)

Yamamoto, Zulham; Jaafar, Ikmal Mohamad; Rohaya, M. A. W.; Abidin, Intan Zarina Zainol; Senafi, Sahidan; Ariffin, Zaidah Zainal; Ariffin, Shahrul Hisham Zainal

2013-11-01

Periodontal tissue changes exerted by external forces in orthodontic treatment allow tooth movement. The changes in periodontal tissues i.e. inflammation can be monitored using gingival crevicular fluid (GCF). GCF is a component of saliva. Saliva could be used to monitor periodontal disease progression. The use of saliva to monitor periodontal tissues changes during orthodontic treatment is still unknown. Therefore, we observed the profiles of inflammatory markers namely creatine kinase ('CK), nitric oxide (NO), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) in saliva of orthodontic patients to evaluate their importance in orthodontic treatment. A total of 21 subjects (13 female and 8 male) participated in this study. Samples were collected from gingival crevicular fluid at three period of archwire changes: baseline (M0), 2 weeks after 0.014" NiTi archwire (M1), and 2 weeks after 0.018" NiTi archwire (M2). All enzyme activities i.e. CK, LDH and AST were measured spectrophotometrically at 340 nm. Griess assay was used to measure nitric oxide level. CK activity, NO level, LDH activity and AST activity in saliva samples did not show significant differences among period of archwire changes. The use of inflammatory marker profiles in saliva may not represent the changes in periodontal tissues during orthodontic treatment.

A Conformal, Bio-interfaced Class of Silicon Electronics for Mapping Cardiac Electrophysiology

PubMed Central

Viventi, Jonathan; Kim, Dae-Hyeong; Moss, Joshua D.; Kim, Yun-Soung; Blanco, Justin A.; Annetta, Nicholas; Hicks, Andrew; Xiao, Jianliang; Huang, Younggang; Callans, David J.; Rogers, John A.; Litt, Brian

2011-01-01

The sophistication and resolution of current implantable medical devices are limited by the need connect each sensor separately to data acquisition systems. The ability of these devices to sample and modulate tissues is further limited by the rigid, planar nature of the electronics and the electrode-tissue interface. Here, we report the development of a class of mechanically flexible silicon electronics for measuring signals in an intimate, conformal integrated mode on the dynamic, three dimensional surfaces of soft tissues in the human body. We illustrate this technology in sensor systems composed of 2016 silicon nanomembrane transistors configured to record electrical activity directly from the curved, wet surface of a beating heart in vivo. The devices sample with simultaneous sub-millimeter and sub-millisecond resolution through 288 amplified and multiplexed channels. We use these systems to map the spread of spontaneous and paced ventricular depolarization in real time, at high resolution, on the epicardial surface in a porcine animal model. This clinical-scale demonstration represents one example of many possible uses of this technology in minimally invasive medical devices. [Conformal electronics and sensors intimately integrated with living tissues enable a new generation of implantable devices capable of addressing important problems in human health.] PMID:20375008

The Structure Of Intact Side Tissue Loss Based On FTIR Spectroscopic Measurements

NASA Astrophysics Data System (ADS)

Hussain, N.; Al-Hadithi, K. O.; Jaafar, M. S.

2009-09-01

Laser applications in dentistry were strongly evolved during the last three decades. Among those applications are laser ablation of dental hard tissue, caries inhibition treatments by localized surface heating, and surface conditioning for bonding. In addition, infra-red lasers are ideally suited for the selective and precise removal of carious dental hard tissue while minimizing the healthy tissue loss. In the present study we applied laser spectroscopy technique FTIR for the study of the structure of intact side tissue of teeth. The aim of the recent work is to study the effect of race and sex (genealogy) on the structure of intact side tissue loss. Our sample consists of twenty Malay females' teeth where the FTIR has been applied. The data show a decrease in the amounts of main substances (like Hydroxyapatite crystals ([Ca5(PO4)3(OH)4], CaF2) than those in healthy teeth. The measured spectra represent the enamel with the characteristic peaks due to the phosphate group in carbonated, hydroxyapatite at 1000 cm-1 and two small peaks near 1500 cm-1 due to the carbonate group. The data explains the effect of the several factors on the intact side tissue loss.

Sinonasal microbiome sampling: a comparison of techniques.

PubMed

Bassiouni, Ahmed; Cleland, Edward John; Psaltis, Alkis James; Vreugde, Sarah; Wormald, Peter-John

2015-01-01

The role of the sino-nasal microbiome in CRS remains unclear. We hypothesized that the bacteria within mucosal-associated biofilms may be different from the more superficial-lying, free-floating bacteria in the sinuses and that this may impact on the microbiome results obtained. This study investigates whether there is a significant difference in the microbiota of a sinonasal mucosal tissue sample versus a swab sample. Cross-sectional study with paired design. Mucosal biopsy and swab samples were obtained intra-operatively from the ethmoid sinuses of 6 patients with CRS. Extracted DNA was sequenced on a Roche-454 sequencer using 16S-rRNA gene targeted primers. Data were analyzed using QIIME 1.8 software package. At a maximum subsampling depth of 1,100 reads, the mean observed species richness was 33.3 species (30.6 for swab, versus 36 for mucosa; p > 0.05). There was no significant difference in phylogenetic and non-phylogenetic alpha diversity metrics (Faith's PD_Whole_Tree and Shannon's index) between the two sampling methods (p > 0.05). The type of sample also had no significant effect on phylogenetic and non-phylogenetic beta diversity metrics (Unifrac and Bray-Curtis; p > 0.05). We observed no significant difference between the microbiota of mucosal tissue and swab samples. This suggests that less invasive swab samples are representative of the sinonasal mucosa microbiome and can be used for future sinonasal microbiome studies.

Expression Analysis of Macrodactyly Identifies Pleiotrophin Upregulation

PubMed Central

Lau, Frank H.; Xia, Fang; Kaplan, Adam; Cerrato, Felecia; Greene, Arin K.; Taghinia, Amir; Cowan, Chad A.; Labow, Brian I.

2012-01-01

Macrodactyly is a rare family of congenital disorders characterized by the diffuse enlargement of 1 or more digits. Multiple tissue types within the affected digits are involved, but skeletal patterning and gross morphological features are preserved. Not all tissues are equally involved and there is marked heterogeneity with respect to clinical phenotype. The molecular mechanisms responsible for these growth disturbances offer unique insight into normal limb growth and development, in general. To date, no genes or loci have been implicated in the development of macrodactyly. In this study, we performed the first transcriptional profiling of macrodactyly tissue. We found that pleiotrophin (PTN) was significantly overexpressed across all our macrodactyly samples. The mitogenic functions of PTN correlate closely with the clinical characteristics of macrodactyly. PTN thus represents a promising target for further investigation into the etiology of overgrowth phenotypes. PMID:22848377

Evaluation of different pulverisation methods for RNA extraction in squash fruit: lyophilisation, cryogenic mill and mortar grinding.

PubMed

Román, Belén; González-Verdejo, Clara I; Peña, Francisco; Nadal, Salvador; Gómez, Pedro

2012-01-01

Quality and integrity of RNA are critical for transcription studies in plant molecular biology. In squash fruit and other high water content crops, the grinding of tissue with mortar and pestle in liquid nitrogen fails to produce a homogeneous and fine powered sample desirable to ensure a good penetration of the extraction reagent. To develop an improved pulverisation method to facilitate the homogenisation process of squash fruit tissue prior to RNA extraction without reducing quality and yield of the extracted RNA. Three methods of pulverisation, each followed by the same extraction protocol, were compared. The first approach consisted of the lyophilisation of the sample in order to remove the excess of water before grinding, the second one used a cryogenic mill and the control one a mortar grinding of frozen tissue. The quality of the isolated RNA was tested by carrying out a quantitative real time downstream amplification. In the three situations considered, mean values for A(260) /A(280) indicated minimal interference by proteins and RNA quality indicator (RQI) values were considered appropriate for quantitative real-time polymerase chain reaction (qRT-PCR) amplification. Successful qRT-PCR amplifications were obtained with cDNA isolated with the three protocols. Both apparatus can improve and facilitate the grinding step in the RNA extraction process in zucchini, resulting in isolated RNA of high quality and integrity as revealed by qRT-PCR downstream application. This is apparently the first time that a cryogenic mill has been used to prepare fruit samples for RNA extraction, thereby improving the sampling strategy because the fine powder obtained represents a homogeneous mix of the organ tissue. Copyright © 2012 John Wiley & Sons, Ltd.

Prevalence of virus-like particles within a staghorn scleractinian coral ( Acropora muricata) from the Great Barrier Reef

NASA Astrophysics Data System (ADS)

Patten, N. L.; Harrison, P. L.; Mitchell, J. G.

2008-09-01

Transmission electron microscopy (TEM) was used to determine whether Acropora muricata coral colonies from the Great Barrier Reef (GBR), Australia, harboured virus-like particles (VLPs). VLPs were present in all coral colonies sampled at Heron Island (southern GBR) and in tagged coral colonies sampled in at least two of the three sampling periods at Lizard Island (northern GBR). VLPs were observed within gastrodermal and epidermal tissues, and on rarer occasions, within the mesoglea. These VLPs had similar morphologies to known prokaryotic and eukaryotic viruses in other systems. Icosahedral VLPs were observed most frequently, however, filamentous VLPs (FVLPs) and phage were also noted. There were no clear differences in VLP size, morphology or location within the tissues with respect to sample date, coral health status or site. The most common VLP morphotype exhibited icosahedral symmetry, 120-150 nm in diameter, with an electron-dense core and an electronlucent membrane. Larger VLPs of similar morphology were also common. VLPs occurred as single entities, in groups, or in dense clusters, either as free particles within coral tissues, or within membrane-bound vacuoles. VLPs were commonly observed within the perinuclear region, with mitochondria, golgi apparatus and crescent-shaped particles frequently observed within close proximity. The host(s) of these observed VLPs was not clear; however, the different sizes and morphologies of VLPs observed within A. muricata tissues suggest that viruses are infecting either the coral animal, zooxanthellae, intracellular bacteria and/or other coral-associated microbiota, or that the one host is susceptible to infection from more than one type of virus. These results add to the limited but emerging body of evidence that viruses represent another potentially important component of the coral holobiont.

Biochemical and molecular characterization of thyroid tissue by micro-Raman spectroscopy and gene expression analysis

NASA Astrophysics Data System (ADS)

Neto, Lázaro P. M.; Martin, Aírton A.; Soto, Claudio A. T.; Santos, André B. O.; Mello, Evandro S.; Pereira, Marina A.; Cernea, Cláudio R.; Brandão, Lenine G.; Canevari, Renata A.

2016-02-01

Thyroid carcinomas represent the main endocrine malignancy and their diagnosis may produce inconclusive results. Raman spectroscopy and gene expression analysis have shown excellent results on the differentiation of carcinomas. This study aimed to improve the discrimination between different thyroid pathologies combining of both analyses. A total of 35 thyroid tissues samples including normal tissue (n=10), goiter (n=10), papillary (n=10) and follicular carcinomas (n=5) were analyzed. Confocal Raman spectra was obtain by using a Rivers Diagnostic System, 785 nm laser excitation and CCD detector. The data was processed by the software Labspec5 and Origin 8.5 and analyzed by Minitab® program. The gene expression analysis was performed by qRT-PCR technique for TG, TPO, PDGFB, SERPINA1, LGALS3 and TFF3 genes and statistically analyzed by Mann-Whitney test. The confocal Raman spectroscopy allowed a maximum discrimination of 91.1% between normal and tumor tissues, 84.8% between benign and malignant pathologies and 84.6% among carcinomas analyzed. Significant differences was observed for TG, LGALS3, SERPINA1 and TFF3 genes between benign lesions and carcinomas, and SERPINA1 and TFF3 genes between papillary and follicular carcinomas. Principal component analysis was performed using PC1 and PC2 in the papillary carcinoma samples that showed over gene expression when compared with normal sample, where 90% of discrimination was observed at the Amide 1 (1655 cm-1), and at the tyrosine spectra region (856 cm-1). The discrimination of tissues thyroid carried out by confocal Raman spectroscopy and gene expression analysis indicate that these techniques are promising tools to be used in the diagnosis of thyroid lesions.

Advanced Mass Spectrometry Technologies for the Study of Microbial Pathogenesis

PubMed Central

Moore, Jessica L.; Caprioli, Richard M.; Skaar, Eric P.

2014-01-01

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been successfully applied to the field of microbial pathogenesis with promising results, principally in diagnostic microbiology to rapidly identify bacteria based on the molecular profiles of small cell populations. Direct profiling of molecules from serum and tissue samples by MALDI MS providesa means to study the pathogen-host interaction and to discover potential markers of infection. Systematic molecular profiling across tissue sections represents a new imaging modality, enabling regiospecific molecular measurements to be made in situ, in both two- and three-dimensional analyses. Herein, we briefly summarize work that employs MALDI MS to study the pathogenesis of microbial infection. PMID:24997399

Lifetime fluorescence spectroscopy for in situ investigation of osteogenic differentiation

NASA Astrophysics Data System (ADS)

Marcu, Laura; Elbarbary, Amir; Zuk, Patricia; De Ugarte, Daniel A.; Benhaim, Prosper; Kurt, Hamza; Hedrick, Marc H.; Ashjian, Peter

2003-07-01

Time-Resolved Laser-Induced Fluorescence Spectroscopy (TR-LIFS) represents a potential tool for the in-situ characterization of bioengineered tissues. In this study, we evaluate the application of TR-LIFS to non-intrusive monitoring of matrix composition during osteogenetic differentiation. Human adipose-derived stem cells, harvested from 3 patients, were induced in osteogenic media for 3, 5, and 7 weeks. Samples were subsequently collected and probed for time-resolved fluorescence emission with a pulsed nitrogen laser. Fluorescence parameters, derived from both spectral- and time-domain, were used for sample characterization. The samples were further analyzed using Western blot analysis and computer-based densitometry. A significant change in the fluorescence parameters was detected for samples beyond 3 weeks of osteogenic differentiation. The spectroscopic observations: 1) show increase of collagen I when contrasted against the time-resolved fluorescence spectra of commercially available collagens; and 2) are in agreement with Western blot analysis that demonstrated significant increase in collagen I content between 3- vs. 5-weeks and 3- vs. 7-weeks and no changes for collagens III, IV, and V. Our results suggest that TR-LIFS can be used as a non-invasive means for the detection of specific collagens in maturing connective tissues.

A Review of Mid-Infrared and Near-Infrared Imaging: Principles, Concepts and Applications in Plant Tissue Analysis.

PubMed

Türker-Kaya, Sevgi; Huck, Christian W

2017-01-20

Plant cells, tissues and organs are composed of various biomolecules arranged as structurally diverse units, which represent heterogeneity at microscopic levels. Molecular knowledge about those constituents with their localization in such complexity is very crucial for both basic and applied plant sciences. In this context, infrared imaging techniques have advantages over conventional methods to investigate heterogeneous plant structures in providing quantitative and qualitative analyses with spatial distribution of the components. Thus, particularly, with the use of proper analytical approaches and sampling methods, these technologies offer significant information for the studies on plant classification, physiology, ecology, genetics, pathology and other related disciplines. This review aims to present a general perspective about near-infrared and mid-infrared imaging/microspectroscopy in plant research. It is addressed to compare potentialities of these methodologies with their advantages and limitations. With regard to the organization of the document, the first section will introduce the respective underlying principles followed by instrumentation, sampling techniques, sample preparations, measurement, and an overview of spectral pre-processing and multivariate analysis. The last section will review selected applications in the literature.

Chromatic confocal microscopy for multi-depth imaging of epithelial tissue

PubMed Central

Olsovsky, Cory; Shelton, Ryan; Carrasco-Zevallos, Oscar; Applegate, Brian E.; Maitland, Kristen C.

2013-01-01

We present a novel chromatic confocal microscope capable of volumetric reflectance imaging of microstructure in non-transparent tissue. Our design takes advantage of the chromatic aberration of aspheric lenses that are otherwise well corrected. Strong chromatic aberration, generated by multiple aspheres, longitudinally disperses supercontinuum light onto the sample. The backscattered light detected with a spectrometer is therefore wavelength encoded and each spectrum corresponds to a line image. This approach obviates the need for traditional axial mechanical scanning techniques that are difficult to implement for endoscopy and susceptible to motion artifact. A wavelength range of 590-775 nm yielded a >150 µm imaging depth with ~3 µm axial resolution. The system was further demonstrated by capturing volumetric images of buccal mucosa. We believe these represent the first microstructural images in non-transparent biological tissue using chromatic confocal microscopy that exhibit long imaging depth while maintaining acceptable resolution for resolving cell morphology. Miniaturization of this optical system could bring enhanced speed and accuracy to endomicroscopic in vivo volumetric imaging of epithelial tissue. PMID:23667789

STED super-resolution microscopy of clinical paraffin-embedded human rectal cancer tissue.

PubMed

Ilgen, Peter; Stoldt, Stefan; Conradi, Lena-Christin; Wurm, Christian Andreas; Rüschoff, Josef; Ghadimi, B Michael; Liersch, Torsten; Jakobs, Stefan

2014-01-01

Formalin fixed and paraffin-embedded human tissue resected during cancer surgery is indispensable for diagnostic and therapeutic purposes and represents a vast and largely unexploited resource for research. Optical microscopy of such specimen is curtailed by the diffraction-limited resolution of conventional optical microscopy. To overcome this limitation, we used STED super-resolution microscopy enabling optical resolution well below the diffraction barrier. We visualized nanoscale protein distributions in sections of well-annotated paraffin-embedded human rectal cancer tissue stored in a clinical repository. Using antisera against several mitochondrial proteins, STED microscopy revealed distinct sub-mitochondrial protein distributions, suggesting a high level of structural preservation. Analysis of human tissues stored for up to 17 years demonstrated that these samples were still amenable for super-resolution microscopy. STED microscopy of sections of HER2 positive rectal adenocarcinoma revealed details in the surface and intracellular HER2 distribution that were blurred in the corresponding conventional images, demonstrating the potential of super-resolution microscopy to explore the thus far largely untapped nanoscale regime in tissues stored in biorepositories.

STED Super-Resolution Microscopy of Clinical Paraffin-Embedded Human Rectal Cancer Tissue

PubMed Central

Wurm, Christian Andreas; Rüschoff, Josef; Ghadimi, B. Michael; Liersch, Torsten; Jakobs, Stefan

2014-01-01

Formalin fixed and paraffin-embedded human tissue resected during cancer surgery is indispensable for diagnostic and therapeutic purposes and represents a vast and largely unexploited resource for research. Optical microscopy of such specimen is curtailed by the diffraction-limited resolution of conventional optical microscopy. To overcome this limitation, we used STED super-resolution microscopy enabling optical resolution well below the diffraction barrier. We visualized nanoscale protein distributions in sections of well-annotated paraffin-embedded human rectal cancer tissue stored in a clinical repository. Using antisera against several mitochondrial proteins, STED microscopy revealed distinct sub-mitochondrial protein distributions, suggesting a high level of structural preservation. Analysis of human tissues stored for up to 17 years demonstrated that these samples were still amenable for super-resolution microscopy. STED microscopy of sections of HER2 positive rectal adenocarcinoma revealed details in the surface and intracellular HER2 distribution that were blurred in the corresponding conventional images, demonstrating the potential of super-resolution microscopy to explore the thus far largely untapped nanoscale regime in tissues stored in biorepositories. PMID:25025184

Automated high-throughput assessment of prostate biopsy tissue using infrared spectroscopic chemical imaging

NASA Astrophysics Data System (ADS)

Bassan, Paul; Sachdeva, Ashwin; Shanks, Jonathan H.; Brown, Mick D.; Clarke, Noel W.; Gardner, Peter

2014-03-01

Fourier transform infrared (FT-IR) chemical imaging has been demonstrated as a promising technique to complement histopathological assessment of biomedical tissue samples. Current histopathology practice involves preparing thin tissue sections and staining them using hematoxylin and eosin (H&E) after which a histopathologist manually assess the tissue architecture under a visible microscope. Studies have shown that there is disagreement between operators viewing the same tissue suggesting that a complementary technique for verification could improve the robustness of the evaluation, and improve patient care. FT-IR chemical imaging allows the spatial distribution of chemistry to be rapidly imaged at a high (diffraction-limited) spatial resolution where each pixel represents an area of 5.5 × 5.5 μm2 and contains a full infrared spectrum providing a chemical fingerprint which studies have shown contains the diagnostic potential to discriminate between different cell-types, and even the benign or malignant state of prostatic epithelial cells. We report a label-free (i.e. no chemical de-waxing, or staining) method of imaging large pieces of prostate tissue (typically 1 cm × 2 cm) in tens of minutes (at a rate of 0.704 × 0.704 mm2 every 14.5 s) yielding images containing millions of spectra. Due to refractive index matching between sample and surrounding paraffin, minimal signal processing is required to recover spectra with their natural profile as opposed to harsh baseline correction methods, paving the way for future quantitative analysis of biochemical signatures. The quality of the spectral information is demonstrated by building and testing an automated cell-type classifier based upon spectral features.

Effects of bioturbation on environmental DNA migration through soil media

PubMed Central

2018-01-01

Extracting and identifying genetic material from environmental media (i.e. water and soil) presents a unique opportunity for researchers to assess biotic diversity and ecosystem health with increased speed and decreased cost as compared to traditional methods (e.g. trapping). The heterogeneity of soil mineralogy, spatial and temporal variations however present unique challenges to sampling and interpreting results. Specifically, fate/transport of genetic material in the terrestrial environment represents a substantial data gap. Here we investigate to what degree, benthic fauna transport genetic material through soil. Using the red worm (Eisenia fetida), we investigate how natural movement through artificial soil affect the transport of genetic material. All experiments were run in Frabill® Habitat® II worm systems with approximately 5 cm depth of artificial soil. We selected an “exotic” source of DNA not expected to be present in soil, zebrafish (Danio rerio) tissue. Experiment groups contained homogenized zebrafish tissue placed in a defined location combined with a varying number of worms (10, 30 or 50 worms per experimental group). Experimental groups comprised two controls and three treatment groups (representing different worm biomass) in triplicate. A total of 210 soil samples were randomly collected over the course of 15 days to investigate the degree of genetic transfer, and the rate of detection. Positive detections were identified in 14% - 38% of samples across treatment groups, with an overall detection rate of 25%. These findings highlight two important issues when utilizing environmental DNA for biologic assessments. First, benthic fauna are capable of redistributing genetic material through a soil matrix. Second, despite a defined sample container and abundance of worm biomass, as many as 86% of the samples were negative. This has substantial implications for researchers and managers who wish to interpret environmental DNA results from terrestrial systems. Studies such as these will aid in future study protocol design and sample collection methodology. PMID:29689092

Gene expression profiling of histologically normal breast tissue in females with human epidermal growth factor receptor 2‑positive breast cancer.

PubMed

Zubor, Pavol; Hatok, Jozef; Moricova, Petra; Kapustova, Ivana; Kajo, Karol; Mendelova, Andrea; Sivonova, Monika Kmetova; Danko, Jan

2015-02-01

Gene expression profile‑based taxonomy of breast cancer (BC) has been described as a significant breakthrough in comprehending the differences in the origin and behavior of cancer to allow individually tailored therapeutic approaches. In line with this, we hypothesized that the gene expression profile of histologically normal epithelium (HNEpi) could harbor certain genetic abnormalities predisposing breast tissue cells to develop human epidermal growth factor receptor 2 (HER2)‑positive BC. Thus, the aim of the present study was to assess gene expression in normal and BC tissue (BCTis) from patients with BC in order to establish its value as a potential diagnostic marker for cancer development. An array study evaluating a panel of 84 pathway‑ and disease‑specific genes in HER2‑positive BC and tumor‑adjacent HNEpi was performed using quantitative polymerase chain reaction in 12 patients using microdissected samples from frozen tissue. Common prognostic and predictive parameters of BC were assessed by immunohistochemistry and in situ hybridization. In the BCTis and HNEpi samples of 12 HER2‑positive subjects with BC, the expression of 2,016 genes was assessed. A total of 39.3% of genes were deregulated at a minimal two‑fold deregulation rate and 10.7% at a five‑fold deregulation rate in samples of HNEpi or BCTis. Significant differences in gene expression between BCTis and HNEpi samples were revealed for BCL2L2, CD44, CTSD, EGFR, ERBB2, ITGA6, NGFB, RPL27, SCBG2A1 and SCGB1D2 genes (P<0.05), as well as GSN, KIT, KLK5, SERPINB5 and STC2 genes (P<0.01). Insignificant differences (P<0.07) were observed for CCNA1, CLU, DLC1, GABRP and IL6 genes. The ontological gene analyses revealed that the majority of the deregulated genes in the HNEpi samples were part of the functional gene group directly associated with BC origin and prognosis. Functional analysis showed that the most frequent gene deregulations occurred in genes associated with apoptosis and cell cycle regulation in BCTis samples, and with angiogenesis, regulation of the cell cycle and transcriptional activity in HNEpi samples. The molecular profiling of HNEpi breast tissue revealed gene expression abnormalities that may represent potential markers of increased risk for HER2‑positive malignant transformation of breast tissue, and may be able to be employed as predictors of prognosis.

Evidence of Nonuniformity in Urothelium Barrier Function between the Upper Urinary Tract and Bladder.

PubMed

Williams, Nicholas A; Barnard, Luke; Allender, Chris J; Bowen, Jenna L; Gumbleton, Mark; Harrah, Tim; Raja, Aditya; Joshi, Hrishi B

2016-03-01

We compared the relative permeability of upper urinary tract and bladder urothelium to mitomycin C. Ex vivo porcine bladder, ureters and kidneys were dissected out and filled with 1 mg ml(-1) mitomycin C. At 60 minutes the organs were emptied and excised tissue samples were sectioned parallel to the urothelium. Sectioned tissue was homogenized and extracted mitomycin C was quantified. Transurothelial permeation across the different urothelia was calculated by normalizing the total amount of drug extracted to the surface area of the tissue sample. Average mitomycin C concentrations at different tissue depths (concentration-depth profiles) were calculated by dividing the total amount of drug recovered by the total weight of tissue. Mitomycin C permeation across the ureteral urothelium was significantly greater than across the bladder and renal pelvis urothelium (9.07 vs 0.94 and 3.61 μg cm(-2), respectively). Concentrations of mitomycin C in the ureter and kidney were markedly higher than those achieved in the bladder at all tissue depths. Average urothelial mitomycin C concentrations were greater than 6.5-fold higher in the ureter and renal pelvis than in the bladder. To our knowledge we report for the first time that the upper urinary tract and bladder show differing permeability to a single drug. Ex vivo porcine ureter is significantly more permeable to mitomycin C than bladder urothelium and consequently higher mitomycin C tissue concentrations can be achieved after topical application. Data in this study correlate with the theory that mammalian upper tract urothelium represents a different cell lineage than that of the bladder and it is innately more permeable to mitomycin C. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Combined quantitative and qualitative two-channel optical biopsy technique for discrimination of tumor borders

NASA Astrophysics Data System (ADS)

Bocher, Thomas; Beuthan, Juergen; Scheller, M.; Hopf, Juergen U. G.; Linnarz, Marietta; Naber, Rolf-Dieter; Minet, Olaf; Becker, Wolfgang; Mueller, Gerhard J.

1995-12-01

Conventional laser-induced fluorescence spectroscopy (LIFS) of endogenous chromophores like NADH (Nicotineamide Adenine Dinucleotide, reduced form) and PP IX (Protoporphyrin IX) provides information about the relative amounts of these metabolites in the observed cells. But for diagnostic applications the concentrations of these chromophores have to be determined quantitatively to establish tissue-independent differentiation criterions. It is well- known that the individually and locally varying optical tissue parameters are major obstacles for the determination of the true chromophore concentrations by simple fluorescence spectroscopy. To overcome these problems a fiber-based, 2-channel technique including a rescaled NADH-channel (delivering quantitative values) and a relative PP IX-channel was developed. Using the accumulated information of both channels can provide good tissue state separation. Ex-vivo studies with resected and frozen samples (with LN2) of squamous cells in the histologically confirmed states: normal, tumor border, inflammation and hyperplasia were performed. Each state was represented in this series with at least 7 samples. At the identical tissue spot both, the rescaled NADH-fluorescence and the relative PP IX- fluorescence, were determined. In the first case a nitrogen laser (337 nm, 500 ps, 200 microjoule, 10 Hz) in the latter case a diode laser (633 nm, 15 mW, cw) were used as excitation sources. In this ex-vivo study a good separation between the different tissue states was achieved. With a device constructed for clinical usage one quantitative, in-vivo NADH- measurement was done recently showing similar separation capabilities.

Three-dimensional spatial localization of thin fluorophore-filled capillaries in thick scattering media

NASA Astrophysics Data System (ADS)

Desrochers, Johanne; Vermette, Patrick; Fontaine, Réjean; Bérubé-Lauzière, Yves

2008-06-01

Fluorescence optical diffuse tomography (fDOT) is of much interest in molecular imaging to retrieve information from fluorescence signals emitted from specifically targeted bioprocesses deep within living tissues. An exciting application of fDOT is in the growing field of tissue engineering, where 3D non-invasive imaging techniques are required to ultimately grow 3D engineered tissues. Via appropriate labelling strategies and fluorescent probes, fDOT has the potential to monitor culture environment and cells viability non-destructively directly within the bioreactor environment where tissues are to be grown. Our ultimate objective is to image the formation of blood vessels in bioreactor conditions. Herein, we use a non-contact setup for small animal fDOT imaging designed for 3D light collection around the sample. We previously presented a time of flight approach using a numerical constant fraction discrimination technique to assign an early photons arrival time to every fluorescence time point-spread function collected around the sample. Towards bioreactor in-situ imaging, we have shown the capability of our approach to localize a fluorophore-filled 500 μm capillary immersed coaxially in a cylindrically shaped bioreactor phantom containing an absorbing/scattering medium representative of experiments on real tissue cultures. Here, we go one step further, and present results for the 3D localization of thinner indocyanine green labelled capillaries (250 μm and 360 μm inner diameter) immersed in the same phantom conditions and geometry but with different spatial configurations (10° and 30° capillary inclination).

Development and evaluation of a real-time RT-qPCR for detection of Crimean-Congo hemorrhagic fever virus representing different genotypes.

PubMed

Jääskeläinen, Anne J; Kallio-Kokko, Hannimari; Ozkul, Aykut; Bodur, Hurrem; Korukruoglu, Gulay; Mousavi, Mehrdad; Pranav, Patel; Vaheri, Antti; Mirazimi, Ali; Vapalahti, Olli

2014-12-01

Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic disease caused by a nairovirus belonging to family Bunyaviridae. The CCHF virus (CCHFV) can be transmitted to humans by Hyalomma ticks as well as by direct contact with infected body fluids or tissues from viremic livestock or humans. Our aim was to set up a fast RT-qPCR for detection of the different CCHFV genotypes in clinical samples, including an inactivation step to make the sample handling possible in lower biosafety levels (BSL) than BSL-4. This method was evaluated against commercial reference assays and international External Quality Assessment (EQA) samples. The analytical limit of detection for the developed CCHFV-S RT-qPCR was 11 CCHFV genomes per reaction. After exclusion of four dubious samples, we studied 38 CCHFV-positive samples (using reference tests) of which 38 were found positive by CCHFV-S RT-qPCR, suggesting a sensitivity of 100%. CCHFV-S RT q-PCR detected all eight different CCHFV strains representing five different CCHFV genotypes. In conclusion, the CCHFV-S RT-qPCR described in this study was evaluated using various sources of CCHFV samples and shown to be an accurate tool to detect human CCHFV infection caused by different genotypes of the virus.

Populational analysis of suspended microtissues for high-throughput, multiplexed 3D tissue engineering

PubMed Central

Chen, Alice A.; Underhill, Gregory H.; Bhatia, Sangeeta N.

2014-01-01

Three-dimensional (3D) tissue models have significantly improved our understanding of structure/function relationships and promise to lead to new advances in regenerative medicine. However, despite the expanding diversity of 3D tissue fabrication methods, approaches for functional assessment have been relatively limited. Here, we describe the fabrication of microtissue (μ-tissue) suspensions and their quantitative evaluation with techniques capable of analyzing large sample numbers and performing multiplexed parallel analysis. We applied this platform to 3D μ-tissues representing multiple stages of liver development and disease including: embryonic stem cells, bipotential hepatic progenitors, mature hepatocytes, and hepatoma cells photoencapsulated in polyethylene glycol hydrogels. Multiparametric μ-tissue cytometry enabled quantitation of fluorescent reporter expression within populations of intact μ-tissues (n≥102-103) and sorting-based enrichment of subsets for subsequent studies. Further, 3D μ-tissues could be implanted in vivo, respond to systemic stimuli, retrieved and quantitatively assessed. In order to facilitate multiplexed ‘pooled’ experimentation, fluorescent labeling strategies were developed and utilized to investigate the impact of μ-tissue composition and exposure to soluble factors. In particular, examination of drug/gene interactions on collections of 3D hepatoma μ-tissues indicated synergistic influence of doxorubicin and knockdown of the anti-apoptotic gene BCL-XL. Collectively, these studies highlight the broad utility of μ-tissue suspensions as an enabling approach for high n, populational analysis of 3D tissue biology in vitro and in vivo. PMID:20820630

Heterogeneous histopathology of cortical microbleeds in cerebral amyloid angiopathy.

PubMed

van Veluw, Susanne J; Biessels, Geert Jan; Klijn, Catharina J M; Rozemuller, Annemieke J M

2016-03-01

To investigate the histopathologic substrate of microbleeds detected on 7T postmortem MRI in autopsy cases with severe cerebral amyloid angiopathy (CAA) and Alzheimer pathology. Five decedents (mean age at death 79.6 ± 5.7 years) with documented severe CAA and Alzheimer pathology on standard neuropathologic examination were selected from a local database. Formalin-fixed coronal brain slices were scanned at 7T MRI, including high-resolution T2- and T2*-weighted sequences. Representative microbleeds from each case were sampled for histopathologic analysis, including the presence of blood, blood breakdown products, and markers of ischemic tissue injury. On MRI, we identified >300 cortical and 4 subcortical microbleeds. Two out of 15 sampled cortical microbleeds corresponded histologically to erythrocytes (suggestive of recent hemorrhages), 4 to vasculopathies (fibrinoid necrosis in 3 and a cavernoma) without substantial parenchymal tissue injury, and 9 to accumulations of iron-positive siderophages without erythrocytes (suggestive of old hemorrhages) combined with mild to moderate degrees of chronic ischemic tissue injury. This study provides evidence for heterogeneous pathologic substrates and possibly different pathophysiologic mechanisms underlying MRI-observed cortical microbleeds in the context of advanced CAA and Alzheimer disease. © 2016 American Academy of Neurology.

Proteomic analysis of human dental cementum and alveolar bone.

PubMed

Salmon, Cristiane R; Tomazela, Daniela M; Ruiz, Karina Gonzales Silvério; Foster, Brian L; Paes Leme, Adriana Franco; Sallum, Enilson Antonio; Somerman, Martha J; Nociti, Francisco H

2013-10-08

Dental cementum (DC) is a bone-like tissue covering the tooth root and responsible for attaching the tooth to the alveolar bone (AB) via the periodontal ligament (PDL). Studies have unsuccessfully tried to identify factors specific to DC versus AB, in an effort to better understand DC development and regeneration. The present study aimed to use matched human DC and AB samples (n=7) to generate their proteomes for comparative analysis. Bone samples were harvested from tooth extraction sites, whereas DC samples were obtained from the apical root portion of extracted third molars. Samples were denatured, followed by protein extraction reduction, alkylation and digestion for analysis by nanoAcquity HPLC system and LTQ-FT Ultra. Data analysis demonstrated that a total of 318 proteins were identified in AB and DC. In addition to shared proteins between these tissues, 105 and 83 proteins exclusive to AB or DC were identified, respectively. This is the first report analyzing the proteomic composition of human DC matrix and identifying putative unique and enriched proteins in comparison to alveolar bone. These findings may provide novel insights into developmental differences between DC and AB, and identify candidate biomarkers that may lead to more efficient and predictable therapies for periodontal regeneration. Periodontal disease is a highly prevalent disease affecting the world population, which involves breakdown of the tooth supporting tissues, the periodontal ligament, alveolar bone, and dental cementum. The lack of knowledge on specific factors that differentiate alveolar bone and dental cementum limits the development of more efficient and predictable reconstructive therapies. In order to better understand cementum development and potentially identify factors to improve therapeutic outcomes, we took the unique approach of using matched patient samples of dental cementum and alveolar bone to generate and compare a proteome list for each tissue. A potential biomarker for dental cementum was identified, superoxide dismutase 3 (SOD3), which is found in cementum and cementum-associated cells in mouse, pig, and human tissues. These findings may provide novel insights into developmental differences between alveolar bone and dental cementum, and represent the basis for improved and more predictable therapies. © 2013.

Temporal and spatial variability in stable isotope compositions of a freshwater mussel: Implications for biomonitoring and ecological studies

USGS Publications Warehouse

Gustafson, L.; Showers, W.; Kwak, T.; Levine, J.; Stoskopf, M.

2007-01-01

Stable isotopes can be used to elucidate ecological relationships in community and trophic studies. Findings are calibrated against baselines, e.g. from a producer or primary consumer, assumed to act as a reference to the isotopic context created by spatio-temporal attributes such as geography, climate, nutrient, and energy sources. The ability of an organism to accurately represent a community base depends on how, and over what time-scale, it assimilates ambient materials. Freshwater mussels have served as references for trophic studies of freshwater communities and as indicators of change in nutrient pollution load or source. Their suitability as reference animals has not yet been fully explored, however. We conducted a series of studies examining the suitability of freshwater mussels as isotopic baselines, using their ability to reflect variation in ambient nutrient loads as a case scenario. (1) We analyzed bivalve foot tissue ??15N and ??13C from 22 stream reaches in the Piedmont region of North Carolina, USA to show that compositions varied substantially among locations. Site mean bivalve ??13C values correlated with site ambient particulate organic matter (POM) ??13C values, and site mean bivalve ??15N values correlated with site ambient water dissolved ??15N-NO3 values. (2) Similarity of results among sample types demonstrated that the minimally invasive hemolymph sample is a suitable substitute for foot tissue in ??15N analyses, and that small sample sizes generate means representative of a larger population. Both findings can help minimize the impact of sampling on imperiled freshwater mussel populations. (3) In a bivalve transplantation study we showed that hemolymph ??15N compositions responded to a shift in ambient dissolved ??15N-NO3, although slowly. The tissue turnover time for bivalve hemolymph was 113 days. We conclude that bivalves serve best as biomonitors of chronic, rather than acute, fluctuations in stream nutrient loads, and provide initial evidence of their suitability as time-integrated isotopic baselines for community studies. ?? 2006 Springer-Verlag.

Label-Free Molecular Imaging of Biological Cells and Tissues by Linear and Nonlinear Raman Spectroscopic Approaches.

PubMed

Krafft, Christoph; Schmitt, Michael; Schie, Iwan W; Cialla-May, Dana; Matthäus, Christian; Bocklitz, Thomas; Popp, Jürgen

2017-04-10

Raman spectroscopy is an emerging technique in bioanalysis and imaging of biomaterials owing to its unique capability of generating spectroscopic fingerprints. Imaging cells and tissues by Raman microspectroscopy represents a nondestructive and label-free approach. All components of cells or tissues contribute to the Raman signals, giving rise to complex spectral signatures. Resonance Raman scattering and surface-enhanced Raman scattering can be used to enhance the signals and reduce the spectral complexity. Raman-active labels can be introduced to increase specificity and multimodality. In addition, nonlinear coherent Raman scattering methods offer higher sensitivities, which enable the rapid imaging of larger sampling areas. Finally, fiber-based imaging techniques pave the way towards in vivo applications of Raman spectroscopy. This Review summarizes the basic principles behind medical Raman imaging and its progress since 2012. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A multiple-well method for immunohistochemical testing of many reagents on a single microscopic slide.

PubMed

McKeever, P E; Letica, L H; Shakui, P; Averill, D R

1988-09-01

Multiple wells (M-wells) have been made over tissue sections on single microscopic slides to simultaneously localize binding specificity of many antibodies. More than 20 individual 4-microliter wells over tissue have been applied/slide, representing more than a 5-fold improvement in wells/slide and a 25-fold reduction in reagent volume over previous methods. More than 30 wells/slide have been applied over cellular monolayers. To produce the improvement, previous strategies of placing specimens into wells were changed to instead create wells over the specimen. We took advantage of the hydrophobic properties of paint to surround the wells and to segregate the various different primary antibodies. Segregation was complete on wells alternating with and without primary monoclonal antibody. The procedure accommodates both frozen and paraffin sections, yielding slides which last more than a year. After monoclonal antibody detection, standard histologic stains can be applied as counterstains. M-wells are suitable for localizing binding of multiple reagents or sample unknowns (polyclonal or monoclonal antibodies, hybridoma supernatants, body fluids, lectins) to either tissues or cells. Their small sample volume and large number of sample wells/slide could be particularly useful for early screening of hybridoma supernatants and for titration curves in immunohistochemistry (McKeever PE, Shakui P, Letica LH, Averill DR: J Histochem Cytochem 36:931, 1988).

EPA Office of Water (OW): Fish Consumption Advisories and Fish Tissue Sampling Stations NHDPlus Indexed Datasets

EPA Pesticide Factsheets

The Fish Consumption Advisories dataset contains information on Fish Advisory events that have been indexed to the EPA Office of Water NHDPlus v2.1 hydrology and stored in the Reach Addressing Database (RAD). NHDPlus is a database that interconnects and uniquely identifies the millions of stream segments or reaches that comprise the Nations' surface water drainage system. NHDPlus provides a national framework for assigning reach addresses to water quality related entities, such as fish advisories locations. Reach addresses establish the locations of these entities relative to one another within the NHD surface water drainage network in a manner similar to street addresses. The assignment of reach addresses is accomplished through a process known as reach indexing. Fish consumption advisories and fish tissue sampling stations are reported to EPA by the states. Sampling stations are the locations where a state has collected fish tissue data for use in advisory determinations. Fish consumption advisory locations are coded onto NHDPlus flowline features to create point and linear events. Fish consumption advisory locations are also coded onto NHDPlus waterbody features to create area events. In addition to NHDPlus-reach indexed data, there may also be custom events (point, line, or area) that are not associated with NHDPlus. Although these Fish consumption advisories are not represented in NHDPlus, the data created for them are in an EPA standard format that is co

Method And Apparatus For Examining A Tissue Using The Spectral Wing Emission Therefrom Induced By Visible To Infrared Photoexcitation.

DOEpatents

Alfano, Robert R.; Demos, Stavros G.; Zhang, Gang

2003-12-16

Method and an apparatus for examining a tissue using the spectral wing emission therefrom induced by visible to infrared photoexcitation. In one aspect, the method is used to characterize the condition of a tissue sample and comprises the steps of (a) photoexciting the tissue sample with substantially monochromatic light having a wavelength of at least 600 nm; and (b) using the resultant far red and near infrared spectral wing emission (SW) emitted from the tissue sample to characterize the condition of the tissue sample. In one embodiment, the substantially monochromatic photoexciting light is a continuous beam of light, and the resultant steady-state far red and near infrared SW emission from the tissue sample is used to characterize the condition of the tissue sample. In another embodiment, the substantially monochromatic photoexciting light is a light pulse, and the resultant time-resolved far red and near infrared SW emission emitted from the tissue sample is used to characterize the condition of the tissue sample. In still another embodiment, the substantially monochromatic photoexciting light is a polarized light pulse, and the parallel and perpendicular components of the resultant polarized time-resolved SW emission emitted from the tissue sample are used to characterize the condition of the tissue sample.

Scanning in situ Spectroscopy platform for imaging surgical breast tissue specimens

PubMed Central

Krishnaswamy, Venkataramanan; Laughney, Ashley M.; Wells, Wendy A.; Paulsen, Keith D.; Pogue, Brian W.

2013-01-01

A non-contact localized spectroscopic imaging platform has been developed and optimized to scan 1x1cm2 square regions of surgically resected breast tissue specimens with ~150-micron resolution. A color corrected, image-space telecentric scanning design maintained a consistent sampling geometry and uniform spot size across the entire imaging field. Theoretical modeling in ZEMAX allowed estimation of the spot size, which is equal at both the center and extreme positions of the field with ~5% variation across the designed waveband, indicating excellent color correction. The spot sizes at the center and an extreme field position were also measured experimentally using the standard knife-edge technique and were found to be within ~8% of the theoretical predictions. Highly localized sampling offered inherent insensitivity to variations in background absorption allowing direct imaging of local scattering parameters, which was validated using a matrix of varying concentrations of Intralipid and blood in phantoms. Four representative, pathologically distinct lumpectomy tissue specimens were imaged, capturing natural variations in tissue scattering response within a given pathology. Variations as high as 60% were observed in the average reflectance and relative scattering power images, which must be taken into account for robust classification performance. Despite this variation, the preliminary data indicates discernible scatter power contrast between the benign vs malignant groups, but reliable discrimination of pathologies within these groups would require investigation into additional contrast mechanisms. PMID:23389199

Recurrence of Marfan syndrome as a result of parental germ-line mosaicism for an FBN1 mutation.

PubMed Central

Rantamäki, T; Kaitila, I; Syvänen, A C; Lukka, M; Peltonen, L

1999-01-01

Mutations in the FBN1 gene cause Marfan syndrome (MFS), a dominantly inherited connective tissue disease. Almost all the identified FBN1mutations have been family specific, and the rate of new mutations is high. We report here a de novo FBN1mutation that was identified in two sisters with MFS born to clinically unaffected parents. The paternity and maternity were unequivocally confirmed by genotyping. Although one of the parents had to be an obligatory carrier for the mutation, we could not detect the mutation in the leukocyte DNA of either parent. To identify which parent was a mosaic for the mutation we analyzed several tissues from both parents, with a quantitative and sensitive solid-phase minisequencing method. The mutation was not, however, detectable in any of the analyzed tissues. Although the mutation could not be identified in a sperm sample from the father or in samples of multiple tissue from the mother, we concluded that the mother was the likely mosaic parent and that the mutation must have occurred during the early development of her germ-line cells. Mosaicism confined to germ-line cells has rarely been reported, and this report of mosaicism for the FBN1 mutation in MFS represents an important case, in light of the evaluation of the recurrence risk in genetic counseling of families with MFS. PMID:10090884

Gene expression in lung adenocarcinomas of smokers and nonsmokers.

PubMed

Powell, Charles A; Spira, Avrum; Derti, Adnan; DeLisi, Charles; Liu, Gang; Borczuk, Alain; Busch, Steve; Sahasrabudhe, Sudhir; Chen, Yangde; Sugarbaker, David; Bueno, Raphael; Richards, William G; Brody, Jerome S

2003-08-01

Adenocarcinoma (AC) has become the most frequent type of lung cancer in men and women, and is the major form of lung cancer in nonsmokers. Our goal in this paper was to determine if AC in smokers and nonsmokers represents the same genetic disease. We compared gene expression profiles in resected samples of nonmalignant lung tissue and tumor tissue in six never-smokers with AC and in six smokers with AC, who were matched for clinical staging and histologic criteria of cell differentiation. Results were analyzed using a variety of bioinformatic tools. Four times as many genes changed expression in the transition from noninvolved lung to tumor in nonsmokers as in smokers, suggesting that AC in nonsmokers evolves locally, whereas AC in smokers evolves in a field of genetically altered tissue. There were some similarities in gene expression in smokers and nonsmokers, but many differences, suggesting different pathways of cell transformation and tumor formation. Gene expression in the noninvolved lungs of smokers differed from that of nonsmokers, and multidimensional scaling showed that noninvolved lungs of smokers groups with tumors rather than noninvolved lungs of nonsmokers. In addition, expression of a number of genes correlated with smoking intensity. Our findings, although limited by small sample size, suggest that additional studies comparing noninvolved to tumor tissue may identify pathogenetic mechanisms and therapeutic targets that differ in AC of smokers and nonsmokers.

A taxonomy of epithelial human cancer and their metastases

PubMed Central

2009-01-01

Background Microarray technology has allowed to molecularly characterize many different cancer sites. This technology has the potential to individualize therapy and to discover new drug targets. However, due to technological differences and issues in standardized sample collection no study has evaluated the molecular profile of epithelial human cancer in a large number of samples and tissues. Additionally, it has not yet been extensively investigated whether metastases resemble their tissue of origin or tissue of destination. Methods We studied the expression profiles of a series of 1566 primary and 178 metastases by unsupervised hierarchical clustering. The clustering profile was subsequently investigated and correlated with clinico-pathological data. Statistical enrichment of clinico-pathological annotations of groups of samples was investigated using Fisher exact test. Gene set enrichment analysis (GSEA) and DAVID functional enrichment analysis were used to investigate the molecular pathways. Kaplan-Meier survival analysis and log-rank tests were used to investigate prognostic significance of gene signatures. Results Large clusters corresponding to breast, gastrointestinal, ovarian and kidney primary tissues emerged from the data. Chromophobe renal cell carcinoma clustered together with follicular differentiated thyroid carcinoma, which supports recent morphological descriptions of thyroid follicular carcinoma-like tumors in the kidney and suggests that they represent a subtype of chromophobe carcinoma. We also found an expression signature identifying primary tumors of squamous cell histology in multiple tissues. Next, a subset of ovarian tumors enriched with endometrioid histology clustered together with endometrium tumors, confirming that they share their etiopathogenesis, which strongly differs from serous ovarian tumors. In addition, the clustering of colon and breast tumors correlated with clinico-pathological characteristics. Moreover, a signature was developed based on our unsupervised clustering of breast tumors and this was predictive for disease-specific survival in three independent studies. Next, the metastases from ovarian, breast, lung and vulva cluster with their tissue of origin while metastases from colon showed a bimodal distribution. A significant part clusters with tissue of origin while the remaining tumors cluster with the tissue of destination. Conclusion Our molecular taxonomy of epithelial human cancer indicates surprising correlations over tissues. This may have a significant impact on the classification of many cancer sites and may guide pathologists, both in research and daily practice. Moreover, these results based on unsupervised analysis yielded a signature predictive of clinical outcome in breast cancer. Additionally, we hypothesize that metastases from gastrointestinal origin either remember their tissue of origin or adapt to the tissue of destination. More specifically, colon metastases in the liver show strong evidence for such a bimodal tissue specific profile. PMID:20017941

A national survey of marine biotoxins in wild-caught abalone in Australia.

PubMed

Malhi, Navreet; Turnbull, Alison; Tan, Jessica; Kiermeier, Andreas; Nimmagadda, Rama; McLeod, Catherine

2014-11-01

The first national survey of Australian wild-caught abalone was conducted between September 2012 and December 2013. The aim of the survey was to determine the presence of paralytic shellfish toxins (PSTs), amnesic shellfish toxins (ASTs), and diarrhetic shellfish toxins (DSTs) in wild-caught abalone at levels above the current Codex marine biotoxin limits during the 2013 fishing season. Abalone (n = 190) were collected from 68 abalone-fishing blocks for which the combined annual harvest accounts for 80 % of Australian production. Concurrent seawater samples were collected and enumerated for potentially toxic phytoplankton. The foot and viscera tissues of each abalone sample were analyzed separately for PSTs, ASTs, and DSTs. No samples (abalone foot or viscera) contained toxins at levels exceeding the marine biotoxin limits stipulated by Codex. The resulting prevalence estimate suggests that less than 1.6 % of the commercially caught wild abalone population in Australia were contaminated with marine biotoxins at levels above the regulatory limit during the survey period. ASTs were detected at very low (trace) levels in the foot and viscera tissue of four and three abalone samples, respectively. To our knowledge, this represents the first reported detection of domoic acid in Australian abalone. PSTs also were detected at very low levels in 17 samples of abalone foot tissue and 6 samples of abalone viscera. The association between the low levels of ASTs and PSTs detected in abalone and the presence of potential toxin-producing phytoplankton in seawater samples was weak. DSTs were not detected in any abalone despite the detection of very low levels of DST-producing phytoplankton in a small number (9 of 77) of seawater samples. The results of this survey should be useful for public health risk assessments and provide additional evidence that the prevalence of marine biotoxins in Australian wild-caught abalone is very low.

Sequencing the transcriptome of milk production: milk trumps mammary tissue.

PubMed

Lemay, Danielle G; Hovey, Russell C; Hartono, Stella R; Hinde, Katie; Smilowitz, Jennifer T; Ventimiglia, Frank; Schmidt, Kimberli A; Lee, Joyce W S; Islas-Trejo, Alma; Silva, Pedro Ivo; Korf, Ian; Medrano, Juan F; Barry, Peter A; German, J Bruce

2013-12-12

Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells. We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat, whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from milk-producing mammary epithelial cells. RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation.

Fascial flap reconstruction of the hand: a single surgeon's 30-year experience.

PubMed

Carty, Matthew J; Taghinia, Amir; Upton, Joseph

2010-03-01

The reconstruction of complex hand wounds is challenging due to the requirements for thin and pliable coverage with a reliable vascular supply, potential for sensibility, and provision of a gliding surface. Fascial flaps represent an excellent option for the reconstruction of these complicated defects. A retrospective review of fascial flap reconstructive procedures to the hand undertaken by a single microsurgeon was performed for operations occurring between 1979 and 2009. Both pedicled and free tissue transfer procedures were included in both pediatric and adult patients. Data were culled from a combination of patient charts, hospital records, radiographic studies, and clinical photographs. Sixty fascial flap reconstructive procedures to the hand were analyzed in 60 patients from the defined 30-year period. The most common pathological process necessitating reconstruction was acute trauma (n = 32, 53 percent). Most of the soft-tissue injuries included in the study sample were located on the dorsal hand and wrist (n = 27, 45 percent). The most commonly utilized reconstructive modality was the temporoparietal fascial flap (n = 35, 58 percent). Most reconstructions were completed as free tissue transfers (n = 46, 77 percent). Perioperative complications were relatively minor; no flap losses were recorded. All cases studied demonstrated excellent long-term coverage with no evidence of underlying tendon adhesion or contracture. Fascial flaps represent an excellent option for coverage of soft-tissue defects of the hand that are not amenable to reconstruction with skin grafting alone, particularly for localized defects with denuded tendons or exposed joints.

PCR-based methods for identification of potentially zoonotic ascaridoid parasites of the dog, fox and cat.

PubMed

Jacobs, D E; Zhu, X; Gasser, R B; Chilton, N B

1997-11-01

Genomic DNA was extracted from ascaridoid nematodes collected from dogs, foxes and cats. A region spanning the second internal transcribed spacer (ITS-2) of the ribosomal DNA of each sample was amplified by PCR. Representative ITS-2 products for each nematode species (Toxocara canis, Toxocara cati and Toxascaris leonina) were sequenced. Restriction sites were identified for use as genetic markers in a PCR-linked RFLP assay. The three species could be differentiated from each other and from other ascaridoids that may be found in human tissues by use of two endonucleases, HinfI and RsaI. Primers were designed to unique regions of the ITS-2 sequences of the three species for use in diagnostic PCR procedures and primer sets evaluated against panels of homologous and heterologous DNA samples. Results suggest that both methods are good candidates for further development for the detection and/or identification of ascaridoid larvae in human tissues.

Subsistence restoration project: Food safety testing. Exxon Valdez Oil Spill Restoration Project. Final report restoration project 94279

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miraglia, R.A.; Chartrand, A.W.

1997-05-01

The goal of this project was to restore the confidence of subsistence users in their abilities to determine the safety of their resources. Methods included community meetings, collection and testing of subsistence resources samples for hydrocarbon contamination, accompanying community representatives on tours of the laboratory where tests were conducted and informational newsletters. Over the two years of the project combined, 228 composite samples of edible tissues from shellfish were tested. The bile of forty rockfish, six sockeye salmon, twelve seals, twenty-three ducks were tested for the presence of hydrocarbon metabolites. Edible tissue (blubber) from seals was also tested. Generally, themore » tests showed such low levels of hydrocarbons and their metabolites, as to be within the test`s margin of error. The project was partly successful in disseminating the subsistence food safety advice of the Oil Spill Health Task Force and in improving the level of trust in the results of hydrocarbon tests on the resources.« less

Biologically relevant effects of mRNA amplification on gene expression profiles.

PubMed

van Haaften, Rachel I M; Schroen, Blanche; Janssen, Ben J A; van Erk, Arie; Debets, Jacques J M; Smeets, Hubert J M; Smits, Jos F M; van den Wijngaard, Arthur; Pinto, Yigal M; Evelo, Chris T A

2006-04-11

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other.Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways.

Biologically relevant effects of mRNA amplification on gene expression profiles

PubMed Central

van Haaften, Rachel IM; Schroen, Blanche; Janssen, Ben JA; van Erk, Arie; Debets, Jacques JM; Smeets, Hubert JM; Smits, Jos FM; van den Wijngaard, Arthur; Pinto, Yigal M; Evelo, Chris TA

2006-01-01

Background Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Results Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other. Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. Conclusion This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways. PMID:16608515

Automated classification of immunostaining patterns in breast tissue from the human protein atlas.

PubMed

Swamidoss, Issac Niwas; Kårsnäs, Andreas; Uhlmann, Virginie; Ponnusamy, Palanisamy; Kampf, Caroline; Simonsson, Martin; Wählby, Carolina; Strand, Robin

2013-01-01

The Human Protein Atlas (HPA) is an effort to map the location of all human proteins (http://www.proteinatlas.org/). It contains a large number of histological images of sections from human tissue. Tissue micro arrays (TMA) are imaged by a slide scanning microscope, and each image represents a thin slice of a tissue core with a dark brown antibody specific stain and a blue counter stain. When generating antibodies for protein profiling of the human proteome, an important step in the quality control is to compare staining patterns of different antibodies directed towards the same protein. This comparison is an ultimate control that the antibody recognizes the right protein. In this paper, we propose and evaluate different approaches for classifying sub-cellular antibody staining patterns in breast tissue samples. The proposed methods include the computation of various features including gray level co-occurrence matrix (GLCM) features, complex wavelet co-occurrence matrix (CWCM) features, and weighted neighbor distance using compound hierarchy of algorithms representing morphology (WND-CHARM)-inspired features. The extracted features are used into two different multivariate classifiers (support vector machine (SVM) and linear discriminant analysis (LDA) classifier). Before extracting features, we use color deconvolution to separate different tissue components, such as the brownly stained positive regions and the blue cellular regions, in the immuno-stained TMA images of breast tissue. We present classification results based on combinations of feature measurements. The proposed complex wavelet features and the WND-CHARM features have accuracy similar to that of a human expert. Both human experts and the proposed automated methods have difficulties discriminating between nuclear and cytoplasmic staining patterns. This is to a large extent due to mixed staining of nucleus and cytoplasm. Methods for quantification of staining patterns in histopathology have many applications, ranging from antibody quality control to tumor grading.

qPCR in gastrointestinal stromal tumors: Evaluation of reference genes and expression analysis of KIT and the alternative receptor tyrosine kinases FLT3, CSF1-R, PDGFRB, MET and AXL

PubMed Central

2010-01-01

Background Gastrointestinal stromal tumors (GIST) represent the most common mesenchymal tumors of the gastrointestinal tract. About 85% carry an activating mutation in the KIT or PDGFRA gene. Approximately 10% of GIST are so-called wild type GIST (wt-GIST) without mutations in the hot spots. In the present study we evaluated appropriate reference genes for the expression analysis of formalin-fixed, paraffin-embedded and fresh frozen samples from gastrointestinal stromal tumors. We evaluated the gene expression of KIT as well as of the alternative receptor tyrosine kinase genes FLT3, CSF1-R, PDGFRB, AXL and MET by qPCR. wt-GIST were compared to samples with mutations in KIT exon 9 and 11 and PDGFRA exon 18 in order to evaluate whether overexpression of these alternative RTK might contribute to the pathogenesis of wt-GIST. Results Gene expression variability of the pooled cDNA samples is much lower than the single reverse transcription cDNA synthesis. By combining the lowest variability values of fixed and fresh tissue, the genes POLR2A, PPIA, RPLPO and TFRC were chosen for further analysis of the GIST samples. Overexpression of KIT compared to the corresponding normal tissue was detected in each GIST subgroup except in GIST with PDGFRA exon 18 mutation. Comparing our sample groups, no significant differences in the gene expression levels of FLT3, CSF1R and AXL were determined. An exception was the sample group with KIT exon 9 mutation. A significantly reduced expression of CSF1R, FLT3 and PDGFRB compared to the normal tissue was detected. GIST with mutations in KIT exon 9 and 11 and in PDGFRA exon 18 showed a significant PDGFRB downregulation. Conclusions As the variability of expression levels for the reference genes is very high comparing fresh frozen and formalin-fixed tissue there is a strong need for validation in each tissue type. None of the alternative receptor tyrosine kinases analyzed is associated with the pathogenesis of wild-type or mutated GIST. It remains to be clarified whether an autocrine or paracrine mechanism by overexpression of receptor tyrosine kinase ligands is responsible for the tumorigenesis of wt-GIST. PMID:21171987

Gene expression profile in mesenchymal stem cells derived from dental tissues and bone marrow

PubMed Central

Kim, Su-Hwan; Kim, Young-Sung; Lee, Su-Yeon; Kim, Kyoung-Hwa; Lee, Yong-Moo; Kim, Won-Kyung

2011-01-01

Purpose The aim of this study is to compare the gene expression profile in mesenchymal stem cells derived from dental tissues and bone marrow for characterization of dental stem cells. Methods We employed GeneChip analysis to the expression levels of approximately 32,321 kinds of transcripts in 5 samples of bone-marrow-derived mesenchymal stem cells (BMSCs) (n=1), periodontal ligament stem cells (PDLSCs) (n=2), and dental pulp stem cells (DPSCs) (n=2). Each cell was sorted by a FACS Vantage Sorter using immunocytochemical staining of the early mesenchymal stem cell surface marker STRO-1 before the microarray analysis. Results We identified 379 up-regulated and 133 down-regulated transcripts in BMSCs, 68 up-regulated and 64 down-regulated transcripts in PDLSCs, and 218 up-regulated and 231 down-regulated transcripts in DPSCs. In addition, anatomical structure development and anatomical structure morphogenesis gene ontology (GO) terms were over-represented in all three different mesenchymal stem cells and GO terms related to blood vessels, and neurons were over-represented only in DPSCs. Conclusions This study demonstrated the genome-wide gene expression patterns of STRO-1+ mesenchymal stem cells derived from dental tissues and bone marrow. The differences among the expression profiles of BMSCs, PDLSCs, and DPSCs were shown, and 999 candidate genes were found to be definitely up- or down-regulated. In addition, GOstat analyses of regulated gene products provided over-represented GO classes. These data provide a first step for discovering molecules key to the characteristics of dental stem cells. PMID:21954424

The proto-oncogene KRAS and BRAF profiles and some clinical characteristics in colorectal cancer in the Turkish population.

PubMed

Ozen, Filiz; Ozdemir, Semra; Zemheri, Ebru; Hacimuto, Gizem; Silan, Fatma; Ozdemir, Ozturk

2013-02-01

The aim of the current study was to investigate the prevalence and predictive significance of the KRAS and BRAF mutations in Turkish patients with colorectal cancer (CRC). Totally, 53 fresh tumoral tissue specimens were investigated in patients with CRC. All specimens were obtained during routine surgery of patients who were histopathologically diagnosed and genotyped for common KRAS and BRAF point mutations. After DNA extraction, the target mutations were analyzed using the AutoGenomics INFINITI(®) assay, and some samples were confirmed by quantitative real-time polymerase chain reaction fluorescence melting curve analyses. KRAS mutations were found in 26 (49.05%) CRC samples. Twenty-seven samples (50.95%) had wild-type profiles for KRAS codon 12, 13, and 61 in the current cohort. In 17 (65.38%) samples, codon 12; in 7 (26.93%) samples, codon 13; and in 2 (7.69%) samples, codon 61 were found to be mutated, particularly in grade 2 of tumoral tissues. No point mutation was detected in BRAF codon Val600Glu for the studied CRC patients. Our study, based on a representative collection of human CRC tumors, indicates that KRAS gene mutations were detected in 49.05% of the samples, and the most frequent mutation was in the G12D codon. Results also showed that codons 12 and 13 of KRAS are relatively frequently without BRAF mutation in a CRC cohort from the Turkish population.

DNA degrades during storage in formalin-fixed and paraffin-embedded tissue blocks.

PubMed

Guyard, Alice; Boyez, Alice; Pujals, Anaïs; Robe, Cyrielle; Tran Van Nhieu, Jeanne; Allory, Yves; Moroch, Julien; Georges, Odette; Fournet, Jean-Christophe; Zafrani, Elie-Serge; Leroy, Karen

2017-10-01

Formalin-fixed paraffin-embedded (FFPE) tissue blocks are widely used to identify clinically actionable molecular alterations or perform retrospective molecular studies. Our goal was to quantify degradation of DNA occurring during mid to long-term storage of samples in usual conditions. We selected 46 FFPE samples of surgically resected carcinomas of lung, colon, and urothelial tract, of which DNA had been previously extracted. We performed a second DNA extraction on the same blocks under identical conditions after a median period of storage of 5.5 years. Quantitation of DNA by fluorimetry showed a 53% decrease in DNA quantity after storage. Quantitative PCR (qPCR) targeting KRAS exon 2 showed delayed amplification of DNA extracted after storage in all samples but one. The qPCR/fluorimetry quantification ratio decreased from 56 to 15% after storage (p < 0.001). Overall, remaining proportion of DNA analyzable by qPCR represented only 11% of the amount obtained at first extraction. Maximal length of amplifiable DNA fragments assessed with a multiplex PCR was reduced in DNA extracted from stored tissue, indicating that DNA fragmentation had increased in the paraffin blocks during storage. Next-generation sequencing was performed on 12 samples and showed a mean 3.3-fold decrease in library yield and a mean 4.5-fold increase in the number of single-nucleotide variants detected after storage. In conclusion, we observed significant degradation of DNA extracted from the same FFPE block after 4 to 6 years of storage. Better preservation strategies should be considered for storage of FFPE biopsy specimens.

De novo metatranscriptome assembly and coral gene expression profile of Montipora capitata with growth anomaly.

PubMed

Frazier, Monika; Helmkampf, Martin; Bellinger, M Renee; Geib, Scott M; Takabayashi, Misaki

2017-09-11

Scleractinian corals are a vital component of coral reef ecosystems, and of significant cultural and economic value worldwide. As anthropogenic and natural stressors are contributing to a global decline of coral reefs, understanding coral health is critical to help preserve these ecosystems. Growth anomaly (GA) is a coral disease that has significant negative impacts on coral biology, yet our understanding of its etiology and pathology is lacking. In this study we used RNA-seq along with de novo metatranscriptome assembly and homology assignment to identify coral genes that are expressed in three distinct coral tissue types: tissue from healthy corals ("healthy"), GA lesion tissue from diseased corals ("GA-affected") and apparently healthy tissue from diseased corals ("GA-unaffected"). We conducted pairwise comparisons of gene expression among these three tissue types to identify genes and pathways that help us to unravel the molecular pathology of this coral disease. The quality-filtered de novo-assembled metatranscriptome contained 76,063 genes, of which 13,643 were identified as putative coral genes. Overall gene expression profiles of coral genes revealed high similarity between healthy tissue samples, in contrast to high variance among diseased samples. This indicates GA has a variety of genetic effects at the colony level, including on seemingly healthy (GA-unaffected) tissue. A total of 105 unique coral genes were found differentially expressed among tissue types. Pairwise comparisons revealed the greatest number of differentially expressed genes between healthy and GA-affected tissue (93 genes), followed by healthy and GA-unaffected tissue (33 genes), and GA-affected and -unaffected tissue (7 genes). The putative function of these genes suggests GA is associated with changes in the activity of genes involved in developmental processes and activation of the immune system. This is one of the first transcriptome-level studies to investigate coral GA, and the first metatranscriptome assembly for the M. capitata holobiont. The gene expression data, metatranscriptome assembly and methodology developed through this study represent a significant addition to the molecular information available to further our understanding of this coral disease.

Development of an Autonomous, Dual Chamber Bioreactor for the Growth of 3-Dimensional Epithelial-Stromal Tissues in Microgravity

NASA Technical Reports Server (NTRS)

Patel, Zarana S.; Wettergreen, Matthew A.; Huff, Janice L.

2014-01-01

We are developing a novel, autonomous bioreactor that can provide for the growth and maintenance in microgravity of 3-D organotypic epithelial-stromal cultures that require an air-liquid interface. These complex 3-D tissue models accurately represent the morphological features, differentiation markers, and growth characteristics observed in normal human epithelial tissues, including the skin, esophagus, lung, breast, pancreas, and colon. However, because of their precise and complex culture requirements, including that of an air-liquid interface, these 3-D models have yet to be utilized for life sciences research aboard the International Space Station. The development of a bioreactor for these cultures will provide the capability to perform biological research on the ISS using these realistic, tissue-like human epithelial-stromal cell models and will contribute significantly to advances in fundamental space biology research on questions regarding microgravity effects on normal tissue development, aging, cancer, and other disease processes. It will also allow for the study of how combined stressors, such as microgravity with radiation and nutritional deficiencies, affect multiple biological processes and will provide a platform for conducting countermeasure investigations on the ISS without the use of animal models. The technology will be autonomous and consist of a cell culture chamber that provides for air-liquid, liquid-liquid, and liquid-air exchanges within the chambers while maintaining the growth and development of the biological samples. The bioreactor will support multiple tissue types and its modular design will provide for incorporation of add-on capabilities such as microfluidics drug delivery, media sampling, and in situ biomarker analysis. Preliminary flight testing of the hardware will be conducted on a parabolic platform through NASA's Flight Opportunities Program.

Tissue grown in space in NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens of cartilage tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. Constructs grown on Mir (A) tended to become more spherical, whereas those grown on Earth (B) maintained their initial disc shape. These findings might be related to differences in cultivation conditions, i.e., videotapes showed that constructs floated freely in microgravity but settled and collided with the rotating vessel wall at 1g (Earth's gravity). In particular, on Mir the constructs were exposed to uniform shear and mass transfer at all surfaces such that the tissue grew equally in all directions, whereas on Earth the settling of discoid constructs tended to align their flat circular areas perpendicular to the direction of motion, increasing shear and mass transfer circumferentially such that the tissue grew preferentially in the radial direction. A and B are full cross sections of constructs from Mir and Earth groups shown at 10-power. C and D are representative areas at the construct surfaces enlarged to 200-power. They are stained red with safranin-O. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). Photo credit: Proceedings of the National Academy of Sciences.

Differential Expression of Cytochrome P450 Enzymes in Normal and Tumor Tissues from Childhood Rhabdomyosarcoma

PubMed Central

Molina-Ortiz, Dora; Camacho-Carranza, Rafael; González-Zamora, José Francisco; Shalkow-Kalincovstein, Jaime; Cárdenas-Cardós, Rocío; Ností-Palacios, Rosario; Vences-Mejía, Araceli

2014-01-01

Intratumoral expression of genes encoding Cytochrome P450 enzymes (CYP) might play a critical role not only in cancer development but also in the metabolism of anticancer drugs. The purpose of this study was to compare the mRNA expression patterns of seven representative CYPs in paired tumor and normal tissue of child patients with rabdomyosarcoma (RMS). Using real time quantitative RT-PCR, the gene expression pattern of CYP1A1, CYP1A2, CYP1B1, CYP2E1, CYP2W1, CYP3A4, and CYP3A5 were analyzed in tumor and adjacent non-tumor tissues from 13 child RMS patients. Protein concentration of CYPs was determined using Western blot. The expression levels were tested for correlation with the clinical and pathological data of the patients. Our data showed that the expression levels of CYP1A1 and CYP1A2 were negligible. Elevated expression of CYP1B1 mRNA and protein was detected in most RMS tumors and adjacent normal tissues. Most cancerous samples exhibit higher levels of both CYP3A4 and CYP3A5 compared with normal tissue samples. Expression of CYP2E1 mRNA was found to be significantly higher in tumor tissue, however no relation was found with protein levels. CYP2W1 mRNA and/or protein are mainly expressed in tumors. In conclusion, we defined the CYP gene expression profile in tumor and paired normal tissue of child patients with RMS. The overexpression of CYP2W1, CYP3A4 and CYP3A5 in tumor tissues suggests that they may be involved in RMS chemoresistance; furthermore, they may be exploited for the localized activation of anticancer prodrugs. PMID:24699256

An abundance of Epsilonproteobacteria revealed in the gut microbiome of the laboratory cultured sea urchin, Lytechinus variegatus

PubMed Central

Hakim, Joseph A.; Koo, Hyunmin; Dennis, Lacey N.; Kumar, Ranjit; Ptacek, Travis; Morrow, Casey D.; Lefkowitz, Elliot J.; Powell, Mickie L.; Bej, Asim K.; Watts, Stephen A.

2015-01-01

In this study, we have examined the bacterial community composition of the laboratory cultured sea urchin Lytechinus variegatus gut microbiome and its culture environment using NextGen amplicon sequencing of the V4 segment of the 16S rRNA gene, and downstream bioinformatics tools. Overall, the gut and tank water was dominated by Proteobacteria, whereas the feed consisted of a co-occurrence of Proteobacteria and Firmicutes at a high abundance. The gut tissue represented Epsilonproteobacteria as dominant, with order Campylobacterales at the highest relative abundance (>95%). However, the pharynx tissue was dominated by class Alphaproteobacteria. The gut digesta and egested fecal pellets had a high abundance of class Gammaproteobacteria, from which Vibrio was found to be the primary genus, and Epsilonproteobacteria, with genus Arcobacter occurring at a moderate level. At the class level, the tank water was dominated by Gammaproteobacteria, and the feed by Alphaproteobacteria. Multi-Dimensional Scaling analysis showed that the microbial community of the gut tissue clustered together, as did the pharynx tissue to the feed. The gut digesta and egested fecal pellets showed a similarity relationship to the tank water. Further analysis of Campylobacterales at a lower taxonomic level using the oligotyping method revealed 37 unique types across the 10 samples, where Oligotype 1 was primarily represented in the gut tissue. BLAST analysis identified Oligotype 1 to be Arcobacter sp., Sulfuricurvum sp., and Arcobacter bivalviorum at an identity level >90%. This study showed that although distinct microbial communities are evident across multiple components of the sea urchin gut ecosystem, there is a noticeable correlation between the overall microbial communities of the gut with the sea urchin L. variegatus culture environment. PMID:26528245

Immune tolerance properties of the testicular tissue as a viral sanctuary site in ART-treated HIV-infected adults.

PubMed

Jenabian, Mohammad-Ali; Costiniuk, Cecilia T; Mehraj, Vikram; Ghazawi, Feras M; Fromentin, Rémi; Brousseau, Joëlle; Brassard, Pierre; Bélanger, Maud; Ancuta, Petronela; Bendayan, Reina; Chomont, Nicolas; Routy, Jean-Pierre

2016-11-28

HIV persistence in long-lived infected cells and in anatomical sanctuary sites are major hurdles to HIV eradication. Testicular tissue may represent a significant viral sanctuary site as it constitutes an immunologically privileged compartment. We assessed immunotolerance properties of the testicular tissue in individuals receiving suppressive antiretroviral therapy (ART). Testicular tissue and matched blood samples were collected from six virally suppressed adults and 10 HIV-uninfected controls prior to sex reassignment surgery. T cells were purified from freshly isolated testicular interstitial cell suspensions. T-cell subsets, expression of immune activation markers and HIV DNA were assessed in matched testicular cells and peripheral blood mononuclear cells (PBMCs). When compared with PBMCs, testes were characterized by a lower CD4 T-cell proportion among total T cells, a decrease in the frequency of naive cells, an increase in the frequency of effector-memory T cells and an increase in CCR5 expression in both the HIV+ and HIV- groups. In HIV-infected individuals on ART, testes displayed higher T-cell immune activation (Coexpression of CD38 and Human Leukocyte Antigen - antigen D Related) than PBMCs. In both groups, testes were characterized by higher frequencies of immunosuppressive CD39 regulatory T cells and a massive increase in CD73 expression on CD8 T cells. In addition, a remarkable increase in indoleamine-pyrrole 2,3-dioxygenase immunosuppressive enzyme involved in tryptophan/kynurenine catabolism was observed in testes versus blood. Rare cells harboring HIV DNA were detected in testes from five out six participants. These findings suggest that the adenosine and tryptophan/kynurenine immune-metabolic pathways contribute to immune tolerance in testicular tissue. Our results suggest that testes may represent a distinctive HIV sanctuary site during ART.

The endothelial lipase protein is promising urinary biomarker for diagnosis of gastric cancer.

PubMed

Dong, Xueyan; Wang, Guoqing; Zhang, Guoqing; Ni, Zhaohui; Suo, Jian; Cui, Juan; Cui, Ai; Yang, Qing; Xu, Ying; Li, Fan

2013-03-19

Gastric cancer is one of the most common malignant tumors in the world. Finding effective diagnostic biomarkers in urine or serum would represent the most ideal solution to detecting gastric cancer during annual physical examination. This study was to evaluate the potential of endothelial lipase (EL) as a urinary biomarker for diagnosis of gastric cancer. The expression levels of EL was measured using Western blotting and immunohistochemical staining experiments on (tissue, serum, and urine) samples of gastric cancer patients versus healthy people. We also checked the EL levels in the urine samples of other cancer types (lung, colon and rectum cancers) and benign lesions (gastritis and gastric leiomyoma) to check if EL was specific to gastric cancer. We observed a clear separation between the EL expression levels in the urine samples of 90 gastric cancer patients and of 57 healthy volunteers. It was approximately 9.9 fold average decrease of the EL expression levels in the urine samples of gastric cancer compared to the healthy controls (P <0.0001), achieving a 0.967 AUC value for the ROC (receiver operating characteristic) curve, demonstrating it's highly accurate as a diagnostic marker for gastric cancer. Interestingly, the expression levels of EL in tissue and serum samples were not nearly as discriminative as in urine samples (P = 0.90 and P = 0.79). In immunohistochemical experiments, positive expression of the EL protein was found in 67% (8/12) of gastric adjacent noncancerous and in 58% (7/12) of gastric cancer samples. There was no significant statistical in the expression levels of this protein between the gastric cancer and the matching noncancerous tissues (P =0.67). The urinary EL as a highly accurate gastric cancer biomarker that is potentially applicable to the general screening with high sensitivity and specificity. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4527331618757552.

The endothelial lipase protein is promising urinary biomarker for diagnosis of gastric cancer

PubMed Central

2013-01-01

Background Gastric cancer is one of the most common malignant tumors in the world. Finding effective diagnostic biomarkers in urine or serum would represent the most ideal solution to detecting gastric cancer during annual physical examination. This study was to evaluate the potential of endothelial lipase (EL) as a urinary biomarker for diagnosis of gastric cancer. Methods The expression levels of EL was measured using Western blotting and immunohistochemical staining experiments on (tissue, serum, and urine) samples of gastric cancer patients versus healthy people. We also checked the EL levels in the urine samples of other cancer types (lung, colon and rectum cancers) and benign lesions (gastritis and gastric leiomyoma) to check if EL was specific to gastric cancer. Result We observed a clear separation between the EL expression levels in the urine samples of 90 gastric cancer patients and of 57 healthy volunteers. It was approximately 9.9 fold average decrease of the EL expression levels in the urine samples of gastric cancer compared to the healthy controls (P <0.0001), achieving a 0.967 AUC value for the ROC (receiver operating characteristic) curve, demonstrating it’s highly accurate as a diagnostic marker for gastric cancer. Interestingly, the expression levels of EL in tissue and serum samples were not nearly as discriminative as in urine samples (P = 0.90 and P = 0.79). In immunohistochemical experiments, positive expression of the EL protein was found in 67% (8/12) of gastric adjacent noncancerous and in 58% (7/12) of gastric cancer samples. There was no significant statistical in the expression levels of this protein between the gastric cancer and the matching noncancerous tissues (P =0.67). Conclusions The urinary EL as a highly accurate gastric cancer biomarker that is potentially applicable to the general screening with high sensitivity and specificity. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4527331618757552 PMID:23510199

Pharmacokinetics and tissue elimination of flunixin in veal calves.

PubMed

Kissell, Lindsey W; Brinson, Patrick D; Gehring, Ronette; Tell, Lisa A; Wetzlich, Scott E; Baynes, Ronald E; Riviere, Jim E; Smith, Geof W

2016-06-01

OBJECTIVE To describe plasma pharmacokinetic parameters and tissue elimination of flunixin in veal calves. ANIMALS 20 unweaned Holstein calves between 3 and 6 weeks old. PROCEDURES Each calf received flunixin (2.2 mg/kg, IV, q 24 h) for 3 days. Blood samples were collected from all calves before the first dose and at predetermined times after the first and last doses. Beginning 24 hours after injection of the last dose, 4 calves were euthanized each day for 5 days. Plasma and tissue samples were analyzed by ultraperformance liquid chromatography. Pharmacokinetic parameters were calculated by compartmental and noncompartmental methods. RESULTS Mean ± SD plasma flunixin elimination half-life, residence time, and clearance were 1.32 ± 0.94 hours, 12.54 ± 10.96 hours, and 64.6 ± 40.7 mL/h/kg, respectively. Mean hepatic and muscle flunixin concentrations decreased to below FDA-established tolerance limits (0.125 and 0.025 μg/mL, respectively) for adult cattle by 3 and 2 days, respectively, after injection of the last dose of flunixin. Detectable flunixin concentrations were present in both the liver and muscle for at least 5 days after injection of the last dose. CONCLUSIONS AND CLINICAL RELEVANCE The labeled slaughter withdrawal interval for flunixin in adult cattle is 4 days. Because administration of flunixin to veal calves represents extralabel drug use, any detectable flunixin concentrations in edible tissues are considered a violation. Results indicated that a slaughter withdrawal interval of several weeks may be necessary to ensure that violative tissue residues of flunixin are not detected in veal calves treated with that drug.

Needle Steering in Biological Tissue using Ultrasound-based Online Curvature Estimation

PubMed Central

Moreira, Pedro; Patil, Sachin; Alterovitz, Ron; Misra, Sarthak

2014-01-01

Percutaneous needle insertions are commonly performed for diagnostic and therapeutic purposes. Accurate placement of the needle tip is important to the success of many needle procedures. The current needle steering systems depend on needle-tissue-specific data, such as maximum curvature, that is unavailable prior to an interventional procedure. In this paper, we present a novel three-dimensional adaptive steering method for flexible bevel-tipped needles that is capable of performing accurate tip placement without previous knowledge about needle curvature. The method steers the needle by integrating duty-cycled needle steering, online curvature estimation, ultrasound-based needle tracking, and sampling-based motion planning. The needle curvature estimation is performed online and used to adapt the path and duty cycling. We evaluated the method using experiments in a homogenous gelatin phantom, a two-layer gelatin phantom, and a biological tissue phantom composed of a gelatin layer and in vitro chicken tissue. In all experiments, virtual obstacles and targets move in order to represent the disturbances that might occur due to tissue deformation and physiological processes. The average targeting error using our new adaptive method is 40% lower than using the conventional non-adaptive duty-cycled needle steering method. PMID:26229729

Bone scaffolds with homogeneous and discrete gradient mechanical properties.

PubMed

Jelen, C; Mattei, G; Montemurro, F; De Maria, C; Mattioli-Belmonte, M; Vozzi, G

2013-01-01

Bone TE uses a scaffold either to induce bone formation from surrounding tissue or to act as a carrier or template for implanted bone cells or other agents. We prepared different bone tissue constructs based on collagen, gelatin and hydroxyapatite using genipin as cross-linking agent. The fabricated construct did not present a release neither of collagen neither of genipin over its toxic level in the surrounding aqueous environment. Each scaffold has been mechanically characterized with compression, swelling and creep tests, and their respective viscoelastic mechanical models were derived. Mechanical characterization showed a practically elastic behavior of all samples and that compressive elastic modulus basically increases as content of HA increases, and it is strongly dependent on porosity and water content. Moreover, by considering that gradients in cellular and extracellular architecture as well as in mechanical properties are readily apparent in native tissues, we developed discrete functionally graded scaffolds (discrete FGSs) in order to mimic the graded structure of bone tissue. These new structures were mechanically characterized showing a marked anisotropy as the native bone tissue. Results obtained have shown FGSs could represent valid bone substitutes. Copyright © 2012 Elsevier B.V. All rights reserved.

Occurrence of Salmonella serotype Typhimurium DT104 on a commercial swine farm before, during, and after depopulation and repopulation.

PubMed

Erdman, Matthew M; Harris, Isabel T; Torremorell, Montserrat; Wilt, Vincil M; Harris, D L Hank

2005-08-01

To determine whether depopulation-repopulation could be used to eradicate Salmonella serotype Typhimurium DT104 from a commercial swine farm in the midwestern United States. Observational study A commercial swine farm undergoing depopulation-repopulation to eliminate porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae. Pooled fecal samples, tissue samples, and serum samples were collected from pigs on the farm before and after depopulation-repopulation. When there were no pigs on the farm, environmental swab specimens were collected for bacterial culture. Serum was analyzed for anti-Salmonella antibodies with an indirect ELISA. Salmonella isolates obtained by bacterial culture of fecal, tissue, and environmental samples were characterized by means of serotyping, phage typing, pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing. 167 Salmonella isolates representing 9 serotypes were recovered from the farm. Results of PFGE and antimicrobial susceptibility testing suggested that S. Typhimurium DT104 strain was not eradicated from the farm. However, seroprevalence of anti-Salmonella antibodies and the percentage of pooled fecal samples positive for Salmonella spp were significantly decreased following repopulation. Results suggested that depopulation-repopulation in conjunction with stringent cleaning and disinfection, attention to biosecurity procedures, control of other diseases, and changes in feed management may reduce the occurrence of, but likely will not eliminate, Salmonella spp in commercial swine herds.

Mining the archives: a cross-platform analysis of gene ...

EPA Pesticide Factsheets

Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for genomic research into the molecular basis of disease. However, use of FFPE samples in gene expression studies has been limited by technical challenges resulting from degradation of nucleic acids. Here we evaluated gene expression profiles derived from fresh-frozen (FRO) and FFPE mouse liver tissues using two DNA microarray protocols and two whole transcriptome sequencing (RNA-seq) library preparation methodologies. The ribo-depletion protocol outperformed the other three methods by having the highest correlations of differentially expressed genes (DEGs) and best overlap of pathways between FRO and FFPE groups. We next tested the effect of sample time in formalin (18 hours or 3 weeks) on gene expression profiles. Hierarchical clustering of the datasets indicated that test article treatment, and not preservation method, was the main driver of gene expression profiles. Meta- and pathway analyses indicated that biological responses were generally consistent for 18-hour and 3-week FFPE samples compared to FRO samples. However, clear erosion of signal intensity with time in formalin was evident, and DEG numbers differed by platform and preservation method. Lastly, we investigated the effect of age in FFPE block on genomic profiles. RNA-seq analysis of 8-, 19-, and 26-year-old control blocks using the ribo-depletion protocol resulted in comparable quality metrics, inc

Presence of archaea and selected bacteria in infected root canal systems.

PubMed

Brzezińska-Błaszczyk, Ewa; Pawłowska, Elżbieta; Płoszaj, Tomasz; Witas, Henryk; Godzik, Urszula; Agier, Justyna

2018-05-01

Infections of the root canal have polymicrobial etiology. The main group of microflora in the infected pulp is bacteria. There is limited data that archaea may be present in infected pulp tissue. The aim of this study was to check the prevalence of archaea in necrotic root canal samples obtained from patients with primary or post-treatment infection. The prevalence of selected bacteria species (Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Synergistes sp.) in necrotic samples was evaluated as well. Sixty-four samples from root canal were collected for DNA and RNA extraction. A PCR assay based on the 16S rRNA gene was used to determine the presence of archaea and selected bacteria. Of the 64 samples, 6 were analyzed by semiquantitative reverse transcription PCR to estimate expression profiles of 16S rRNA, and another 9 were selected for direct sequencing. Archaea were detected in 48.4% samples. Statistical analysis indicated a negative association in coexistence between archaea and Treponema denticola (P < 0.05; Pearson's χ 2 test). The main representative of the Archaea domain found in infected pulp tissue was Methanobrevibacter oralis. Archaea 16S rRNA gene expression was significantly lower than Synergistes sp., Porphyromonas gingivalis, and Tannerella forsythia (P < 0.05; Student's t test). Thus, it can be hypothesized that archaea may participate in the endodontic microbial community.

Significance of histopathology in pulsed NMR studies on cancer.

PubMed

Ranade, S S; Bharade, S H; Talwalkar, G V; Sujata, G K; Shrinivasan, V T; Singh, B B

1985-04-01

Characterization of tissue by pulsed nuclear magnetic resonance spectrometry opened a new area of research. The differences in the NMR parameters T1 and T2 of normal and malignant tissues constitute the basis for their distinction by pulsed NMR spectrometry and also by NMR imaging in vivo. The present studies were undertaken to correlate the role of constituent histological elements encountered in various malignancy-associated changes and T1 variations and are based on evaluation of samples taken from surgically resected specimens of carcinoma of the esophagus, comprising the uninvolved portions of the esophagus and the gastric end on gross examination. The uninvolved and involved regions showed low and high T1 values, respectively. High T1 values were also encountered in the zones of samples of uninvolved esophagus which histologically revealed areas with dysplasia. This feature, viz., dysplasia representing malignancy-associated changes, has been found to recur in many samples. Detailed histological studies provided further evidence confirming that areas with dysplasia contribute to an increase in T1 values whereas in zones at the gastric end metaplasia and hyperplasia are more common. The results are of value for demarcation of tumor area by in vivo NMR imaging.

CD13 as target for tissue factor induced tumor vascular infarction in small cell lung cancer.

PubMed

Schmidt, Lars Henning; Stucke-Ring, Janine; Brand, Caroline; Schliemann, Christoph; Harrach, Saliha; Muley, Thomas; Herpel, Esther; Kessler, Torsten; Mohr, Michael; Görlich, Dennis; Kreuter, Michael; Lenz, Georg; Wardelmann, Eva; Thomas, Michael; Berdel, Wolfgang E; Schwöppe, Christian; Hartmann, Wolfgang

2017-11-01

Zinc-binding protease aminopeptidase N (CD13) is expressed on tumor vascular cells and tumor cells. It represents a potential candidate for molecular targeted therapy, e.g. employing truncated tissue factor (tTF)-NGR, which can bind CD13 and thereby induce tumor vascular infarction. We performed a comprehensive analysis of CD13 expression in a clinically well characterized cohort of patients with small cell lung cancer (SCLC) to evaluate its potential use for targeted therapies in this disease. CD13 expression was analyzed immunohistochemically in 27 SCLC patients and correlated with clinical course and outcome. In CD-1 nude mice bearing human HTB119 SCLC xenotransplants, the systemic effects of the CD13-targeting fusion protein tTF-NGR on tumor growth were tested. In 52% of the investigated SCLC tissue samples, CD13 was expressed in tumor stroma cells, while the tumor cells were negative for CD13. No prognostic effect was found in the investigated SCLC study collective with regard to overall survival (p>0.05). In CD-1 nude mice, xenografts of CD13 negative HTB119 SCLC cells showed CD13 expression in the intratumoral vascular and perivascular cells, and the systemic application of CD13-targeted tissue factor tTF-NGR led to a significant reduction of tumor growth. We here present first data on the expression of CD13 in SCLC tumor samples. Our results strongly recommend the further investigation of tTF-NGR and other molecules targeted by NGR-peptides in SCLC patients. Considering the differential expression of CD13 in SCLC samples pre-therapeutic CD13 analysis is proposed for testing as investigational predictive biomarker for patient selection. Copyright © 2017 Elsevier B.V. All rights reserved.

Improving accuracy of DNA diet estimates using food tissue control materials and an evaluation of proxies for digestion bias.

PubMed

Thomas, Austen C; Jarman, Simon N; Haman, Katherine H; Trites, Andrew W; Deagle, Bruce E

2014-08-01

Ecologists are increasingly interested in quantifying consumer diets based on food DNA in dietary samples and high-throughput sequencing of marker genes. It is tempting to assume that food DNA sequence proportions recovered from diet samples are representative of consumer's diet proportions, despite the fact that captive feeding studies do not support that assumption. Here, we examine the idea of sequencing control materials of known composition along with dietary samples in order to correct for technical biases introduced during amplicon sequencing and biological biases such as variable gene copy number. Using the Ion Torrent PGM(©) , we sequenced prey DNA amplified from scats of captive harbour seals (Phoca vitulina) fed a constant diet including three fish species in known proportions. Alongside, we sequenced a prey tissue mix matching the seals' diet to generate tissue correction factors (TCFs). TCFs improved the diet estimates (based on sequence proportions) for all species and reduced the average estimate error from 28 ± 15% (uncorrected) to 14 ± 9% (TCF-corrected). The experimental design also allowed us to infer the magnitude of prey-specific digestion biases and calculate digestion correction factors (DCFs). The DCFs were compared with possible proxies for differential digestion (e.g. fish protein%, fish lipid%) revealing a strong relationship between the DCFs and percent lipid of the fish prey, suggesting prey-specific corrections based on lipid content would produce accurate diet estimates in this study system. These findings demonstrate the value of parallel sequencing of food tissue mixtures in diet studies and offer new directions for future research in quantitative DNA diet analysis. © 2013 John Wiley & Sons Ltd.

Comparison of Follicular and Luteal Phase Mucosal Markers of HIV Susceptibility in Healthy Women

PubMed Central

Chandra, Neelima; Yousefieh, Nazita; Zalenskaya, Irina; Kimble, Thomas; Asin, Susana; Rollenhagen, Christiane; Anderson, Sharon M.; Herold, Betsy; Mesquita, Pedro M.M.; Richardson-Harman, Nicola; Cunningham, Tina; Schwartz, Jill L.; Doncel, Gustavo F.

2016-01-01

Abstract The purpose of this study was to evaluate differences in vaginal immune cell populations, vaginal tissue gene expression, antimicrobial activity of the cervicovaginal (CV) lavage (CVL), vaginal flora, and p24 antigen production from CV tissues after ex vivo human immunodeficiency virus (HIV) infection between follicular (FOL) and luteal (LUT) phases of the menstrual cycle. CV tissue biopsies, CV secretions, and blood samples were obtained as part of two longitudinal clinical trials of healthy women (CONRAD D11-119 and A12-124 studies). Participants (n = 39) were HIV-seronegative women not using exogenous hormone supplementation, with normal menstrual cycles, who were screened to exclude sexually transmitted and reproductive tract infections. Serum levels of estradiol and progesterone were significantly higher in the LUT versus the FOL phase of the menstrual cycle. Controlling for race, reported contraceptive use/sexual practices, and clinical trial, we found no differences in vaginal tissue immune cell populations and activation status, transcriptomes, inhibition of HIV, herpes simplex virus type 2 and Escherichia coli by the CVL, vaginal pH or Nugent score, or production of p24 antigen after ex vivo infection by HIV-1BaL between CV samples obtained in the FOL phase versus the LUT phase of the menstrual cycle. There were no significant correlations between serum estradiol and progesterone levels and CV endpoints. The hypothesis that the LUT phase of the menstrual cycle represents a more vulnerable stage for mucosal infection with HIV was not supported by data from samples obtained from the lower genital tract (ectocervix and vagina) from these two clinical trials. PMID:26750085

A Stochastic Polygons Model for Glandular Structures in Colon Histology Images.

PubMed

Sirinukunwattana, Korsuk; Snead, David R J; Rajpoot, Nasir M

2015-11-01

In this paper, we present a stochastic model for glandular structures in histology images of tissue slides stained with Hematoxylin and Eosin, choosing colon tissue as an example. The proposed Random Polygons Model (RPM) treats each glandular structure in an image as a polygon made of a random number of vertices, where the vertices represent approximate locations of epithelial nuclei. We formulate the RPM as a Bayesian inference problem by defining a prior for spatial connectivity and arrangement of neighboring epithelial nuclei and a likelihood for the presence of a glandular structure. The inference is made via a Reversible-Jump Markov chain Monte Carlo simulation. To the best of our knowledge, all existing published algorithms for gland segmentation are designed to mainly work on healthy samples, adenomas, and low grade adenocarcinomas. One of them has been demonstrated to work on intermediate grade adenocarcinomas at its best. Our experimental results show that the RPM yields favorable results, both quantitatively and qualitatively, for extraction of glandular structures in histology images of normal human colon tissues as well as benign and cancerous tissues, excluding undifferentiated carcinomas.

Determination of a tissue-level failure evaluation standard for rat femoral cortical bone utilizing a hybrid computational-experimental method.

PubMed

Fan, Ruoxun; Liu, Jie; Jia, Zhengbin; Deng, Ying; Liu, Jun

2018-01-01

Macro-level failure in bone structure could be diagnosed by pain or physical examination. However, diagnosing tissue-level failure in a timely manner is challenging due to the difficulty in observing the interior mechanical environment of bone tissue. Because most fractures begin with tissue-level failure in bone tissue caused by continually applied loading, people attempt to monitor the tissue-level failure of bone and provide corresponding measures to prevent fracture. Many tissue-level mechanical parameters of bone could be predicted or measured; however, the value of the parameter may vary among different specimens belonging to a kind of bone structure even at the same age and anatomical site. These variations cause difficulty in representing tissue-level bone failure. Therefore, determining an appropriate tissue-level failure evaluation standard is necessary to represent tissue-level bone failure. In this study, the yield and failure processes of rat femoral cortical bones were primarily simulated through a hybrid computational-experimental method. Subsequently, the tissue-level strains and the ratio between tissue-level failure and yield strains in cortical bones were predicted. The results indicated that certain differences existed in tissue-level strains; however, slight variations in the ratio were observed among different cortical bones. Therefore, the ratio between tissue-level failure and yield strains for a kind of bone structure could be determined. This ratio may then be regarded as an appropriate tissue-level failure evaluation standard to represent the mechanical status of bone tissue.

Pharmacokinetics and tissue distribution of five active ingredients of Eucommiae cortex in normal and ovariectomized mice by UHPLC-MS/MS.

PubMed

An, Jing; Hu, Fangdi; Wang, Changhong; Zhang, Zijia; Yang, Li; Wang, Zhengtao

2016-09-01

1. Pinoresinol di-O-β-d-glucopyranoside (PDG), geniposide (GE), geniposidic acid (GA), aucubin (AN) and chlorogenic acid (CA) are the representative active ingredients in Eucommiae cortex (EC), which may be estrogenic. 2. The ultra high-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of the five ingredients showed good linearity, low limits of quantification and high extraction recoveries, as well as acceptable precision, accuracy and stability in mice plasma and tissue samples (liver, spleen, kidney and uterus). It was successfully applied to the comparative study on pharmacokinetics and tissue distribution of PDG, GE, GA, AN and CA between normal and ovariectomized (OVX) mice. 3. The results indicated that except CA, the plasma and tissue concentrations of PDG, GE, GA in OVX mice were all greater than those in normal mice. AN could only be detected in the plasma and liver homogenate of normal mice, which was poorly absorbed in OVX mice and low in other measured tissues. PDG, GE and GA seem to be better absorbed in OVX mice than in normal mice proved by the remarkable increased value of AUC0-∞ and Cmax. It is beneficial that PDG, GE, GA have better plasma absorption and tissue distribution in pathological state.

Using synchrotron X-ray phase-contrast micro-computed tomography to study tissue damage by laser irradiation.

PubMed

Robinson, Alan M; Stock, Stuart R; Soriano, Carmen; Xiao, Xianghui; Richter, Claus-Peter

2016-11-01

The aim of this study was to determine if X-ray micro-computed tomography could be used to locate and characterize tissue damage caused by laser irradiation and to describe its advantages over classical histology for this application. A surgical CO 2 laser, operated in single pulse mode (100 milliseconds) at different power settings, was used to ablate different types of cadaveric animal tissues. Tissue samples were then harvested and imaged with synchrotron X-ray phase-contrast and micro-computed tomography to generate stacks of virtual sections of the tissues. Subsequently, Fiji (ImageJ) software was used to locate tissue damage, then to quantify volumes of laser ablation cones and thermal coagulation damage from 3D renderings of tissue image stacks. Visual comparisons of tissue structures in X-ray images with those visible by classic light microscopy histology were made. We demonstrated that micro-computed tomography could be used to rapidly identify areas of surgical laser ablation, vacuolization, carbonization, and thermally coagulated tissue. Quantification and comparison of the ablation crater, which represents the volume of ablated tissue, and the thermal coagulation zone volumes were performed faster than we could by classical histology. We demonstrated that these procedures can be performed on fresh hydrated and non-sectioned plastic embedded tissue. We demonstrated that the application of non-destructive micro-computed tomography to the visualization and analysis of laser induced tissue damage without tissue sectioning is possible. This will improve evaluation of new surgical lasers and their corresponding effect on tissues. Lasers Surg. Med. 48:866-877, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Novel and differential accumulation of mitochondrial DNA deletions in Swedish and vietnamese patients with colorectal cancer.

PubMed

Dimberg, Jan; Hong, Thai Trinh; Skarstedt, Marita; Löfgren, Sture; Zar, Niklas; Matussek, Andreas

2014-01-01

Mitochondrial DNA (mtDNA) has been proposed to be involved in carcinogenesis and aging. The mtDNA 4977 bp deletion is one of the most frequently observed mtDNA mutations in human tissues and may play a role in colorectal cancer (CRC). In the present study, we aimed to evaluate the frequency of mtDNA 4977 bp deletion in CRC tissues and its association with clinical factors. We determined the presence of the 4977 bp common deletion in cancer and normal paired tissue samples from 105 Swedish and 88 Vietnamese patients with CRC using polymerase chain reaction (PCR) assays. The mtDNA 4977 bp deletion was shown to be significantly more frequent in normal tissues in comparison with paired cancer tissues in both Swedish and Vietnamese patients. The 4977 bp common deletion was significantly more frequent in cancer tissues of the Vietnamese patients compared to the Swedish patients, and in Vietnamese cancer tissues, the 4977 bp deletion was significantly over represented in those with localized disease compared to those with disseminated disease. Moreover, we detected nine novel mtDNA deletions and found a significantly higher rate of these in CRC tissues in Swedish in comparison to Vietnamese patients. The mtDNA 4977 bp deletion seems to have an impact on the clinical outcome of CRC in Vietnamese patients, that the Swedish patients accumulate more of the detected novel deletions in CRC tissue compared to Vietnamese patients probably indicates divergent mechanisms in colorectal carcinogenesis.

Biophysical interpretation and ex-vivo characterization of scattered light from tumor-associated breast stroma

NASA Astrophysics Data System (ADS)

Laughney, Ashley; Krishnaswamy, Venkat; Schwab, Mary; Wells, Wendy A.; Paulsen, Keith D.; Pogue, Brian W.

2009-02-01

The purpose of this study was to extract scatter parameters related to tissue ultra-structures from freshly excised breast tissue and to assess whether evident changes in scatter across diagnostic categories is primarily influenced by variation in the composition of each tissues subtypes or by physical remodeling of the extra-cellular environment. Pathologists easily distinguish between epithelium, stroma and adipose tissues, so this classification was adopted for macroscopic subtype classification. Micro-sampling reflectance spectroscopy was used to characterize single-backscattered photons from fresh, excised tumors and normal reduction specimens with sub-millimeter resolution. Phase contrast microscopy (sub-micron resolution) was used to characterize forward-scattered light through frozen tissue from the DHMC Tissue Bank, representing normal, benign and malignant breast tissue, sectioned at 10 microns. The packing density and orientation of collagen fibers in the extracellular matrix (ECM) associated with invasive, normal and benign epithelium was evaluated using transmission electron microscopy (TEM). Regions of interest (ROIs) in the H&E stained tissues were identified for analysis, as outlined by a pathologist as the gold standard. We conclude that the scatter parameters associated with tumor specimens (Npatients=6, Nspecimens=13) significantly differs from that of normal reductions (Npatients=6, Nspecimens=10). Further, tissue subtypes may be identified by their scatter spectra at sub-micron resolution. Stromal tissue scatters significantly more than the epithelial cells embedded in its ECM and adipose tissue scatters much less. However, the scatter signature of the stroma at the sub-micron level is not particularly differentiating in terms of a diagnosis.

Tumoural specimens for forensic purposes: comparison of genetic alterations in frozen and formalin-fixed paraffin-embedded tissues.

PubMed

Ananian, Viviana; Tozzo, Pamela; Ponzano, Elena; Nitti, Donato; Rodriguez, Daniele; Caenazzo, Luciana

2011-05-01

In certain circumstances, tumour tissue specimens are the only DNA resource available for forensic DNA analysis. However, cancer tissues can show microsatellite instability and loss of heterozygosity which, if concerning the short tandem repeats (STRs) used in the forensic field, can cause misinterpretation of the results. Moreover, though formalin-fixed paraffin-embedded tissues (FFPET) represent a large resource for these analyses, the quality of the DNA obtained from this kind of specimen can be an important limit. In this study, we evaluated the use of tumoural tissue as biological material for the determination of genetic profiles in the forensic field, highlighting which STR polymorphisms are more susceptible to tumour genetic alterations and which of the analysed tumours show a higher genetic variability. The analyses were conducted on samples of the same tissues conserved in different storage conditions, to compare genetic profiles obtained by frozen tissues and formalin-fixed paraffin-embedded tissues. The importance of this study is due to the large number of specimens analysed (122), the large number of polymorphisms analysed for each specimen (39), and the possibility to compare, many years after storage, the same tissue frozen and formalin-fixed paraffin-embedded. In the comparison between the genetic profiles of frozen tumour tissues and FFPET, the same genetic alterations have been reported in both kinds of specimens. However, FFPET showed new alterations. We conclude that the use of FFPET requires greater attention than frozen tissues in the results interpretation and great care in both pre-extraction and extraction processes.

Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients.

PubMed

Siravegna, Giulia; Mussolin, Benedetta; Buscarino, Michela; Corti, Giorgio; Cassingena, Andrea; Crisafulli, Giovanni; Ponzetti, Agostino; Cremolini, Chiara; Amatu, Alessio; Lauricella, Calogero; Lamba, Simona; Hobor, Sebastijan; Avallone, Antonio; Valtorta, Emanuele; Rospo, Giuseppe; Medico, Enzo; Motta, Valentina; Antoniotti, Carlotta; Tatangelo, Fabiana; Bellosillo, Beatriz; Veronese, Silvio; Budillon, Alfredo; Montagut, Clara; Racca, Patrizia; Marsoni, Silvia; Falcone, Alfredo; Corcoran, Ryan B; Di Nicolantonio, Federica; Loupakis, Fotios; Siena, Salvatore; Sartore-Bianchi, Andrea; Bardelli, Alberto

2015-07-01

Colorectal cancers (CRCs) evolve by a reiterative process of genetic diversification and clonal evolution. The molecular profile of CRC is routinely assessed in surgical or bioptic samples. Genotyping of CRC tissue has inherent limitations; a tissue sample represents a single snapshot in time, and it is subjected to spatial selection bias owing to tumor heterogeneity. Repeated tissue samples are difficult to obtain and cannot be used for dynamic monitoring of disease progression and response to therapy. We exploited circulating tumor DNA (ctDNA) to genotype colorectal tumors and track clonal evolution during treatment with the epidermal growth factor receptor (EGFR)-specific antibodies cetuximab or panitumumab. We identified alterations in ctDNA of patients with primary or acquired resistance to EGFR blockade in the following genes: KRAS, NRAS, MET, ERBB2, FLT3, EGFR and MAP2K1. Mutated KRAS clones, which emerge in blood during EGFR blockade, decline upon withdrawal of EGFR-specific antibodies, indicating that clonal evolution continues beyond clinical progression. Pharmacogenomic analysis of CRC cells that had acquired resistance to cetuximab reveals that upon antibody withdrawal KRAS clones decay, whereas the population regains drug sensitivity. ctDNA profiles of individuals who benefit from multiple challenges with anti-EGFR antibodies exhibit pulsatile levels of mutant KRAS. These results indicate that the CRC genome adapts dynamically to intermittent drug schedules and provide a molecular explanation for the efficacy of rechallenge therapies based on EGFR blockade.

Genome-wide DNA methylation sequencing reveals miR-663a is a novel epimutation candidate in CIMP-high endometrial cancer

PubMed Central

Yanokura, Megumi; Banno, Kouji; Adachi, Masataka; Aoki, Daisuke; Abe, Kuniya

2017-01-01

Aberrant DNA methylation is widely observed in many cancers. Concurrent DNA methylation of multiple genes occurs in endometrial cancer and is referred to as the CpG island methylator phenotype (CIMP). However, the features and causes of CIMP-positive endometrial cancer are not well understood. To investigate DNA methylation features characteristic to CIMP-positive endometrial cancer, we first classified samples from 25 patients with endometrial cancer based on the methylation status of three genes, i.e. MLH1, CDH1 (E-cadherin) and APC: CIMP-high (CIMP-H, 2/25, 8.0%), CIMP-low (CIMP-L, 7/25, 28.0%) and CIMP-negative (CIMP(-), 16/25, 64.0%). We then selected two samples each from CIMP-H and CIMP(-) classes, and analyzed DNA methylation status of both normal (peripheral blood cells: PBCs) and cancer tissues by genome-wide, targeted bisulfite sequencing. Genomes of the CIMP-H cancer tissues were significantly hypermethylated compared to those of the CIMP(-). Surprisingly, in normal tissues of the CIMP-H patients, promoter region of the miR-663a locus is hypermethylated relative to CIMP(-) samples. Consistent with this finding, miR-663a expression was lower in the CIMP-H PBCs than in the CIMP(-) PBCs. The same region of the miR663a locus is found to be highly methylated in cancer tissues of both CIMP-H and CIMP(-) cases. This is the first report showing that aberrant DNA methylation of the miR-663a promoter can occur in normal tissue of the cancer patients, suggesting a possible link between this epigenetic abnormality and endometrial cancer. This raises the possibility that the hypermethylation of the miR-663a promoter represents an epimutation associated with the CIMP-H endometrial cancers. Based on these findings, relationship of the aberrant DNA methylation and CIMP-H phenotype is discussed. PMID:28440489

Genome-wide DNA methylation sequencing reveals miR-663a is a novel epimutation candidate in CIMP-high endometrial cancer.

PubMed

Yanokura, Megumi; Banno, Kouji; Adachi, Masataka; Aoki, Daisuke; Abe, Kuniya

2017-06-01

Aberrant DNA methylation is widely observed in many cancers. Concurrent DNA methylation of multiple genes occurs in endometrial cancer and is referred to as the CpG island methylator phenotype (CIMP). However, the features and causes of CIMP-positive endometrial cancer are not well understood. To investigate DNA methylation features characteristic to CIMP-positive endometrial cancer, we first classified samples from 25 patients with endometrial cancer based on the methylation status of three genes, i.e. MLH1, CDH1 (E-cadherin) and APC: CIMP-high (CIMP-H, 2/25, 8.0%), CIMP-low (CIMP-L, 7/25, 28.0%) and CIMP-negative (CIMP(-), 16/25, 64.0%). We then selected two samples each from CIMP-H and CIMP(-) classes, and analyzed DNA methylation status of both normal (peripheral blood cells: PBCs) and cancer tissues by genome-wide, targeted bisulfite sequencing. Genomes of the CIMP-H cancer tissues were significantly hypermethylated compared to those of the CIMP(-). Surprisingly, in normal tissues of the CIMP-H patients, promoter region of the miR-663a locus is hypermethylated relative to CIMP(-) samples. Consistent with this finding, miR-663a expression was lower in the CIMP-H PBCs than in the CIMP(-) PBCs. The same region of the miR663a locus is found to be highly methylated in cancer tissues of both CIMP-H and CIMP(-) cases. This is the first report showing that aberrant DNA methylation of the miR-663a promoter can occur in normal tissue of the cancer patients, suggesting a possible link between this epigenetic abnormality and endometrial cancer. This raises the possibility that the hypermethylation of the miR-663a promoter represents an epimutation associated with the CIMP-H endometrial cancers. Based on these findings, relationship of the aberrant DNA methylation and CIMP-H phenotype is discussed.

Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples

PubMed Central

Zubakov, Dmitry; Hanekamp, Eline; Kokshoorn, Mieke; van IJcken, Wilfred

2007-01-01

Human body fluids such as blood and saliva represent the most common source of biological material found at a crime scene. Reliable tissue identification in forensic science can reveal significant insights into crime scene reconstruction and can thus contribute toward solving crimes. Limitations of existing presumptive tests for body fluid identification in forensics, which are usually based on chemoluminescence or protein analysis, are expected to be overcome by RNA-based methods, provided that stable RNA markers with tissue-specific expression patterns are available. To generate sets of stable RNA markers for reliable identification of blood and saliva stains we (1) performed whole-genome gene expression analyses on a series of time-wise degraded blood and saliva stain samples using the Affymetrix U133 plus2 GeneChip, (2) consulted expression databases to obtain additional information on tissue specificity, and (3) confirmed expression patterns of the most promising candidate genes by quantitative real-time polymerase chain reaction including additional forensically relevant tissues such as semen and vaginal secretion. Overall, we identified nine stable mRNA markers for blood and five stable mRNA markers for saliva detection showing tissue-specific expression signals in stains aged up to 180 days of age, expectedly older. Although, all of the markers were able to differentiate blood/saliva from semen samples, none of them could differentiate vaginal secretion because of the complex nature of vaginal secretion and the biological similarity of buccal and vaginal mucosa. We propose the use of these 14 stable mRNA markers for identification of blood and saliva stains in future forensic practice. Electronic supplementary material The online version of this article (doi:10.1007/s00414-007-0182-6) contains supplementary material, which is available to authorized users. PMID:17579879

Fungal Endophyte Diversity and Bioactivity in the Indian Medicinal Plant Ocimum sanctum Linn

PubMed Central

Chowdhary, Kanika; Kaushik, Nutan

2015-01-01

Endophytic mycopopulation isolated from India’s Queen of herbs Tulsi (Ocimum sanctum) were explored and investigated for their diversity and antiphytopathogenic activity against widespread plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani and Fusarium oxysporum. 90 fungal isolates, representing 17 genera were recovered from 313 disease-free and surface sterilised plant segments (leaf and stem tissues) from three different geographic locations (Delhi, Hyderabad and Mukteshwar) during distinct sampling times in consequent years 2010 and 2011 in India. Fungal endophytes were subjected to molecular identification based on rDNA ITS sequence analysis. Plant pathogens such as F. verticillioides, B. maydis, C. coarctatum, R. bataticola, Hypoxylon sp., Diaporthe phaseolorum, Alternaria tenuissima and A. alternata have occurred as endophyte only during second sampling (second sampling in 2011) in the present study. Bi-plot generated by principal component analysis suggested tissue specificity of certain fungal endophytes. Dendrogram revealed species abundance as a function of mean temperature of the location at the time of sampling. Shannon diversity in the first collection is highest in Hyderabad leaf tissues (H' = 1.907) whereas in second collection it was highest from leaf tissues of Delhi (H' = 1.846). Mukteshwar (altitude: 7500 feet) reported least isolation rate in second collection. Nearly 23% of the total fungal isolates were considered as potent biocontrol agent. Hexane extract of M. phaseolina recovered from Hyderabad in first collection demonstrated highest activity against S. sclerotiorum with IC50 value of 0.38 mg/ml. Additionally, its components 2H-pyran-2-one, 5,6-dihydro-6-pentyl and palmitic acid, methyl ester as reported by GC-MS Chromatogram upon evaluation for their antiphytopathogenic activity exhibited IC50 value of 1.002 and 0.662 against respectively S. sclerotiorum indicating their significant role in antiphytopathogenic activity of hexane extract. The production of 2H-pyran-2-one, 5,6-dihydro-6-pentyl from M. phaseolina, an endophytic fungus is being reported for the first time. PMID:26529087

Fast widefield techniques for fluorescence and phase endomicroscopy

NASA Astrophysics Data System (ADS)

Ford, Tim N.

Endomicroscopy is a recent development in biomedical optics which gives researchers and physicians microscope-resolution views of intact tissue to complement macroscopic visualization during endoscopy screening. This thesis presents HiLo endomicroscopy and oblique back-illumination endomicroscopy, fast wide-field imaging techniques with fluorescence and phase contrast, respectively. Fluorescence imaging in thick tissue is often hampered by strong out-of-focus background signal. Laser scanning confocal endomicroscopy has been developed for optically-sectioned imaging free from background, but reliance on mechanical scanning fundamentally limits the frame rate and represents significant complexity and expense. HiLo is a fast, simple, widefield fluorescence imaging technique which rejects out-of-focus background signal without the need for scanning. It works by acquiring two images of the sample under uniform and structured illumination and synthesizing an optically sectioned result with real-time image processing. Oblique back-illumination microscopy (OBM) is a label-free technique which allows, for the first time, phase gradient imaging of sub-surface morphology in thick scattering tissue with a reflection geometry. OBM works by back-illuminating the sample with the oblique diffuse reflectance from light delivered via off-axis optical fibers. The use of two diametrically opposed illumination fibers allows simultaneous and independent measurement of phase gradients and absorption contrast. Video-rate single-exposure operation using wavelength multiplexing is demonstrated.

Comparative Proteomics of Tumor and Paired Normal Breast Tissue Highlights Potential Biomarkers in Breast Cancer.

PubMed

Da Costa, Gustavo Góes; Gomig, Talita Helen Bombardelli; Kaviski, Rodrigo; Santos Sousa, Karla; Kukolj, Caroline; De Lima, Rubens Silveira; De Andrade Urban, Cicero; Cavalli, Iglenir J; Ribeiro, Enilze M S F

2015-01-01

Breast cancer is the most common type of cancer among women worldwide, and about 57,000 new cases are expected for the Brazilian population in 2015. Elucidation of protein expression and modification is essential for the biological understanding, early diagnosis and therapeutics of breast cancer. The main objectives of the study are comparison between the proteome of tumor and paired non-tumor breast cancer tissues, describing all identified proteins, highlighting the ones most differentially expressed and comparing the data with existing literature. The five paired samples from patients with invasive ductal carcinoma were analyzed by 2-DE and MS. We collected 161 identified spots corresponding to 110 distinct proteins. Forty-three differentially-expressed spots were common to at least two samples, and the ten proteins with the highest-fold changes were CASPE, ENOG, TPM1, CAPG, VIME, TPM3, TRFE, PDIA6, WDR61 and PDIA3. Metabolic enzymes and proteins with binding functions were the most representative functional classes of proteins with increased and decreased expression in tumor tissue respectively. Taking the fold change as a parameter, we point to future targets to be studied by functional methods in a search for biomarkers for initiation and progress of breast cancer. Copyright© 2015, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Biopsy in idiopathic pulmonary fibrosis: back to the future.

PubMed

Rossi, Giulio; Spagnolo, Paolo

2017-09-01

Idiopathic Pulmonary Fibrosis (IPF) is a relentlessly progressive, fibrosing interstitial pneumonia characterized by a radiologic and/or histologic pattern of usual interstitial pneumonia (UIP). The availability of two effective anti-fibrotic drugs in IPF has encouraged the identification and treatment of patients in early stages in order to maximize clinical benefit. The ability of high-resolution computed tomography (HRCT) to identify a 'definite' UIP pattern is suboptimal, particularly in the absence of honeycombing. Therefore, radiologic criteria for UIP are currently being redefined. Histology represents the major source of information to define a UIP pattern. Novel and less invasive approaches (particularly cryobiopsy) to sample interstitial lung diseases have demonstrated high sensitivity and specificity. In parallel, researchers are focusing on molecular mechanisms underlying IPF with the aim to identify more specific druggable targets. Lung tissue is therefore essential for diagnostic, pathogenetic and therapeutic purposes. Areas covered: We identified and critically reviewed the most relevant recent literature related to the limitations of current radiologic criteria, new lung sampling procedures, and molecular pathways in support of the need of lung tissue to better understand IPF. Expert commentary: The development of truly effective treatments for IPF requires the identification of key pathogenetic molecules and pathways. To this end, the availability of lung tissue is vital.

Upregulation of angiogenesis in oral lichen planus.

PubMed

Al-Hassiny, A; Friedlander, L T; Parachuru, V P B; Seo, B; Hussaini, H M; Rich, A M

2018-02-01

As angiogenesis is fundamental to the pathogenesis of many chronic inflammatory disorders, this study investigated the expression of various vascular markers in oral lichen planus and non-specific oral mucosal inflammatory tissues. Archival specimens of oral lichen planus (n = 15) and inflamed tissues (n = 13) were stained using immunohistochemistry with antibodies to CD34, vascular endothelial growth factor, vascular endothelial growth factor receptor and vasohibin. Nine representative sites at the epithelial-connective tissue junction and through the fibrous connective tissue were selected, and automated analysis techniques were used to determine the extent of positivity expressed as the percentage of positive cells. Significance was denoted when P < .05. The expression of pro-angiogenic factors was higher in lichen planus samples compared with inflamed controls. A higher level of CD34 was observed in the deeper parts of the connective tissue of Oral lichen planus (OLP) (P = .04), whereas VEGF and VEGFR2 expressions were higher all through the tissues (respectively, P < .02 and P < .01). The expression of the anti-angiogenic VASH1 was higher in inflamed tissue compared with lichen planus in all sites evaluated (P < .01). The findings indicate that angiogenic factors are differentially expressed in oral lichen planus compared with inflamed controls, with increased expression of pro-angiogenic factors and decreased anti-angiogenic expression. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Future role of MR elastography in tissue engineering and regenerative medicine.

PubMed

Othman, Shadi F; Xu, Huihui; Mao, Jeremy J

2015-05-01

Tissue engineering (TE) has been introduced for more than 25 years without a boom in clinical trials. More than 70 TE-related start-up companies spent more than $600 million/year, with only two FDA-approved tissue-engineered products. Given the modest performance in clinically approved organs, TE is a tenaciously promising field. The TE community is advocating the application of clinically driven methodologies in large animal models enabling clinical translation. This challenge is hindered by the scarcity of tissue biopsies and the absence of standardized evaluation tools, but can be negated through non-invasive assessment of growth and integration, with reduced sample size and low cost. Solving this issue will speed the transition to cost-efficient clinical studies. In this paper we: (a) introduce magnetic resonance elastography to the tissue-engineering and regenerative medicine (TERM) community; (b) review recent MRE applications in TERM; and (c) discuss future directions of MRE in TERM. We have used MRE to study engineered tissues both in vitro and in vivo, where the mechanical properties of mesenchymally derived constructs were progressively monitored before and after tissues were implanted in mouse models. This study represents a stepping stone toward the applications of MRE in directing clinical trials with low cost and likely expediting the translation to more relevantly large animal models and clinical trials. Copyright © 2013 John Wiley & Sons, Ltd.

Quantitative measurement of essential and not essential metals in muscular, hepatic and renal tissue of horses by means of PIXE technique

NASA Astrophysics Data System (ADS)

Balzan, S.; Buoso, M. C.; Ceccato, D.; De Poli, M.; Giaccone, V.; Moschini, G.; Novelli, E.; Olabanji, S. O.; Passi, P.; Tepedino, V.

2004-06-01

Nowadays, the food safety and the related health risks for humans are a major issue. It has become a priority to ensure the health and well-being of the population through the better scientific understanding of the food intake influence. A frequently found harmful feature of human diet is the simultaneous presence in food stuff of both essential and toxic elements. A significant example of this situation is represented by horse meat. The aim of this survey is to evaluate the elemental content of muscular, hepatic and renal tissues of horses bred in different countries of the East of Europe and slaughtered in Italy. The studied population consisted of about 100 individuals; the sampling period, coinciding with the highest market request, was extended from November to March. Freeze dried tissues were pelletized and carbon coated prior to analysis. The samples were analyzed using Particle Induced X-ray Emission (PIXE) technique at the AN2000 van de Graaff accelerator of the INFN-National Laboratories of Legnaro. The accuracy of the results was verified using certified standard reference materials prepared and measured in the same experimental conditions. Results have highlighted the noble nutritional values of horse muscular tissue in terms of iron, zinc and calcium content. Cadmium was staidly identified in horse kidneys, with an average concentration of 257 μg/g on dried matter, while in liver it was sporadically present.

Skin-infiltrating, interleukin-22-producing T cells differentiate pediatric psoriasis from adult psoriasis.

PubMed

Cordoro, Kelly M; Hitraya-Low, Maria; Taravati, Keyon; Sandoval, Priscila Munoz; Kim, Esther; Sugarman, Jeffrey; Pauli, Mariela L; Liao, Wilson; Rosenblum, Michael D

2017-09-01

Evidence from adult psoriasis studies implicates an imbalance between regulatory and effector T cells, particularly T H -17-producing T cells, in the pathogenesis of psoriasis. Little is known about the immunopathology of psoriasis in children. We sought to functionally characterize the inflammatory cell profiles of psoriatic plaques from pediatric patients and compare them with healthy, age-matched controls and adult psoriasis patients. Skin samples from pediatric psoriasis patients and healthy controls were analyzed by multiparameter flow cytometry to determine the dominant immune cell subsets present and cytokines produced. Lesional tissue from pediatric psoriasis patients had significantly increased interleukin (IL) 22 derived from CD4 + and CD8 + cells compared with the tissues from healthy pediatric controls and adult psoriasis patients. Tissue from pediatric psoriasis patients had significantly less elevation of IL-17 derived from CD4 + and CD8 + cells compared with the tissue from adult psoriasis patients. In contrast with the lesions from adult patients, lesional skin in pediatric patients with psoriasis did not have increases in regulatory T cells. This is a pilot study, thus the sample size is small. Significant differences in IL-17 and IL-22 expression were observed in the pediatric psoriasis patients compared with pediatric healthy controls and adult psoriasis patients. IL-22 might be relevant in the pathogenesis of pediatric psoriasis and represents a potential treatment target unique to pediatric psoriasis. Copyright © 2017 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

Cartilage biomarkers in the osteoarthropathy of alkaptonuria reveal low turnover and accelerated ageing.

PubMed

Taylor, Adam M; Hsueh, Ming-Feng; Ranganath, Lakshminarayan R; Gallagher, James A; Dillon, Jane P; Huebner, Janet L; Catterall, Jon B; Kraus, Virginia B

2017-01-01

Alkaptonuria (AKU) is a rare autosomal recessive disease resulting from a single enzyme deficiency in tyrosine metabolism. As a result, homogentisic acid cannot be metabolized, causing systemic increases. Over time, homogentisic acid polymerizes and deposits in collagenous tissues, leading to ochronosis. Typically, this occurs in joint cartilages, leading to an early onset, rapidly progressing osteoarthropathy. The aim of this study was to examine tissue turnover in cartilage affected by ochronosis and its role in disease initiation and progression. With informed patient consent, hip and knee cartilages were obtained at surgery for arthropathy due to AKU (n = 6; 2 knees/4 hips) and OA (n = 12; 5 knees/7 hips); healthy non-arthritic (non-OA n = 6; 1 knee/5 hips) cartilages were obtained as waste from trauma surgery. We measured cartilage concentrations (normalized to dry weight) of racemized aspartate, GAG, COMP and deamidated COMP (D-COMP). Unpaired AKU, OA and non-OA samples were compared by non-parametric Mann-Whitney U test. Despite more extractable total protein being obtained from AKU cartilage than from OA or non-OA cartilage, there was significantly less extractable GAG, COMP and D-COMP in AKU samples compared with OA and non-OA comparators. Racemized Asx (aspartate and asparagine) was significantly enriched in AKU cartilage compared with in OA cartilage. These novel data represent the first examination of cartilage matrix components in a sample of patients with AKU, representing almost 10% of the known UK alkaptonuric population. Compared with OA and non-OA, AKU cartilage demonstrates a very low turnover state and has low levels of extractable matrix proteins. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cartilage biomarkers in the osteoarthropathy of alkaptonuria reveal low turnover and accelerated ageing

PubMed Central

Hsueh, Ming-Feng; Ranganath, Lakshminarayan R.; Gallagher, James A.; Dillon, Jane P.; Huebner, Janet L.; Catterall, Jon B.; Kraus, Virginia B.

2017-01-01

Objective. Alkaptonuria (AKU) is a rare autosomal recessive disease resulting from a single enzyme deficiency in tyrosine metabolism. As a result, homogentisic acid cannot be metabolized, causing systemic increases. Over time, homogentisic acid polymerizes and deposits in collagenous tissues, leading to ochronosis. Typically, this occurs in joint cartilages, leading to an early onset, rapidly progressing osteoarthropathy. The aim of this study was to examine tissue turnover in cartilage affected by ochronosis and its role in disease initiation and progression. Methods. With informed patient consent, hip and knee cartilages were obtained at surgery for arthropathy due to AKU (n = 6; 2 knees/4 hips) and OA (n = 12; 5 knees/7 hips); healthy non-arthritic (non-OA n = 6; 1 knee/5 hips) cartilages were obtained as waste from trauma surgery. We measured cartilage concentrations (normalized to dry weight) of racemized aspartate, GAG, COMP and deamidated COMP (D-COMP). Unpaired AKU, OA and non-OA samples were compared by non-parametric Mann–Whitney U test. Results. Despite more extractable total protein being obtained from AKU cartilage than from OA or non-OA cartilage, there was significantly less extractable GAG, COMP and D-COMP in AKU samples compared with OA and non-OA comparators. Racemized Asx (aspartate and asparagine) was significantly enriched in AKU cartilage compared with in OA cartilage. Conclusions. These novel data represent the first examination of cartilage matrix components in a sample of patients with AKU, representing almost 10% of the known UK alkaptonuric population. Compared with OA and non-OA, AKU cartilage demonstrates a very low turnover state and has low levels of extractable matrix proteins. PMID:28028161

DNA Methylation as a Biomarker for Preeclampsia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anderson, Cindy M.; Ralph, Jody L.; Wright, Michelle L.

Background: Preeclampsia contributes significantly to pregnancy-associated morbidity and mortality as well as future risk of cardiovascular disease in mother and offspring, and preeclampsia in offspring. The lack of reliable methods for early detection limits the opportunities for prevention, diagnosis, and timely treatment. Purpose: The purpose of this study was to explore distinct DNA methylation patterns associated with preeclampsia in both maternal cells and fetal-derived tissue that represent potential biomarkers to predict future preeclampsia and inheritance in children. Method: A convenience sample of nulliparous women (N = 55) in the first trimester of pregnancy was recruited for this prospective study. Genome-widemore » DNA methylation was quantified in first-trimester maternal peripheral white blood cells and placental chorionic tissue from normotensive women and those with preeclampsia (n = 6/group). Results: Late-onset preeclampsia developed in 12.7% of women. Significant differences in DNA methylation were identified in 207 individual linked cytosine and guanine (CpG) sites in maternal white blood cells collected in the first trimester (132 sites with gain and 75 sites with loss of methylation), which were common to approximately 75% of the differentially methylated CpG sites identified in chorionic tissue of fetal origin. Conclusion: This study is the first to identify maternal epigenetic targets and common targets in fetal-derived tissue that represent putative biomarkers for early detection and heritable risk of preeclampsia. Findings may pave the way for diagnosis of preeclampsia prior to its clinical presentation and acute damaging effects, and the potential for prevention of the detrimental long-term sequelae.« less

Sequencing the transcriptome of milk production: milk trumps mammary tissue

PubMed Central

2013-01-01

Background Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells. Results We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat, whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from milk-producing mammary epithelial cells. Conclusions RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation. PMID:24330573

Learning semantic histopathological representation for basal cell carcinoma classification

NASA Astrophysics Data System (ADS)

Gutiérrez, Ricardo; Rueda, Andrea; Romero, Eduardo

2013-03-01

Diagnosis of a histopathology glass slide is a complex process that involves accurate recognition of several structures, their function in the tissue and their relation with other structures. The way in which the pathologist represents the image content and the relations between those objects yields a better and accurate diagnoses. Therefore, an appropriate semantic representation of the image content will be useful in several analysis tasks such as cancer classification, tissue retrieval and histopahological image analysis, among others. Nevertheless, to automatically recognize those structures and extract their inner semantic meaning are still very challenging tasks. In this paper we introduce a new semantic representation that allows to describe histopathological concepts suitable for classification. The approach herein identify local concepts using a dictionary learning approach, i.e., the algorithm learns the most representative atoms from a set of random sampled patches, and then models the spatial relations among them by counting the co-occurrence between atoms, while penalizing the spatial distance. The proposed approach was compared with a bag-of-features representation in a tissue classification task. For this purpose, 240 histological microscopical fields of view, 24 per tissue class, were collected. Those images fed a Support Vector Machine classifier per class, using 120 images as train set and the remaining ones for testing, maintaining the same proportion of each concept in the train and test sets. The obtained classification results, averaged from 100 random partitions of training and test sets, shows that our approach is more sensitive in average than the bag-of-features representation in almost 6%.

Trace elements distribution and post-mortem intake in human bones from Middle Age by total reflection X-ray fluorescence*1

NASA Astrophysics Data System (ADS)

Carvalho, M. L.; Marques, A. F.; Lima, M. T.; Reus, U.

2004-08-01

The purpose of the present work is to investigate the suitability of TXRF technique to study the distribution of trace elements along human bones of the 13th century, to conclude about environmental conditions and dietary habits of old populations and to study the uptake of some elements from the surrounding soil. In this work, we used TXRF to quantify and to make profiles of the elements through long bones. Two femur bones, one from a man and another from a woman, buried in the same grave were cross-sectioned in four different points at a distance of 1 cm. Microsamples of each section were taken at a distance of 1 mm from each other. Quantitative analysis was performed for Ca, Mn, Fe, Cu, Zn, Sr, Ba and Pb. Very high concentrations of Mn and Fe were obtained in the whole analysed samples, reaching values higher than 2% in some samples of trabecular tissue, very much alike to the concentrations in the burial soil. A sharp decrease for both elements was observed in cortical tissue. Zn and Sr present steady concentration levels in both kinds of bone tissues. Pb and Cu show very low concentrations in the inner tissue of cortical bone. However, these concentrations increase in the regions in contact to trabecular tissue and external surface in contact with the soil, where high levels of both elements were found. We suggest that contamination from the surrounding soil exists for Mn and Fe in the whole bone tissue. Pb can be both from post-mortem and ante-mortem origin. Inner compact tissue might represent in vivo accumulation and trabecular one corresponds to uptake during burial. The steady levels of Sr and Zn together with soil concentration lower levels for these elements may allow us to conclude that they are originated from in vivo incorporation in the hydroxyapatite bone matrix.

Seabird tissue archival and monitoring project: Protocol for collecting and banking seabird eggs

USGS Publications Warehouse

Weston-York, Geoff; Porter, Barbara J.; Pugh, Rebecca S.; Roseneau, David G.; Simac, Kristin S.; Becker, Paul R.; Thorsteinson, Lyman K.; Wise, Stephen A.

2001-01-01

Archiving biological and environmental samples for retrospective analysis is a major component of systematic environmental monitoring. The long-term storage of carefully selected, representative samples in an environmental specimen bank is an important complement to the real-time monitoring of the environment. These archived samples permit:The use of subsequently developed innovative analytical technology that was not available at the time the samples were archived, for clear state-of-art identification an~ quantification of analytes of interest,The identification and quantification of analytes that are of subsequent interest but that were not of interest at the time the samples were archived, andThe comparison of present and past analytical techniques and values, providing continued credibility of past analytical values, and allowing flexibility in environmental monitoring programs.Seabirds, including albatrosses, pelicans, cormorants, terns, kittiwakes, murres, guillemots, and puffins spend most of their lives at sea and have special adaptations for feeding in the marine environment, including the ability to excrete the excess salt obtained from ingesting seawater. Many species nest in dense groups (colonies) on steep, precipitous sea-cliffs and headlands.Seabirds are long-lived and slow to mature. They occupy high positions in the marine food web and are considered sensitive indicators for the marine environment (prey includes krill, small fish, and squid). Breeding success, timing of nesting, diets, and survival rates may provide early indications of changing environmental conditions (e.g., see Hatch et aI., 1993). Chemical analysis of seabird tissues, including egg contents, can be particularly useful in determining whether contaminants (and potential biological effects) associated with human industrial activities, such as offshore petroleum and mineral exploration and development, are accumulating in marine environments. The collection and archival of seabird tissues over a period of several years will be a resource for future analyses, providing samples that can be used to determine historical baseline contaminant levels.

Automated quantification of renal interstitial fibrosis for computer-aided diagnosis: A comprehensive tissue structure segmentation method.

PubMed

Tey, Wei Keat; Kuang, Ye Chow; Ooi, Melanie Po-Leen; Khoo, Joon Joon

2018-03-01

Interstitial fibrosis in renal biopsy samples is a scarring tissue structure that may be visually quantified by pathologists as an indicator to the presence and extent of chronic kidney disease. The standard method of quantification by visual evaluation presents reproducibility issues in the diagnoses. This study proposes an automated quantification system for measuring the amount of interstitial fibrosis in renal biopsy images as a consistent basis of comparison among pathologists. The system extracts and segments the renal tissue structures based on colour information and structural assumptions of the tissue structures. The regions in the biopsy representing the interstitial fibrosis are deduced through the elimination of non-interstitial fibrosis structures from the biopsy area and quantified as a percentage of the total area of the biopsy sample. A ground truth image dataset has been manually prepared by consulting an experienced pathologist for the validation of the segmentation algorithms. The results from experiments involving experienced pathologists have demonstrated a good correlation in quantification result between the automated system and the pathologists' visual evaluation. Experiments investigating the variability in pathologists also proved the automated quantification error rate to be on par with the average intra-observer variability in pathologists' quantification. Interstitial fibrosis in renal biopsy samples is a scarring tissue structure that may be visually quantified by pathologists as an indicator to the presence and extent of chronic kidney disease. The standard method of quantification by visual evaluation presents reproducibility issues in the diagnoses due to the uncertainties in human judgement. An automated quantification system for accurately measuring the amount of interstitial fibrosis in renal biopsy images is presented as a consistent basis of comparison among pathologists. The system identifies the renal tissue structures through knowledge-based rules employing colour space transformations and structural features extraction from the images. In particular, the renal glomerulus identification is based on a multiscale textural feature analysis and a support vector machine. The regions in the biopsy representing interstitial fibrosis are deduced through the elimination of non-interstitial fibrosis structures from the biopsy area. The experiments conducted evaluate the system in terms of quantification accuracy, intra- and inter-observer variability in visual quantification by pathologists, and the effect introduced by the automated quantification system on the pathologists' diagnosis. A 40-image ground truth dataset has been manually prepared by consulting an experienced pathologist for the validation of the segmentation algorithms. The results from experiments involving experienced pathologists have demonstrated an average error of 9 percentage points in quantification result between the automated system and the pathologists' visual evaluation. Experiments investigating the variability in pathologists involving samples from 70 kidney patients also proved the automated quantification error rate to be on par with the average intra-observer variability in pathologists' quantification. The accuracy of the proposed quantification system has been validated with the ground truth dataset and compared against the pathologists' quantification results. It has been shown that the correlation between different pathologists' estimation of interstitial fibrosis area has significantly improved, demonstrating the effectiveness of the quantification system as a diagnostic aide. Copyright © 2017 Elsevier B.V. All rights reserved.

Study of Biological Pigments by Single Specimen Derivative Spectrophotometry

PubMed Central

Goldstein, Jack M.

1970-01-01

The single specimen derivative (SSD) method provides an absolute absorption spectrum of a substance in the absence of a suitable reference. Both a reference and a measuring monochromatic beam pass through a single sample, and the specimen itself acts as its own reference. The two monochromatic beams maintain a fixed wavelength difference upon scanning, and the difference in absorbance of the two beams is determined. Thus, the resulting spectrum represents the first derivative of the conventional type absorption spectrum. Tissues and cell fractions have been examined at room and liquid N2 temperature and chromophoric molecules such as the mitochondrial cytochromes and blood pigments have been detectable in low concentrations. In the case of isolated cellular components, the observed effects of substrates and inhibitors confirm similar studies by conventional spectrophotometry. The extension of the SSD concept to the microscopic level has permitted the study of the tissue compartmentalization and function of cytochromes and other pigments within layered tissue. PMID:4392452

Poster — Thur Eve — 03: Application of the non-negative matrix factorization technique to [{sup 11}C]-DTBZ dynamic PET data for the early detection of Parkinson's disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Dong-Chang; Jans, Hans; McEwan, Sandy

2014-08-15

In this work, a class of non-negative matrix factorization (NMF) technique known as alternating non-negative least squares, combined with the projected gradient method, is used to analyze twenty-five [{sup 11}C]-DTBZ dynamic PET/CT brain data. For each subject, a two-factor model is assumed and two factors representing the striatum (factor 1) and the non-striatum (factor 2) tissues are extracted using the proposed NMF technique and commercially available factor analysis software “Pixies”. The extracted factor 1 and 2 curves represent the binding site of the radiotracer and describe the uptake and clearance of the radiotracer by soft tissues in the brain, respectively.more » The proposed NMF technique uses prior information about the dynamic data to obtain sample time-activity curves representing the striatum and the non-striatum tissues. These curves are then used for “warm” starting the optimization. Factor solutions from the two methods are compared graphically and quantitatively. In healthy subjects, radiotracer uptake by factors 1 and 2 are approximately 35–40% and 60–65%, respectively. The solutions are also used to develop a factor-based metric for the detection of early, untreated Parkinson's disease. The metric stratifies healthy subjects from suspected Parkinson's patients (based on the graphical method). The analysis shows that both techniques produce comparable results with similar computational time. The “semi-automatic” approach used by the NMF technique allows clinicians to manually set a starting condition for “warm” starting the optimization in order to facilitate control and efficient interaction with the data.« less

Novel biomarker identification using metabolomic profiling to differentiate radiation necrosis and recurrent tumor following Gamma Knife radiosurgery.

PubMed

Lu, Alex Y; Turban, Jack L; Damisah, Eyiyemisi C; Li, Jie; Alomari, Ahmed K; Eid, Tore; Vortmeyer, Alexander O; Chiang, Veronica L

2017-08-01

OBJECTIVE Following an initial response of brain metastases to Gamma Knife radiosurgery, regrowth of the enhancing lesion as detected on MRI may represent either radiation necrosis (a treatment-related inflammatory change) or recurrent tumor. Differentiation of radiation necrosis from tumor is vital for management decision making but remains difficult by imaging alone. In this study, gas chromatography with time-of-flight mass spectrometry (GC-TOF) was used to identify differential metabolite profiles of the 2 tissue types obtained by surgical biopsy to find potential targets for noninvasive imaging. METHODS Specimens of pure radiation necrosis and pure tumor obtained from patient brain biopsies were flash-frozen and validated histologically. These formalin-free tissue samples were then analyzed using GC-TOF. The metabolite profiles of radiation necrosis and tumor samples were compared using multivariate and univariate statistical analysis. Statistical significance was defined as p ≤ 0.05. RESULTS For the metabolic profiling, GC-TOF was performed on 7 samples of radiation necrosis and 7 samples of tumor. Of the 141 metabolites identified, 17 (12.1%) were found to be statistically significantly different between comparison groups. Of these metabolites, 6 were increased in tumor, and 11 were increased in radiation necrosis. An unsupervised hierarchical clustering analysis found that tumor had elevated levels of metabolites associated with energy metabolism, whereas radiation necrosis had elevated levels of metabolites that were fatty acids and antioxidants/cofactors. CONCLUSIONS To the authors' knowledge, this is the first tissue-based metabolomics study of radiation necrosis and tumor. Radiation necrosis and recurrent tumor following Gamma Knife radiosurgery for brain metastases have unique metabolite profiles that may be targeted in the future to develop noninvasive metabolic imaging techniques.

The presence of enterovirus, adenovirus, and parvovirus B19 in myocardial tissue samples from autopsies: an evaluation of their frequencies in deceased individuals with myocarditis and in non-inflamed control hearts.

PubMed

Nielsen, Trine Skov; Hansen, Jakob; Nielsen, Lars Peter; Baandrup, Ulrik Thorngren; Banner, Jytte

2014-09-01

Multiple viruses have been detected in cardiac tissue, but their role in causing myocarditis remains controversial. Viral diagnostics are increasingly used in forensic medicine, but the interpretation of the results can sometimes be challenging. In this study, we examined the prevalence of adenovirus, enterovirus, and parvovirus B19 (PVB) in myocardial autopsy samples from myocarditis related deaths and in non-inflamed control hearts in an effort to clarify their significance as the causes of myocarditis in a forensic material. We collected all autopsy cases diagnosed with myocarditis from 1992 to 2010. Eighty-four suicidal deaths with morphologically normal hearts served as controls. Polymerase chain reaction was used for the detection of the viral genomes (adenovirus, enterovirus, and PVB) in myocardial tissue specimens. The distinction between acute and persistent PVB infection was made by the serological determination of PVB-specific immunoglobulins M and G. PVB was detected in 33 of 112 (29 %) myocarditis cases and 37 of 84 (44 %) control cases. All of the samples were negative for the presence of adenovirus and enterovirus. Serological evidence of an acute PVB infection, determined by the presence of immunoglobulin M, was only present in one case. In the remaining cases, PVB was considered to be a bystander with no or limited association to myocardial inflammation. In this study, adenovirus, enterovirus, and PVB were found to be rare causes of myocarditis. The detection of PVB in myocardial autopsy samples most likely represents a persistent infection with no or limited association with myocardial inflammation. The forensic investigation of myocardial inflammation demands a thorough examination, including special attention to non-viral causes and requires a multidisciplinary approach.

Testing an aflatoxin B1 gene signature in rat archival tissues.

PubMed

Merrick, B Alex; Auerbach, Scott S; Stockton, Patricia S; Foley, Julie F; Malarkey, David E; Sills, Robert C; Irwin, Richard D; Tice, Raymond R

2012-05-21

Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c, and the C8orf46 homologue. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 μg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p ≤ 0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and β-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25-500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in the liver, the primary target for AFB1-induced tumors. We conclude that an evaluation of gene signatures in archival tissues can be an important toxicological tool for evaluating critical molecular events associated with chemical exposures.

Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue

PubMed Central

2010-01-01

Background The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months. Methods RNA from fresh frozen (FF) and FFPE tumor samples of 82 patients were used for quality control, and independent FFPE tissues of 342 postmenopausal participants of BIG 1-98 with ER-positive cancer were analyzed by measuring prospectively selected genes and computing scores representing the functions of the estrogen receptor (eight genes, ER_8), the progesterone receptor (five genes, PGR_5), Her2 (two genes, HER2_2), and proliferation (ten genes, PRO_10) by quantitative reverse transcription PCR (qRT-PCR) on TaqMan Low Density Arrays. Molecular scores were computed for each category and ER_8, PGR_5, HER2_2, and PRO_10 scores were combined into a RISK_25 score. Results Pearson correlation coefficients between FF- and FFPE-derived scores were at least 0.94 and high concordance was observed between molecular scores and immunohistochemical data. The HER2_2, PGR_5, PRO_10 and RISK_25 scores were significant predictors of disease free-survival (DFS) in univariate Cox proportional hazard regression. PRO_10 and RISK_25 scores predicted DFS in patients with histological grade II breast cancer and in lymph node positive disease. The PRO_10 and PGR_5 scores were independent predictors of DFS in multivariate Cox regression models incorporating clinical risk indicators; PRO_10 outperformed Ki-67 labeling index in multivariate Cox proportional hazard analyses. Conclusions Scores representing the endocrine responsiveness and proliferation status of breast cancers were developed from gene expression analyses based on RNA derived from FFPE tissues. The validation of the molecular scores with tumor samples of participants of the BIG 1-98 trial demonstrates that such scores can serve as independent prognostic factors to estimate disease free survival (DFS) in postmenopausal patients with estrogen receptor positive breast cancer. Trial Registration Current Controlled Trials: NCT00004205 PMID:20144231

A systematic surveillance programme for infectious salmon anaemia virus supports its absence in the Pacific Northwest of the United States

USGS Publications Warehouse

Gustafson, Lori L.; Creekmore, Lynn H.; Snekvik, Kevin R.; Ferguson, Jayde A.; Warg, Janet V.; Blair, Marilyn; Meyers, Theodore R.; Stewart, Bruce; Warheit, Kenneth I.; Kerwin, John; Goodwin, Andrew E.; Rhodes, Linda D.; Whaley, Janet E.; Purcell, Maureen K.; Bentz, Collette; Shasa, Desiree; Bader, Joel; Winton, James R.

2018-01-01

In response to reported findings of infectious salmon anaemia virus (ISAV) in British Columbia (BC), Canada, in 2011, U.S. national, state and tribal fisheries managers and fish health specialists developed and implemented a collaborative ISAV surveillance plan for the Pacific Northwest region of the United States. Accordingly, over a 3-1/2-year period, 4,962 salmonids were sampled and successfully tested by real-time reverse-transcription PCR. The sample set included multiple tissues from free-ranging Pacific salmonids from coastal regions of Alaska and Washington and farmed Atlantic salmon (Salmo salar L.) from Washington, all representing fish exposed to marine environments. The survey design targeted physiologically compromised or moribund animals more vulnerable to infection as well as species considered susceptible to ISAV. Samples were handled with a documented chain of custody and testing protocols, and criteria for interpretation of test results were defined in advance. All 4,962 completed tests were negative for ISAV RNA. Results of this surveillance effort provide sound evidence to support the absence of ISAV in represented populations of free-ranging and marine-farmed salmonids on the northwest coast of the United States.

A systematic surveillance programme for infectious salmon anaemia virus supports its absence in the Pacific Northwest of the United States.

PubMed

Gustafson, L L; Creekmore, L H; Snekvik, K R; Ferguson, J A; Warg, J V; Blair, M; Meyers, T R; Stewart, B; Warheit, K I; Kerwin, J; Goodwin, A E; Rhodes, L D; Whaley, J E; Purcell, M K; Bentz, C; Shasa, D; Bader, J; Winton, J R

2018-02-01

In response to reported findings of infectious salmon anaemia virus (ISAV) in British Columbia (BC), Canada, in 2011, U.S. national, state and tribal fisheries managers and fish health specialists developed and implemented a collaborative ISAV surveillance plan for the Pacific Northwest region of the United States. Accordingly, over a 3-1/2-year period, 4,962 salmonids were sampled and successfully tested by real-time reverse-transcription PCR. The sample set included multiple tissues from free-ranging Pacific salmonids from coastal regions of Alaska and Washington and farmed Atlantic salmon (Salmo salar L.) from Washington, all representing fish exposed to marine environments. The survey design targeted physiologically compromised or moribund animals more vulnerable to infection as well as species considered susceptible to ISAV. Samples were handled with a documented chain of custody and testing protocols, and criteria for interpretation of test results were defined in advance. All 4,962 completed tests were negative for ISAV RNA. Results of this surveillance effort provide sound evidence to support the absence of ISAV in represented populations of free-ranging and marine-farmed salmonids on the northwest coast of the United States. © 2017 John Wiley & Sons Ltd.

[Cowpox virus infection in an alpaca (Vicugna pacos) - clinical symptoms, laboratory diagnostic findings and pathological changes].

PubMed

Goerigk, D; Theuß, T; Pfeffer, M; Konrath, A; Kalthoff, D; Woll, D; Vahlenkamp, T W; Beer, M; Starke, A

2014-01-01

Orthopoxvirus infections appear to be rare in South American Camelids, because only a few cases have been reported in the literature. Based on a generalized infection with cowpox virus in an alpaca, the clinical symptoms, laboratory diagnostic findings and the pathological changes are described. The case history showed a long treatment because of chronic skin lesions. The main clinical symptom was miliary papules over the entire skin. Furthermore, a bilateral mucopurulent conjunctivitis occurred as well as excessive salivation due to a severe erosive-ulcerative stomatitis. Although the animal received intensive treatment, it died 8 days after admission to the clinic. During necropsy, an erosive-ulcerative laryngitis as well as a necrotising pneumonia and lymphadenitis were observed. Histopathological examination of representative organ samples led to the diagnosis of a suspected orthopoxvirus infection. Electron microscopy and quantitative polymerase chain reaction (qPCR) of tissue samples confirmed this diagnosis. The virus could be isolated in tissue culture and a PCR with subsequent nucleotide sequencing identified cowpox virus as the causative agent for this generalised infection.

Evaluation of Two Enzyme-Linked Immunosorbent Assay Kits for Chikungunya Virus IgM Using Samples from Deceased Organ and Tissue Donors

PubMed Central

Altrich, Michelle L.; Nowicki, Marek J.

2016-01-01

The identification of nearly 3,500 cases of chikungunya virus (CHIKV) infection in U.S. residents returning in 2014 and 2015 from areas in which it is endemic has raised concerns within the transplant community that, should recently infected individuals become organ and/or tissue donors, CHIKV would be transmitted to transplant recipients. Thus, tests designed to detect recent CHIKV infection among U.S. organ and tissue donors may become necessary in the future. Accordingly, we evaluated 2 enzyme-linked immunosorbent assays (ELISAs) for CHIKV IgM readily available in the United States using 1,000 deidentified serum or plasma specimens collected from donors between November 2014 and March 2015. The Euroimmun indirect ELISA identified 38 reactive specimens; however, all 38 were negative for CHIKV IgG and IgM in immunofluorescence assays (IFAs) conducted at a reference laboratory and, thus, were falsely reactive in the Euroimmun CHIKV IgM assay. The InBios IgM-capture ELISA identified 26 reactive samples, and one was still reactive (index ≥ 1.00) when retested using the InBios kit with a background subtraction modification to identify false reactivity. This reactive specimen was CHIKV IgM negative but IgG positive by IFAs at two reference laboratories; plaque reduction neutralization testing (PRNT) demonstrated CHIKV-specific reactivity. The IgG and PRNT findings strongly suggest that the InBios CHIKV IgM-reactive result represents true reactivity, even though the IgM IFA result was negative. If testing organ/tissue donors for CHIKV IgM becomes necessary, the limitations of the currently available CHIKV IgM ELISAs and options for their optimization must be understood to avoid organ/tissue wastage due to falsely reactive results. PMID:27535838

Microgravity

NASA Image and Video Library

1998-01-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens of cartilage tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. Constructs grown on Mir (A) tended to become more spherical, whereas those grown on Earth (B) maintained their initial disc shape. These findings might be related to differences in cultivation conditions, i.e., videotapes showed that constructs floated freely in microgravity but settled and collided with the rotating vessel wall at 1g (Earth's gravity). In particular, on Mir the constructs were exposed to uniform shear and mass transfer at all surfaces such that the tissue grew equally in all directions, whereas on Earth the settling of discoid constructs tended to align their flat circular areas perpendicular to the direction of motion, increasing shear and mass transfer circumferentially such that the tissue grew preferentially in the radial direction. A and B are full cross sections of constructs from Mir and Earth groups shown at 10-power. C and D are representative areas at the construct surfaces enlarged to 200-power. They are stained red with safranin-O. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). Photo credit: Proceedings of the National Academy of Sciences.

Dynamic landscape and regulation of RNA editing in mammals

PubMed Central

Tan, Meng How; Li, Qin; Shanmugam, Raghuvaran; Piskol, Robert; Kohler, Jennefer; Young, Amy N.; Liu, Kaiwen Ivy; Zhang, Rui; Ramaswami, Gokul; Ariyoshi, Kentaro; Gupte, Ankita; Keegan, Liam P.; George, Cyril X.; Ramu, Avinash; Huang, Ni; Pollina, Elizabeth A.; Leeman, Dena S.; Rustighi, Alessandra; Sharon Goh, Y. P.; Chawla, Ajay; Del Sal, Giannino; Peltz, Gary; Brunet, Anne; Conrad, Donald F.; Samuel, Charles E.; O’Connell, Mary A.; Walkley, Carl R.; Nishikura, Kazuko; Li, Jin Billy

2017-01-01

Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules1. Although many editing sites have recently been discovered2–7, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood8–10. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing. PMID:29022589

Identification of viral infections in the prostate and evaluation of their association with cancer

PubMed Central

2010-01-01

Background Several viruses with known oncogenic potential infect prostate tissue, among these are the polyomaviruses BKV, JCV, and SV40; human papillomaviruses (HPVs), and human cytomegalovirus (HCMV) infections. Recently, the Xenotropic Murine Leukemia Virus-related gammaretrovirus (XMRV) was identified in prostate tissue with a high prevalence observed in prostate cancer (PC) patients homozygous for the glutamine variant of the RNASEL protein (462Q/Q). Association studies with the R462Q allele and non-XMRV viruses have not been reported. We assessed associations between prostate cancer, prostate viral infections, and the RNASEL 462Q allele in Mexican cancer patients and controls. Methods 130 subjects (55 prostate cancer cases and 75 controls) were enrolled in the study. DNA and RNA isolated from prostate tissues were screened for the presence of viral genomes. Genotyping of the RNASEL R462Q variant was performed by Taqman method. Results R/R, R/Q, and Q/Q frequencies for R462Q were 0.62, 0.38, and 0.0 for PC cases and 0.69, 0.24, and 0.07 for controls, respectively. HPV sequences were detected in 11 (20.0%) cases and 4 (5.3%) controls. XMRV and HCMV infections were detected in one and six control samples, respectively. The risk of PC was significantly increased (Odds Ratio = 3.98; 95% CI: 1.17-13.56, p = 0.027) by infection of the prostatic tissue with HPV. BKV, JCV, and SV40 sequences were not detected in any of the tissue samples examined. Conclusions We report a positive association between PC and HPV infection. The 462Q/Q RNASEL genotype was not represented in our PC cases; thus, its interaction with prostate viral infections and cancer could not be evaluated. PMID:20576103

Dynamic landscape and regulation of RNA editing in mammals.

PubMed

Tan, Meng How; Li, Qin; Shanmugam, Raghuvaran; Piskol, Robert; Kohler, Jennefer; Young, Amy N; Liu, Kaiwen Ivy; Zhang, Rui; Ramaswami, Gokul; Ariyoshi, Kentaro; Gupte, Ankita; Keegan, Liam P; George, Cyril X; Ramu, Avinash; Huang, Ni; Pollina, Elizabeth A; Leeman, Dena S; Rustighi, Alessandra; Goh, Y P Sharon; Chawla, Ajay; Del Sal, Giannino; Peltz, Gary; Brunet, Anne; Conrad, Donald F; Samuel, Charles E; O'Connell, Mary A; Walkley, Carl R; Nishikura, Kazuko; Li, Jin Billy

2017-10-11

Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.

Determination of diagnostic standards on saturated soil extracts for cut roses grown in greenhouses.

PubMed

Franco-Hermida, John Jairo; Quintero, María Fernanda; Cabrera, Raúl Iskander; Guzman, José Miguel

2017-01-01

This work comprises the theoretical determination and validation of diagnostic standards for the analysis of saturated soil extracts for cut rose flower crops (Rosa spp.) growing in the Bogota Plateau, Colombia. The data included 684 plant tissue analyses and 684 corresponding analyses of saturated soil extracts, all collected between January 2009 and June 2013. The tissue and soil samples were selected from 13 rose farms, and from cultivars grafted on the 'Natal Briar' rootstock. These concurrent samples of soil and plant tissues represented 251 production units (locations) of approximately 10,000 m2 distributed across the study area. The standards were conceived as a tool to improve the nutritional balance in the leaf tissue of rose plants and thereby define the norms for expressing optimum productive potential relative to nutritional conditions in the soil. To this end, previously determined diagnostic standard for rose leaf tissues were employed to obtain rates of foliar nutritional balance at each analyzed location and as criteria for determining the diagnostic norms for saturated soil extracts. Implementing this methodology to foliar analysis, showed a higher significant correlation for diagnostic indices. A similar behavior was observed in saturated soil extracts analysis, becoming a powerful tool for integrated nutritional diagnosis. Leaf analyses determine the most limiting nutrients for high yield and analyses of saturated soil extracts facilitate the possibility of correcting the fertigation formulations applied to soils or substrates. Recommendations are proposed to improve the balance in soil-plant system with which the possibility of yield increase becomes more probable. The main recommendations to increase and improve rose crop flower yields would be: continuously check pH values of SSE, reduce the amounts of P, Fe, Zn and Cu in fertigation solutions and carefully analyze the situation of Mn in the soil-plant system.

An expanded maize gene expression atlas based on RNA sequencing and its use to explore root development

DOE PAGES

Stelpflug, Scott C.; Sekhon, Rajandeep S.; Vaillancourt, Brieanne; ...

2015-12-30

Comprehensive and systematic transcriptome profiling provides valuable insight into biological and developmental processes that occur throughout the life cycle of a plant. We have enhanced our previously published microarray-based gene atlas of maize ( Zea mays L.) inbred B73 to now include 79 distinct replicated samples that have been interrogated using RNA sequencing (RNA-seq). The current version of the atlas includes 50 original array-based gene atlas samples, a time-course of 12 stalk and leaf samples postflowering, and an additional set of 17 samples from the maize seedling and adult root system. The entire dataset contains 4.6 billion mapped reads, withmore » an average of 20.5 million mapped reads per biological replicate, allowing for detection of genes with lower transcript abundance. As the new root samples represent key additions to the previously examined tissues, we highlight insights into the root transcriptome, which is represented by 28,894 (73.2%) annotated genes in maize. Additionally, we observed remarkable expression differences across both the longitudinal (four zones) and radial gradients (cortical parenchyma and stele) of the primary root supported by fourfold differential expression of 9353 and 4728 genes, respectively. Among the latter were 1110 genes that encode transcription factors, some of which are orthologs of previously characterized transcription factors known to regulate root development in Arabidopsis thaliana (L.) Heynh., while most are novel, and represent attractive targets for reverse genetics approaches to determine their roles in this important organ. As a result, this comprehensive transcriptome dataset is a powerful tool toward understanding maize development, physiology, and phenotypic diversity.« less

An expanded maize gene expression atlas based on RNA sequencing and its use to explore root development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stelpflug, Scott C.; Sekhon, Rajandeep S.; Vaillancourt, Brieanne

Comprehensive and systematic transcriptome profiling provides valuable insight into biological and developmental processes that occur throughout the life cycle of a plant. We have enhanced our previously published microarray-based gene atlas of maize ( Zea mays L.) inbred B73 to now include 79 distinct replicated samples that have been interrogated using RNA sequencing (RNA-seq). The current version of the atlas includes 50 original array-based gene atlas samples, a time-course of 12 stalk and leaf samples postflowering, and an additional set of 17 samples from the maize seedling and adult root system. The entire dataset contains 4.6 billion mapped reads, withmore » an average of 20.5 million mapped reads per biological replicate, allowing for detection of genes with lower transcript abundance. As the new root samples represent key additions to the previously examined tissues, we highlight insights into the root transcriptome, which is represented by 28,894 (73.2%) annotated genes in maize. Additionally, we observed remarkable expression differences across both the longitudinal (four zones) and radial gradients (cortical parenchyma and stele) of the primary root supported by fourfold differential expression of 9353 and 4728 genes, respectively. Among the latter were 1110 genes that encode transcription factors, some of which are orthologs of previously characterized transcription factors known to regulate root development in Arabidopsis thaliana (L.) Heynh., while most are novel, and represent attractive targets for reverse genetics approaches to determine their roles in this important organ. As a result, this comprehensive transcriptome dataset is a powerful tool toward understanding maize development, physiology, and phenotypic diversity.« less

Evaluating hepatocellular carcinoma cell lines for tumour samples using within-sample relative expression orderings of genes.

PubMed

Ao, Lu; Guo, You; Song, Xuekun; Guan, Qingzhou; Zheng, Weicheng; Zhang, Jiahui; Huang, Haiyan; Zou, Yi; Guo, Zheng; Wang, Xianlong

2017-11-01

Concerns are raised about the representativeness of cell lines for tumours due to the culture environment and misidentification. Liver is a major metastatic destination of many cancers, which might further confuse the origin of hepatocellular carcinoma cell lines. Therefore, it is of crucial importance to understand how well they can represent hepatocellular carcinoma. The HCC-specific gene pairs with highly stable relative expression orderings in more than 99% of hepatocellular carcinoma but with reversed relative expression orderings in at least 99% of one of the six types of cancer, colorectal carcinoma, breast carcinoma, non-small-cell lung cancer, gastric carcinoma, pancreatic carcinoma and ovarian carcinoma, were identified. With the simple majority rule, the HCC-specific relative expression orderings from comparisons with colorectal carcinoma and breast carcinoma could exactly discriminate primary hepatocellular carcinoma samples from both primary colorectal carcinoma and breast carcinoma samples. Especially, they correctly classified more than 90% of liver metastatic samples from colorectal carcinoma and breast carcinoma to their original tumours. Finally, using these HCC-specific relative expression orderings from comparisons with six cancer types, we identified eight of 24 hepatocellular carcinoma cell lines in the Cancer Cell Line Encyclopedia (Huh-7, Huh-1, HepG2, Hep3B, JHH-5, JHH-7, C3A and Alexander cells) that are highly representative of hepatocellular carcinoma. Evaluated with a REOs-based prognostic signature for hepatocellular carcinoma, all these eight cell lines showed the same metastatic properties of the high-risk metastatic hepatocellular carcinoma tissues. Caution should be taken for using hepatocellular carcinoma cell lines. Our results should be helpful to select proper hepatocellular carcinoma cell lines for biological experiments. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Spermine oxidase (SMO) activity in breast tumor tissues and biochemical analysis of the anticancer spermine analogues BENSpm and CPENSpm.

PubMed

Cervelli, Manuela; Bellavia, Gabriella; Fratini, Emiliano; Amendola, Roberto; Polticelli, Fabio; Barba, Marco; Federico, Rodolfo; Signore, Fabrizio; Gucciardo, Giacomo; Grillo, Rosalba; Woster, Patrick M; Casero, Robert A; Mariottini, Paolo

2010-10-14

Polyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates the expression of spermine oxidase (SMO) and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme. BC tissue samples were analyzed for SMO transcript level and SMO activity. Student's t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined. Both the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors. This study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials.

Spermine oxidase (SMO) activity in breast tumor tissues and biochemical analysis of the anticancer spermine analogues BENSpm and CPENSpm

PubMed Central

2010-01-01

Background Polyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates the expression of spermine oxidase (SMO) and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme. Methods BC tissue samples were analyzed for SMO transcript level and SMO activity. Student's t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined. Results Both the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors. Conclusions This study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials. PMID:20946629

Survey of the Heritability and Sparse Architecture of Gene Expression Traits across Human Tissues.

PubMed

Wheeler, Heather E; Shah, Kaanan P; Brenner, Jonathon; Garcia, Tzintzuni; Aquino-Michaels, Keston; Cox, Nancy J; Nicolae, Dan L; Im, Hae Kyung

2016-11-01

Understanding the genetic architecture of gene expression traits is key to elucidating the underlying mechanisms of complex traits. Here, for the first time, we perform a systematic survey of the heritability and the distribution of effect sizes across all representative tissues in the human body. We find that local h2 can be relatively well characterized with 59% of expressed genes showing significant h2 (FDR < 0.1) in the DGN whole blood cohort. However, current sample sizes (n ≤ 922) do not allow us to compute distal h2. Bayesian Sparse Linear Mixed Model (BSLMM) analysis provides strong evidence that the genetic contribution to local expression traits is dominated by a handful of genetic variants rather than by the collective contribution of a large number of variants each of modest size. In other words, the local architecture of gene expression traits is sparse rather than polygenic across all 40 tissues (from DGN and GTEx) examined. This result is confirmed by the sparsity of optimal performing gene expression predictors via elastic net modeling. To further explore the tissue context specificity, we decompose the expression traits into cross-tissue and tissue-specific components using a novel Orthogonal Tissue Decomposition (OTD) approach. Through a series of simulations we show that the cross-tissue and tissue-specific components are identifiable via OTD. Heritability and sparsity estimates of these derived expression phenotypes show similar characteristics to the original traits. Consistent properties relative to prior GTEx multi-tissue analysis results suggest that these traits reflect the expected biology. Finally, we apply this knowledge to develop prediction models of gene expression traits for all tissues. The prediction models, heritability, and prediction performance R2 for original and decomposed expression phenotypes are made publicly available (https://github.com/hakyimlab/PrediXcan).

Adipose tissue metabolic and inflammatory responses to a mixed meal in lean, overweight and obese men.

PubMed

Travers, Rebecca L; Motta, Alexandre C; Betts, James A; Thompson, Dylan

2017-02-01

Most of what we know about adipose tissue is restricted to observations derived after an overnight fast. However, humans spend the majority of waking hours in a postprandial (fed) state, and it is unclear whether increasing adiposity impacts adipose tissue responses to feeding. The aim of this research was to investigate postprandial responses in adipose tissue across varying degrees of adiposity. Thirty males aged 35-55 years with waist circumference 81-118 cm were divided equally into groups categorized as either lean, overweight or obese. Participants consumed a meal and insulinaemic, glycaemic and lipidaemic responses were monitored over 6 h. Subcutaneous adipose tissue samples were obtained at baseline and after 6 h to examine changes in gene expression and adipose tissue secretion of various adipokines. Following consumption of the meal, insulin and glucose responses were higher with increased adiposity (total AUC effects of group; p = 0.058 and p = 0.027, respectively). At 6 h, significant time effects reflected increases in IL-6 (F = 14.7, p = 0.001) and MCP-1 (F = 10.7, p = 0.003) and reduction in IRS2 adipose tissue gene expression (F = 24.6, p < 0.001), all independent of adiposity. Ex vivo adipokine secretion from adipose tissue explants remained largely unchanged after feeding. Increased systemic measures of postprandial metabolism with greater adiposity do not translate into increased inflammatory responses within adipose tissue. Instead, postprandial adipose tissue changes may represent a normal response to feeding or a (relatively) normalized response with increased adiposity due to either similar net exposure (i.e. per g of adipose) or reduced adipose tissue responsiveness.

Determination of toxic metals, trace and essentials, and macronutrients in Sarpa salpa and Chelon labrosus: risk assessment for the consumers.

PubMed

Afonso, Aridani; Gutiérrez, Angel J; Lozano, Gonzalo; González-Weller, Dailos; Rubio, Carmen; Caballero, José M; Hardisson, Arturo; Revert, Consuelo

2017-04-01

Due to increased environmental pollution, monitoring of contaminants in the environment and marine organisms is a fundamental tool for assessing the existence of risk from their consumption to human health. The levels of toxic heavy metals (Cd, Pb, and Al), trace and essential metals (B, Ba, Co, Cu, Cr, Fe, Li, Mn, Mo, Ni, Sr, V, and Zn), and macronutrients (Ca, K, Mg, Na) in two species of fish for human consumption were quantified in the present study. Eighty samples of muscle tissue and 80 samples of liver tissue belonging to two species of Osteichthyes fish; Sarpa salpa and Chelon labrosus were analyzed. The studied specimens were caught on the northern coast of Gran Canaria (Canary Islands) with fishing rods. As they caught from the shore, they are suitable samples for assessing the toxic levels of representative species caught by local amateur fishermen. The results show that both species are fit for human consumption since they have toxic levels of heavy metals (Cd, Pb, and Al) which are below the maximum established levels; however, the toxic levels of the liver samples are several orders of magnitude higher than the muscle samples, so we discourage their regular consumption. The risk assessment indicated that the two species of fish are safe for the average consumer; however, if the livers of these species are consumed, there could be risks because they exceed the PTWI for Pb and the TWI for Cd.

Fungal endophytes of aquatic macrophytes: diverse host-generalists characterized by tissue preferences and geographic structure

PubMed Central

Sandberg, Dustin C.; Battista, Lorna J.; Arnold, A. Elizabeth

2014-01-01

Most studies of endophytic symbionts have focused on terrestrial plants, neglecting the ecologically and economically important plants present in aquatic ecosystems. We evaluated the diversity, composition, host- and tissue affiliations, and geographic structure of fungal endophytes associated with common aquatic plants in northern Arizona, USA. Endophytes were isolated in culture from roots and photosynthetic tissues during two growing seasons. A total of 226 isolates representing 60 putative species was recovered from 9,600 plant tissue segments. Although isolation frequency was low, endophytes were phylogenetically diverse and species-rich. Comparisons among the most thoroughly sampled species and reservoirs revealed that isolation frequency and diversity did not differ significantly between collection periods, among species, among reservoirs, or as a function of depth. However, community structure differed significantly among reservoirs and tissue types. Phylogenetic analyses of a focal genus (Penicillium) corroborated estimates of species boundaries and informed community analyses, highlighting clade- and genotype-level affiliations of aquatic endophytes with both sediment- and waterborne fungi, and endophytes of proximate terrestrial plants. Together these analyses provide a first quantitative examination of endophytic associations in roots and foliage of aquatic plants and can be used to optimize survey strategies for efficiently capturing fungal biodiversity at local and regional scales. PMID:24402358

A histological atlas of the tissues and organs of neotenic and metamorphosed axolotl.

PubMed

Demircan, Turan; İlhan, Ayşe Elif; Aytürk, Nilüfer; Yıldırım, Berna; Öztürk, Gürkan; Keskin, İlknur

2016-09-01

Axolotl (Ambystoma Mexicanum) has been emerging as a promising model in stem cell and regeneration researches due to its exceptional regenerative capacity. Although it represents lifelong lasting neoteny, induction to metamorphosis with thyroid hormones (THs) treatment advances the utilization of Axolotl in various studies. It has been reported that amphibians undergo anatomical and histological remodeling during metamorphosis and this transformation is crucial for adaptation to terrestrial conditions. However, there is no comprehensive histological investigation regarding the morphological alterations of Axolotl organs and tissues throughout the metamorphosis. Here, we reveal the histological differences or resemblances between the neotenic and metamorphic axolotl tissues. In order to examine structural features and cellular organization of Axolotl organs, we performed Hematoxylin & Eosin, Luxol-Fast blue, Masson's trichrome, Alcian blue, Orcein and Weigart's staining. Stained samples from brain, gallbladder, heart, intestine, liver, lung, muscle, skin, spleen, stomach, tail, tongue and vessel were analyzed under the light microscope. Our findings contribute to the validation of the link between newly acquired functions and structural changes of tissues and organs as observed in tail, skin, gallbladder and spleen. We believe that this descriptive work provides new insights for a better histological understanding of both neotenic and metamorphic Axolotl tissues. Copyright © 2016 Elsevier GmbH. All rights reserved.

Quantification of HCV RNA in Liver Tissue by bDNA Assay.

PubMed

Dailey, P J; Collins, M L; Urdea, M S; Wilber, J C

1999-01-01

With this statement, Sherlock and Dooley have described two of the three major challenges involved in quantitatively measuring any analyte in tissue samples: the distribution of the analyte in the tissue; and the standard of reference, or denominator, with which to make comparisons between tissue samples. The third challenge for quantitative measurement of an analyte in tissue is to ensure reproducible and quantitative recovery of the analyte on extraction from tissue samples. This chapter describes a method that can be used to measure HCV RNA quantitatively in liver biopsy and tissue samples using the bDNA assay. All three of these challenges-distribution, denominator, and recovery-apply to the measurement of HCV RNA in liver biopsies.

Concordance of human papillomavirus types detected on the surface and in the tissue of genital lesions in men.

PubMed

Anic, Gabriella M; Messina, Jane L; Stoler, Mark H; Rollison, Dana E; Stockwell, Heather; Villa, Luisa L; Lazcano-Ponce, Eduardo; Gage, Christine; Silva, Roberto Jose C; Baggio, Maria L; Salmerón, Jorge; Giuliano, Anna R

2013-09-01

Swabbing the surface of a genital lesion to obtain a sample for HPV DNA testing is less invasive than a biopsy, but may not represent HPV types present in the lesion tissue. The objective of this study was to examine the concordance of HPV types detected in swab and biopsy samples from 165 genital lesions from men ages 18-70. Lesions included 90 condyloma, 10 penile intraepithelial neoplasia (PeIN), 23 non-condyloma with a known histology, and 42 lesions with an undetermined histology. All lesions were sampled by swabbing the surface of the lesion with a pre-wetted Dacron swab and taking a shave biopsy. HPV genotyping was performed using Linear Array for swab samples and INNO-LiPA for biopsy samples. The kappa and McNemar statistics were used to compare the concordance of detecting HPV types in swab and biopsy samples. Both sampling methods had high agreement for detection of HPV DNA in condyloma (87.8% agreement) and PeIN (100% agreement). There was also high concordance for detection of HPV16 (kappa = 1.00) and HPV18 (kappa = 1.00) in PeIN, however, agreement was low to moderate for detecting HPV6 (kappa = 0.31) and HPV11 (kappa = 0.56) in condyloma. Low to moderate agreement was also observed between sampling methods for detecting individual HPV types in the non-condyloma and lesions with an indefinite histology. The results suggest that obtaining a biopsy in addition to swabbing the surface of a lesion may provide additional information about specific HVP types associated with male genital lesions. Copyright © 2013 Wiley Periodicals, Inc.

Taenia sp. in human burial from Kan River, East Siberia.

PubMed

Slepchenko, Sergey Mikhailovich; Ivanov, Sergey Nikolaevich; Vybornov, Anton Vasilevich; Alekseevich, Tsybankov Alexander; Sergeyevich, Slavinsky Vyacheslav; Lysenko, Danil Nikolaevich; Matveev, Vyacheslav Evgenievich

2017-05-01

We present an arhaeoparasitological analysis of a unique burial from the Neftprovod II burial ground in East Siberia, which dated from the Bronze Age. Analysis of a sediment sample from the sacral region of the pelvis revealed the presence of Taenia sp. eggs. Because uncooked animal tissue is the primary source of Taenia, this indicated that the individual was likely consuming raw or undercooked meat of roe deer, red deer, or elk infected with Taenia. This finding represents the oldest case of a human infected with Taenia sp. from Eastern Siberia and Russia.

Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats

NASA Technical Reports Server (NTRS)

Mednieks, M. I.; Popova, I.; Grindeland, R. E.

1992-01-01

Photoaffinity labeling in heart tissue of rats flown on Cosmos 2044 was used to measure the regulatory (R) subunits of adenosine monophosphate-dependent protein kinase. A significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) was observed when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was observed to be unaffected by spaceflight or any of the simulation conditions. Proteins of the S2 fraction constitute a minor (less than 10 percent) component of the total, whereas the S1 fraction contained most of the cell proteins. Changes in a relatively minor aspect of adenosine monophosphate-mediated reactions are considered to be representative of a metabolic effect.

MicroRNA expression in melanocytic nevi: the usefulness of formalin-fixed, paraffin-embedded material for miRNA microarray profiling.

PubMed

Glud, Martin; Klausen, Mikkel; Gniadecki, Robert; Rossing, Maria; Hastrup, Nina; Nielsen, Finn C; Drzewiecki, Krzysztof T

2009-05-01

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate cellular differentiation, proliferation, and apoptosis. MiRNAs are expressed in a developmentally regulated and tissue-specific manner. Aberrant expression may contribute to pathological processes such as cancer, and miRNA may therefore serve as biomarkers that may be useful in a clinical environment for diagnosis of various diseases. Most miRNA profiling studies have used fresh tissue samples. However, in some types of cancer, including malignant melanoma, fresh material is difficult to obtain from primary tumors, and most surgical specimens are formalin fixed and paraffin embedded (FFPE). To explore whether FFPE material would be suitable for miRNA profiling in melanocytic lesions, we compared miRNA expression patterns in FFPE versus fresh frozen samples, obtained from 15 human melanocytic nevi. Out of microarray data, we identified 84 miRNAs that were expressed in both types of samples and represented an miRNA profile of melanocytic nevi. Our results showed a high correlation in miRNA expression (Spearman r-value of 0.80) between paired FFPE and fresh frozen material. The data were further validated by quantitative RT-PCR. In conclusion, FFPE specimens of melanocytic lesions are suitable as a source for miRNA microarray profiling.

High-throughput SNP genotyping of historical and modern samples of five bird species via sequence capture of ultraconserved elements.

PubMed

Lim, Haw Chuan; Braun, Michael J

2016-09-01

Sample availability limits population genetics research on many species, especially taxa from regions with high diversity. However, many such species are well represented in museum collections assembled before the molecular era. Development of techniques to recover genetic data from these invaluable specimens will benefit biodiversity science. Using a mixture of freshly preserved and historical tissue samples, and a sequence capture probe set targeting >5000 loci, we produced high-confidence genotype calls on thousands of single nucleotide polymorphisms (SNPs) in each of five South-East Asian bird species and their close relatives (N = 27-43). On average, 66.2% of the reads mapped to the pseudo-reference genome of each species. Of these mapped reads, an average of 52.7% was identified as PCR or optical duplicates. We achieved deeper effective sequencing for historical samples (122.7×) compared to modern samples (23.5×). The number of nucleotide sites with at least 8× sequencing depth was high, with averages ranging from 0.89 × 10(6)  bp (Arachnothera, modern samples) to 1.98 × 10(6)  bp (Stachyris, modern samples). Linear regression revealed that the amount of sequence data obtained from each historical sample (represented by per cent of the pseudo-reference genome recovered with ≥8× sequencing depth) was positively and significantly (P ≤ 0.013) related to how recently the sample was collected. We observed characteristic post-mortem damage in the DNA of historical samples. However, we were able to reduce the error rate significantly by truncating ends of reads during read mapping (local alignment) and conducting stringent SNP and genotype filtering. © 2016 John Wiley & Sons Ltd.

Usefulness of tissue autofluorescence imaging in actinic cheilitis diagnosis.

PubMed

Takahama Junior, Ademar; Kurachi, Cristina; Cosci, Alessandro; Pereira Faustino, Isabel Schausltz; Camisasca, Danielle Resende; da Costa Fontes, Karla Bianca Fernandes; Pires, Fábio Ramôa; Azevedo, Rebeca Souza

2013-07-01

Actinic cheilitis (AC) is a potentially malignant disorder of the lips. Because of its heterogeneous clinical aspect, it is difficult to indicate representative biopsy area. The purpose of this study was to evaluate the usefulness of tissue autofluorescence in AC diagnosis. The system was composed of a 405-nm light-emitting diode, sent to the sample by a dichroic, that allows the fluorescence signal to reach a camera directly plugged in the system. Fifty-seven patients with clinical diagnosis of AC and 45 normal volunteers were selected. According to clinical and fluorescence features, one or more areas were selected for biopsies in the AC group and epithelial dysplasia (ED) grades were established. The autofluorescence images were processed by a clustering algorithm for AC automated diagnosis. The tissue autofluorescence image revealed a heterogeneous pattern of loss and increase of fluorescence in patients with AC. ED was found in 93% of the cases, and most of the areas graded as moderate or severe ED were chosen with the aid of autofluorescence. The processed autofluorescence images from AC patients showed a higher number of spots in an irregular pattern. Tissue autofluorescence image system is a useful technique in association with clinical examination for AC diagnosis.

Total Mercury in Squalid Callista Megapitaria squalida from the SW Gulf of California, Mexico: Tissue Distribution and Human Health Risk.

PubMed

Romo-Piñera, Abril K; Escobar-Sánchez, Ofelia; Ruelas-Inzunza, Jorge; Frías-Espericueta, Martín G

2018-03-01

We evaluated the total Hg concentration in different tissues of squalid callista Megapitaria squalida in order to measure Hg distribution in tissue and to estimate human health risk. Samples were obtained by free diving in the SW Gulf of California, Mexico. Concentrations are given on a wet weight basis. A total of 89 squalid callista specimens were obtained, presenting an average Hg concentration of 0.07 ± 0.04 µg g -1 . There were no significant differences (p > 0.05) in Hg concentration between tissues (visceral mass = 0.09 ± 0.08 µg g -1 ; mantle = 0.06 ± 0.07 µg g -1 ; muscle = 0.06 ± 0.04 µg g -1 ). The low Hg values found in squalid callista and its low risk quotient (HQ = 0.03) suggest that the consumption of squalid callista does not represent a human health risk. However, HQ calculated using MeHg was > 1, it which could indicate a potential risk related to consumption of clams.

Combined Bisulfite Restriction Analysis for brain tissue identification.

PubMed

Samsuwan, Jarunya; Muangsub, Tachapol; Yanatatsaneejit, Pattamawadee; Mutirangura, Apiwat; Kitkumthorn, Nakarin

2018-05-01

According to the tissue-specific methylation database (doi: 10.1016/j.gene.2014.09.060), methylation at CpG locus cg03096975 in EML2 has been preliminarily proven to be specific to brain tissue. In this study, we enlarged sample size and developed a technique for identifying brain tissue in aged samples. Combined Bisulfite Restriction Analysis-for EML2 (COBRA-EML2) technique was established and validated in various organ samples obtained from 108 autopsies. In addition, this technique was also tested for its reliability, minimal DNA concentration detected, and use in aged samples and in samples obtained from specific brain compartments and spinal cord. COBRA-EML2 displayed 100% sensitivity and specificity for distinguishing brain tissue from other tissues, showed high reliability, was capable of detecting minimal DNA concentration (0.015ng/μl), could be used for identifying brain tissue in aged samples. In summary, COBRA-EML2 is a technique to identify brain tissue. This analysis is useful in criminal cases since it can identify the vital organ tissues from small samples acquired from criminal scenes. The results from this analysis can be counted as a medical and forensic marker supporting criminal investigations, and as one of the evidences in court rulings. Copyright © 2018 Elsevier B.V. All rights reserved.

Long term organ culture of human prostate tissue in a NASA-designed rotating wall bioreactor

NASA Technical Reports Server (NTRS)

Margolis, L.; Hatfill, S.; Chuaqui, R.; Vocke, C.; Emmert-Buck, M.; Linehan, W. M.; Duray, P. H.

1999-01-01

PURPOSE: To maintain ex vivo integral prostatic tissue including intact stromal and ductal elements using the NASA-designed Rotating Wall Vessel (RWV) which maintains colocalized cells in an environment that promotes both three-dimensional cellular interactions together with the uniform mass transfer of nutrients and metabolic wastes. MATERIALS AND METHODS: Samples of normal prostate were obtained as a byproduct of transurethral prostatectomy or needle biopsy. Prostatic tissue dissected into small 1 x 1 mm. blocks was cultured in the Rotating Wall Vessel (RWV) Bioreactor for various time periods and analyzed using histological, immunochemical, and total cell RNA assays. RESULTS: We report the long term maintenance of benign explanted human prostate tissue grown in simple culture medium, under the simulated microgravity conditions afforded by the RWV bioreactor. Mesenchymal stromal elements including blood vessels and architecturally preserved tubuloglandular acini were maintained for a minimum of 28 days. Cytokeratins, vimentin and TGF-beta2 receptor and ligand were preserved through the entire culture period as revealed by immunocytochemistry. Prostatic acid phosphatase (PAP) was continuously expressed during the culture period, although somewhat decreased. Prostatic specific antigen (PSA) and its transcript were down regulated over time of culture. Prostatic carcinoma cells from the TSU cell line were able to invade RWV-cultured benign prostate tissue explants. CONCLUSIONS: The RWV bioreactor represents an additional new technology for culturing prostate tissue for further investigations concerning the basic physiology and pathobiology of this clinically important tissue.

Tissue spray ionization mass spectrometry for rapid recognition of human lung squamous cell carcinoma

NASA Astrophysics Data System (ADS)

Wei, Yiping; Chen, Liru; Zhou, Wei; Chingin, Konstantin; Ouyang, Yongzhong; Zhu, Tenggao; Wen, Hua; Ding, Jianhua; Xu, Jianjun; Chen, Huanwen

2015-05-01

Tissue spray ionization mass spectrometry (TSI-MS) directly on small tissue samples has been shown to provide highly specific molecular information. In this study, we apply this method to the analysis of 38 pairs of human lung squamous cell carcinoma tissue (cancer) and adjacent normal lung tissue (normal). The main components of pulmonary surfactants, dipalmitoyl phosphatidylcholine (DPPC, m/z 757.47), phosphatidylcholine (POPC, m/z 782.52), oleoyl phosphatidylcholine (DOPC, m/z 808.49), and arachidonic acid stearoyl phosphatidylcholine (SAPC, m/z 832.43), were identified using high-resolution tandem mass spectrometry. Monte Carlo sampling partial least squares linear discriminant analysis (PLS-LDA) was used to distinguish full-mass-range mass spectra of cancer samples from the mass spectra of normal tissues. With 5 principal components and 30 - 40 Monte Carlo samplings, the accuracy of cancer identification in matched tissue samples reached 94.42%. Classification of a tissue sample required less than 1 min, which is much faster than the analysis of frozen sections. The rapid, in situ diagnosis with minimal sample consumption provided by TSI-MS is advantageous for surgeons. TSI-MS allows them to make more informed decisions during surgery.

Functional integrative levels in the human interactome recapitulate organ organization.

PubMed

Souiai, Ouissem; Becker, Emmanuelle; Prieto, Carlos; Benkahla, Alia; De las Rivas, Javier; Brun, Christine

2011-01-01

Interactome networks represent sets of possible physical interactions between proteins. They lack spatio-temporal information by construction. However, the specialized functions of the differentiated cell types which are assembled into tissues or organs depend on the combinatorial arrangements of proteins and their physical interactions. Is tissue-specificity, therefore, encoded within the interactome? In order to address this question, we combined protein-protein interactions, expression data, functional annotations and interactome topology. We first identified a subnetwork formed exclusively of proteins whose interactions were observed in all tested tissues. These are mainly involved in housekeeping functions and are located at the topological center of the interactome. This 'Largest Common Interactome Network' represents a 'functional interactome core'. Interestingly, two types of tissue-specific interactions are distinguished when considering function and network topology: tissue-specific interactions involved in regulatory and developmental functions are central whereas tissue-specific interactions involved in organ physiological functions are peripheral. Overall, the functional organization of the human interactome reflects several integrative levels of functions with housekeeping and regulatory tissue-specific functions at the center and physiological tissue-specific functions at the periphery. This gradient of functions recapitulates the organization of organs, from cells to organs. Given that several gradients have already been identified across interactomes, we propose that gradients may represent a general principle of protein-protein interaction network organization.

The evaluation of risk-benefit ratio for gut tissue sampling in HIV cure research.

PubMed

Mehraj, Vikram; Ghali, Peter; Ramendra, Rayoun; Costiniuk, Cecilia; Lebouché, Bertrand; Ponte, Rosalie; Reinhard, Robert; Sousa, Jose; Chomont, Nicolas; Cohen, Eric A; Ancuta, Petronela; Routy, Jean-Pierre

2017-10-01

Antiretroviral therapy (ART) does not cure HIV infection due to the persistence of HIV reservoirs in long-lived memory CD4 T cells present in the blood, lymph nodes, intestinal tract, and other tissues. Interest grows in obtaining gut-tissue samples for HIV persistence studies, which poses an ethical challenge to provide study volunteers with adequate information on risks and benefits. Herein we assess the risks and benefits of undergoing gut biopsy procedures for HIV pathogenesis and reservoir studies. A group discussion was organised with physicians and community representatives on performing either a flexible sigmoidoscopy or a colonoscopy. Consensus was reached on conducting colonoscopy in persons ≥50 years. Thirty HIV-infected, ART-treated and nine uninfected participants were recruited. Colonoscopy was performed to collect 30 gut mucosal biopsies. When present, polyps were removed and abnormal mucosal findings were biopsied for pathological analysis. Participants were interviewed on potential discomfort following colonoscopic examination. The HIV-infected and uninfected groups were comparable in terms of age and gender with more men who have sex with men (MSM) in the former group. Abnormal colonoscopic findings were observed in 43.6% of all the participants and did not differ by HIV status. In total, 24 polyps were removed with a higher mean number of polyps removed in HIV-infected versus uninfected participants (1.7 vs 1.0, P =0.013). The number of polyps marginally correlated with inverted CD4:CD8 ratio. Based on our findings, colonoscopic examination was safe to use for gut biopsy procedures where almost half of the participants had polyps removed. Participation in the study provided colon cancer screening as an ancillary benefit that participants could have received in standard medical care, thus mitigating burdens of invasive procedures. Dialogue between community representatives and clinical researchers can increase participation and advance HIV cure research.

Application of ribonucleoside vanadyl complex (RVC) for developing a multifunctional tissue preservative solution

PubMed Central

Yu, Cheng-Chia; Chen, Chin-Chuan

2018-01-01

The quality of biological samples greatly affects the accuracy of scientific results. However, RNA in cryopreserved tissues gradually degrades during storage, leading to errors in the results of subsequent experiments. A suitable sample preservative solution can prolong storage and enhance the research value of samples. Here, we developed a sample preservative solution using the properties of the ribonucleoside vanadyl complex (RVC) and compared its effects on RNA and DNA quality, protein activity, and tissue morphology with the commercially available and widely used RNAlater® Stabilization Solution. The results showed that both the RVC-based preservative solution and RNAlater can effectively delay RNA degradation in tissue samples stored at 4°C or −80°C compared with samples stored without any preservative solution. In contrast to RNAlater, the RVC-based preservative solution did not result in damage to the tissue morphology or a loss of protein activity. Additionally, the RVC-based preservative solution did not affect the RNA and genomic DNA contents of the tissue samples or the results of subsequent experimental analyses. An RVC-based reagent can be used as a multifunctional yet relatively inexpensive tissue preservative solution to provide a comprehensive and cost-effective method for preserving samples for tissue banks. PMID:29538436

A deep learning approach to estimate chemically-treated collagenous tissue nonlinear anisotropic stress-strain responses from microscopy images.

PubMed

Liang, Liang; Liu, Minliang; Sun, Wei

2017-11-01

Biological collagenous tissues comprised of networks of collagen fibers are suitable for a broad spectrum of medical applications owing to their attractive mechanical properties. In this study, we developed a noninvasive approach to estimate collagenous tissue elastic properties directly from microscopy images using Machine Learning (ML) techniques. Glutaraldehyde-treated bovine pericardium (GLBP) tissue, widely used in the fabrication of bioprosthetic heart valves and vascular patches, was chosen to develop a representative application. A Deep Learning model was designed and trained to process second harmonic generation (SHG) images of collagen networks in GLBP tissue samples, and directly predict the tissue elastic mechanical properties. The trained model is capable of identifying the overall tissue stiffness with a classification accuracy of 84%, and predicting the nonlinear anisotropic stress-strain curves with average regression errors of 0.021 and 0.031. Thus, this study demonstrates the feasibility and great potential of using the Deep Learning approach for fast and noninvasive assessment of collagenous tissue elastic properties from microstructural images. In this study, we developed, to our best knowledge, the first Deep Learning-based approach to estimate the elastic properties of collagenous tissues directly from noninvasive second harmonic generation images. The success of this study holds promise for the use of Machine Learning techniques to noninvasively and efficiently estimate the mechanical properties of many structure-based biological materials, and it also enables many potential applications such as serving as a quality control tool to select tissue for the manufacturing of medical devices (e.g. bioprosthetic heart valves). Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Geometric Triangular Chiral Hexagon Crystal-Like Complexes Organization in Pathological Tissues Biological Collision Order

PubMed Central

Díaz, Jairo A.; Jaramillo, Natalia A.; Murillo, Mauricio F.

2007-01-01

The present study describes and documents self-assembly of geometric triangular chiral hexagon crystal like complex organizations (GTCHC) in human pathological tissues.The authors have found this architectural geometric expression at macroscopic and microscopic levels mainly in cancer processes. This study is based essentially on macroscopic and histopathologic analyses of 3000 surgical specimens: 2600 inflammatory lesions and 400 malignant tumours. Geometric complexes identified photographically at macroscopic level were located in the gross surgical specimen, and these areas were carefully dissected. Samples were taken to carry out histologic analysis. Based on the hypothesis of a collision genesis mechanism and because it is difficult to carry out an appropriate methodological observation in biological systems, the authors designed a model base on other dynamic systems to obtain indirect information in which a strong white flash wave light discharge, generated by an electronic device, hits over the lines of electrical conductance structured in helicoidal pattern. In their experimental model, the authors were able to reproduce and to predict polarity, chirality, helicoid geometry, triangular and hexagonal clusters through electromagnetic sequential collisions. They determined that similar events among constituents of extracelular matrix which drive and produce piezoelectric activity are responsible for the genesis of GTCHC complexes in pathological tissues. This research suggests that molecular crystals represented by triangular chiral hexagons derived from a collision-attraction event against collagen type I fibrils emerge at microscopic and macroscopic scales presenting a lateral assembly of each side of hypertrophy helicoid fibers, that represent energy flow in cooperative hierarchically chiral electromagnetic interaction in pathological tissues and arises as a geometry of the equilibrium in perturbed biological systems. Further interdisciplinary studies must be carried out to reproduce, manipulate and amplify their activity and probably use them as a base to develop new therapeutic strategies in cancer. PMID:18074008

Assessment of the accuracy of an ultrasound elastography liver scanning system using a PVA-cryogel phantom with optimal acoustic and mechanical properties

NASA Astrophysics Data System (ADS)

Cournane, S.; Cannon, L.; Browne, J. E.; Fagan, A. J.

2010-10-01

The accuracy of a transient elastography liver-scanning ultrasound system was assessed using a novel application of PVA-cryogel as a tissue-mimicking material with acoustic and shear elasticity properties optimized to best represent those of liver tissue. Although the liver-scanning system has been shown to offer a safer alternative for diagnosing liver cirrhosis through stiffness measurement, as compared to the liver needle biopsy exam, the scanner's accuracy has not been fully established. Young's elastic modulus values of 5-6 wt% PVA-cryogel phantoms, also containing glycerol and 0.3 µm Al2O3 and 3 µm Al2O3, were measured using a 'gold standard' mechanical testing technique and transient elastography. The mechanically measured values and acoustic velocities of the phantoms ranged between 1.6 and 16.1 kPa and 1540 and 1570 m s-1, respectively, mimicking those observed in liver tissue. The values reported by the transient elastography system overestimated Young's elastic modulus values representative of the progressive stages of liver fibrosis by up to 32%. These results were attributed to the relative rather than absolute nature of the measurement arising from the single-point acoustic velocity calibration of the system, rendering the measurements critically dependent on the speed of sound of the sample under investigation. Given the wide range of acoustic velocities which exist in the liver, spanning healthy tissue to cirrhotic pathology, coupled with the system's assumption that the liver is approximately elastic when it is rather highly viscoelastic, care should be exercised when interpreting the results from this system in patient groups.

System and method for controlling depth of imaging in tissues using fluorescence microscopy under ultraviolet excitation following staining with fluorescing agents

DOE Office of Scientific and Technical Information (OSTI.GOV)

Demos, Stavros; Levenson, Richard

The present disclosure relates to a method for analyzing tissue specimens. In one implementation the method involves obtaining a tissue sample and exposing the sample to one or more fluorophores as contrast agents to enhance contrast of subcellular compartments of the tissue sample. The tissue sample is illuminated by an ultraviolet (UV) light having a wavelength between about 200 nm to about 400 nm, with the wavelength being selected to result in penetration to only a specified depth below a surface of the tissue sample. Inter-image operations between images acquired under different imaging parameters allow for improvement of the imagemore » quality via removal of unwanted image components. A microscope may be used to image the tissue sample and provide the image to an image acquisition system that makes use of a camera. The image acquisition system may create a corresponding image that is transmitted to a display system for processing and display.« less

Periapical fluid RANKL and IL-8 are differentially regulated in pulpitis and apical periodontitis.

PubMed

Rechenberg, Dan-K; Bostanci, Nagihan; Zehnder, Matthias; Belibasakis, Georgios N

2014-09-01

The dental pulp space can become infected due to a breach in the surrounding hard tissues. This leads to inflammation of the pulp (pulpitis), soft tissue breakdown, and finally to bone loss around the root apex (apical periodontitis). The succession of the molecular events leading to apical periodontitis is currently not known. The main inflammatory mediator associated with neutrophil chemotaxis is interleukin-8 (IL-8), and with bone resorption the dyad of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG). The levels of RANKL, OPG and IL-8 were studied in periapical tissue fluid of human teeth (n = 48) diagnosed with symptomatic irreversible pulpitis (SIP) and asymptomatic apical periodontitis (AAP). SIP represents the starting point, and AAP an established steady state of the disease. Periapical tissue fluid samples were collected using paper points and then evaluated using enzyme-linked immunosorbent assays (ELISAs). Target protein levels per case were calibrated against the corresponding total protein content, as determined fluorometrically. RANKL was expressed at significantly higher levels in SIP compared to AAP (P < 0.05), whereas OPG was under the detection limit in most samples. In contrast, IL-8 levels were significantly lower in SIP compared to AAP (P < 0.05). Spearman's correlation analysis between RANKL and IL-8 revealed a significantly (P < 0.05) negative correlation between the two measures (rho = -.44). The results of this study suggest that, in the development of apical periodontitis, periapical bone resorption signaling, as determined by RANKL, occurs prior to inflammatory cell recruitment signaling, as determined by IL-8. Copyright © 2014 Elsevier Ltd. All rights reserved.

Corals Form Characteristic Associations with Symbiotic Nitrogen-Fixing Bacteria

PubMed Central

Lema, Kimberley A.; Willis, Bette L.

2012-01-01

The complex symbiotic relationship between corals and their dinoflagellate partner Symbiodinium is believed to be sustained through close associations with mutualistic bacterial communities, though little is known about coral associations with bacterial groups able to fix nitrogen (diazotrophs). In this study, we investigated the diversity of diazotrophic bacterial communities associated with three common coral species (Acropora millepora, Acropora muricata, and Pocillopora damicormis) from three midshelf locations of the Great Barrier Reef (GBR) by profiling the conserved subunit of the nifH gene, which encodes the dinitrogenase iron protein. Comparisons of diazotrophic community diversity among coral tissue and mucus microenvironments and the surrounding seawater revealed that corals harbor diverse nifH phylotypes that differ between tissue and mucus microhabitats. Coral mucus nifH sequences displayed high heterogeneity, and many bacterial groups overlapped with those found in seawater. Moreover, coral mucus diazotrophs were specific neither to coral species nor to reef location, reflecting the ephemeral nature of coral mucus. In contrast, the dominant diazotrophic bacteria in tissue samples differed among coral species, with differences remaining consistent at all three reefs, indicating that coral-diazotroph associations are species specific. Notably, dominant diazotrophs for all coral species were closely related to the bacterial group rhizobia, which represented 71% of the total sequences retrieved from tissue samples. The species specificity of coral-diazotroph associations further supports the coral holobiont model that bacterial groups associated with corals are conserved. Our results suggest that, as in terrestrial plants, rhizobia have developed a mutualistic relationship with corals and may contribute fixed nitrogen to Symbiodinium. PMID:22344646

Next generation sequencing techniques in liquid biopsy: focus on non-small cell lung cancer patients.

PubMed

Malapelle, Umberto; Pisapia, Pasquale; Rocco, Danilo; Smeraglio, Riccardo; di Spirito, Maria; Bellevicine, Claudio; Troncone, Giancarlo

2016-10-01

The advent of genomic based personalized medicine has led to multiple advances in the molecular characterization of many tumor types, such as non-small cell lung cancer (NSCLC). NSCLC is diagnosed in most cases on small tissue samples that may be not always sufficient for EGFR mutational assessment to select patients for first and second generations' tyrosine kinase inhibitors (TKIs) therapy. In patients without tissue availability at presentation, the analysis of cell free DNA (cfDNA) derived from liquid biopsy samples, in particular from plasma, represent an established alternative to provide EGFR mutational testing for treatment decision making. In addition, a new paradigm for TKIs resistance management was recently approved by Food and Drug Administration, supporting the liquid biopsy based genotyping prior to tissue based genotyping for the detection of T790M mutation to select patients for third generation TKIs. In these settings, real time PCR (RT-PCR) and digital PCR 'targeted' methods, which detect known mutations by specific probes, have extensively been adopted. Taking into account the restricted reference range and the limited multiplexing power of these targeted methods, the performance of liquid biopsy analyses may be further improved by next generation sequencing (NGS). While most tissue based NGS genotyping is well established, liquid biopsy NGS application is challenging, requiring a careful validation of the whole process, from blood collection to variant calling. Here we review this evolving field, highlighting those methodological points that are crucial to accurately select NSCLC patients for TKIs treatment administration by NGS on cfDNA.

Proteomic analysis of corneal endothelial cell-descemet membrane tissues reveals influence of insulin dependence and disease severity in type 2 diabetes mellitus.

PubMed

Skeie, Jessica M; Aldrich, Benjamin T; Goldstein, Andrew S; Schmidt, Gregory A; Reed, Cynthia R; Greiner, Mark A

2018-01-01

The objective of this study was to characterize the proteome of the corneal endothelial cell layer and its basement membrane (Descemet membrane) in humans with various severities of type II diabetes mellitus compared to controls, and identify differentially expressed proteins across a range of diabetic disease severities that may influence corneal endothelial cell health. Endothelium-Descemet membrane complex tissues were peeled from transplant suitable donor corneas. Protein fractions were isolated from each sample and subjected to multidimensional liquid chromatography and tandem mass spectrometry. Peptide spectra were matched to the human proteome, assigned gene ontology, and grouped into protein signaling pathways unique to each of the disease states. We identified an average of 12,472 unique proteins in each of the endothelium-Descemet membrane complex tissue samples. There were 2,409 differentially expressed protein isoforms that included previously known risk factors for type II diabetes mellitus related to metabolic processes, oxidative stress, and inflammation. Gene ontology analysis demonstrated that diabetes progression has many protein footprints related to metabolic processes, binding, and catalysis. The most represented pathways involved in diabetes progression included mitochondrial dysfunction, cell-cell junction structure, and protein synthesis regulation. This proteomic dataset identifies novel corneal endothelial cell and Descemet membrane protein expression in various stages of diabetic disease. These findings give insight into the mechanisms involved in diabetes progression relevant to the corneal endothelium and its basement membrane, prioritize new pathways for therapeutic targeting, and provide insight into potential biomarkers for determining the health of this tissue.

How to count cells: the advantages and disadvantages of the isotropic fractionator compared with stereology.

PubMed

Herculano-Houzel, Suzana; von Bartheld, Christopher S; Miller, Daniel J; Kaas, Jon H

2015-04-01

The number of cells comprising biological structures represents fundamental information in basic anatomy, development, aging, drug tests, pathology and genetic manipulations. Obtaining unbiased estimates of cell numbers, however, was until recently possible only through stereological techniques, which require specific training, equipment, histological processing and appropriate sampling strategies applied to structures with a homogeneous distribution of cell bodies. An alternative, the isotropic fractionator (IF), became available in 2005 as a fast and inexpensive method that requires little training, no specific software and only a few materials before it can be used to quantify total numbers of neuronal and non-neuronal cells in a whole organ such as the brain or any dissectible regions thereof. This method entails transforming a highly anisotropic tissue into a homogeneous suspension of free-floating nuclei that can then be counted under the microscope or by flow cytometry and identified morphologically and immunocytochemically as neuronal or non-neuronal. We compare the advantages and disadvantages of each method and provide researchers with guidelines for choosing the best method for their particular needs. IF is as accurate as unbiased stereology and faster than stereological techniques, as it requires no elaborate histological processing or sampling paradigms, providing reliable estimates in a few days rather than many weeks. Tissue shrinkage is also not an issue, since the estimates provided are independent of tissue volume. The main disadvantage of IF, however, is that it necessarily destroys the tissue analyzed and thus provides no spatial information on the cellular composition of biological regions of interest.

Investigation of real tissue water equivalent path lengths using an efficient dose extinction method

NASA Astrophysics Data System (ADS)

Zhang, Rongxiao; Baer, Esther; Jee, Kyung-Wook; Sharp, Gregory C.; Flanz, Jay; Lu, Hsiao-Ming

2017-07-01

For proton therapy, an accurate conversion of CT HU to relative stopping power (RSP) is essential. Validation of the conversion based on real tissue samples is more direct than the current practice solely based on tissue substitutes and can potentially address variations over the population. Based on a novel dose extinction method, we measured water equivalent path lengths (WEPL) on animal tissue samples to evaluate the accuracy of CT HU to RSP conversion and potential variations over a population. A broad proton beam delivered a spread out Bragg peak to the samples sandwiched between a water tank and a 2D ion-chamber detector. WEPLs of the samples were determined from the transmission dose profiles measured as a function of the water level in the tank. Tissue substitute inserts and Lucite blocks with known WEPLs were used to validate the accuracy. A large number of real tissue samples were measured. Variations of WEPL over different batches of tissue samples were also investigated. The measured WEPLs were compared with those computed from CT scans with the Stoichiometric calibration method. WEPLs were determined within  ±0.5% percentage deviation (% std/mean) and  ±0.5% error for most of the tissue surrogate inserts and the calibration blocks. For biological tissue samples, percentage deviations were within  ±0.3%. No considerable difference (<1%) in WEPL was observed for the same type of tissue from different sources. The differences between measured WEPLs and those calculated from CT were within 1%, except for some bony tissues. Depending on the sample size, each dose extinction measurement took around 5 min to produce ~1000 WEPL values to be compared with calculations. This dose extinction system measures WEPL efficiently and accurately, which allows the validation of CT HU to RSP conversions based on the WEPL measured for a large number of samples and real tissues.

The Diagnostic Accuracy of Incisional Biopsy in the Oral Cavity.

PubMed

Chen, Sara; Forman, Michael; Sadow, Peter M; August, Meredith

2016-05-01

To determine the accuracy of incisional biopsy examination to diagnose oral lesions. This retrospective cohort study was performed to determine the concordance rate between incisional biopsy examination and definitive resection diagnosis for different oral lesions. The study sample was derived from the population of patients who presented to the Department of Oral and Maxillofacial Surgery, Massachusetts General Hospital (Boston, MA) from January 2005 through December 2012. Inclusion criteria were the diagnosis of an oral lesion from an incisional biopsy examination, subsequent diagnosis from the definitive resection of the same lesion, and complete clinical and pathologic patient records. The predictor variables were the origin and size of the lesion. The primary outcome variable was concordance between the provisional incisional biopsy diagnosis and definitive pathologic resection diagnosis. The secondary outcome variable was type of biopsy error for the discordant cases. Incisional biopsy errors were assessed and grouped into 5 categories: 1) sampling error; 2) insufficient tissue for diagnosis; 3) presence of inflammation making diagnosis difficult; 4) artifact; and 5) pathologist discordance. A total of 272 patients met the inclusion criteria. The study sample had a mean age of 47.4 years and 55.7% were women. Of these cases, 242 (88.9%) were concordant when comparing the biopsy and final resection pathology reports. At histologic evaluation, 60.0% of discordant findings were attributed to sampling error, 23.3% to pathologist discrepancy, 13.3% to insufficient tissue provided in the biopsy specimen, and 3.4% to inflammation obscuring diagnosis. Overall, concordant cases had a larger average biopsy volume (1.53 cm(3)) than discordant cases (0.42 cm(3)). The data collected indicate an 88.9% diagnostic concordance with final pathologic results for incisional oral biopsy diagnoses. Sixty percent of discordance was attributed to sampling error when sampled tissue was not representative of the lesion in toto. Multiple-site biopsy specimens and larger-volume samples allowed for a more accurate diagnosis. Copyright © 2016 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.

Micro-organisms isolated from cadaveric samples of allograft musculoskeletal tissue.

PubMed

Varettas, Kerry

2013-12-01

Allograft musculoskeletal tissue is commonly used in orthopaedic surgical procedures. Cadaveric donors of musculoskeletal tissue supply multiple allografts such as tendons, ligaments and bone. The microbiology laboratory of the South Eastern Area Laboratory Services (SEALS, Australia) has cultured cadaveric allograft musculoskeletal tissue samples for bacterial and fungal isolates since 2006. This study will retrospectively review the micro-organisms isolated over a 6-year period, 2006-2011. Swab and tissue samples were received for bioburden testing and were inoculated onto agar and/or broth culture media. Growth was obtained from 25.1 % of cadaveric allograft musculoskeletal tissue samples received. The predominant organisms isolated were coagulase-negative staphylococci and coliforms, with the heaviest bioburden recovered from the hemipelvis. The rate of bacterial and fungal isolates from cadaveric allograft musculoskeletal tissue samples is higher than that from living donors. The type of organism isolated may influence the suitability of the allograft for transplant.

CODIFI (Concordance in Diabetic Foot Ulcer Infection): a cross-sectional study of wound swab versus tissue sampling in infected diabetic foot ulcers in England.

PubMed

Nelson, Andrea; Wright-Hughes, Alexandra; Backhouse, Michael Ross; Lipsky, Benjamin A; Nixon, Jane; Bhogal, Moninder S; Reynolds, Catherine; Brown, Sarah

2018-01-31

To determine the extent of agreement and patterns of disagreement between wound swab and tissue samples in patients with an infected diabetic foot ulcer (DFU). Multicentre, prospective, cross-sectional study. Primary and secondary care foot ulcer/diabetic outpatient clinics and hospital wards across England. Inclusion criteria: consenting patients aged ≥18 years; diabetes mellitus; suspected infected DFU. clinically inappropriate to take either sample. Wound swab obtained using Levine's technique; tissue samples collected using a sterile dermal curette or scalpel. Coprimary: reported presence, and number, of pathogens per sample; prevalence of resistance to antimicrobials among likely pathogens. Secondary: recommended change in antibiotic therapy based on blinded clinical review; adverse events; sampling costs. 400 consenting patients (79% male) from 25 centres.Most prevalent reported pathogens were Staphylococcus aureus (43.8%), Streptococcus (16.7%) and other aerobic Gram-positive cocci (70.6%). At least one potential pathogen was reported from 70.1% of wound swab and 86.1% of tissue samples. Pathogen results differed between sampling methods in 58% of patients, with more pathogens and fewer contaminants reported from tissue specimens.The majority of pathogens were reported significantly more frequently in tissue than wound swab samples (P<0.01), with equal disagreement for S. aureus and Pseudomonas aeruginosa. Blinded clinicians more often recommended a change in antibiotic regimen based on tissue compared with wound swab results (increase of 8.9%, 95% CI 2.65% to 15.3%). Ulcer pain and bleeding occurred more often after tissue collection versus wound swabs (pain: 9.3%, 1.3%; bleeding: 6.8%, 1.5%, respectively). Reports of tissue samples more frequently identified pathogens, and less frequently identified non-pathogens compared with wound swab samples. Blinded clinicians more often recommended changes in antibiotic therapy based on tissue compared with wound swab specimens. Further research is needed to determine the effect of the additional information provided by tissue samples. ISRCTN52608451. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Fate of Salmonella enterica and Enterohemorrhagic Escherichia coli Cells Artificially Internalized into Vegetable Seeds during Germination.

PubMed

Liu, Da; Cui, Yue; Walcott, Ronald; Chen, Jinru

2018-01-01

Vegetable seeds contaminated with bacterial pathogens have been linked to fresh-produce-associated outbreaks of gastrointestinal infections. This study was undertaken to observe the physiological behavior of Salmonella enterica and enterohemorrhagic Escherichia coli (EHEC) cells artificially internalized into vegetable seeds during the germination process. Surface-decontaminated seeds of alfalfa, fenugreek, lettuce, and tomato were vacuum-infiltrated with four individual strains of Salmonella or EHEC. Contaminated seeds were germinated at 25°C for 9 days, and different sprout/seedling tissues were microbiologically analyzed every other day. The internalization of Salmonella and EHEC cells into vegetable seeds was confirmed by the absence of pathogens in seed-rinsing water and the presence of pathogens in seed homogenates after postinternalization seed surface decontamination. Results show that 317 (62%) and 343 (67%) of the 512 collected sprout/seedling tissue samples were positive for Salmonella and EHEC, respectively. The average Salmonella populations were significantly larger ( P < 0.05) than the EHEC populations. Significantly larger Salmonella populations were recovered from the cotyledon and seed coat tissues, followed by the root tissues, but the mean EHEC populations from all sampled tissue sections were statistically similar, except in pregerminated seeds. Three Salmonella and two EHEC strains had significantly larger cell populations on sprout/seedling tissues than other strains used in the study. Salmonella and EHEC populations from fenugreek and alfalfa tissues were significantly larger than those from tomato and lettuce tissues. The study showed the fate of internalized human pathogens on germinating vegetable seeds and sprout/seedling tissues and emphasized the importance of using pathogen-free seeds for sprout production. IMPORTANCE The internalization of microorganisms into vegetable seeds could occur naturally and represents a possible pathway of vegetable seed contamination by human pathogens. The present study investigated the ability of two important bacterial pathogens, Salmonella and enterohemorrhagic Escherichia coli (EHEC), when artificially internalized into vegetable seeds, to grow and disseminate along vegetable sprouts/seedlings during germination. The data from the study revealed that the pathogen cells artificially internalized into vegetable seeds caused the contamination of different tissues of sprouts/seedlings and that pathogen growth on germinating seeds is bacterial species and vegetable seed-type dependent. These results further stress the necessity of using pathogen-free vegetable seeds for edible sprout production. Copyright © 2017 American Society for Microbiology.

Summary of information on synthetic organic compounds and trace elements in tissue of aquatic biota, Clark Fork-Pend Oreille and Spokane River basins, Montana, Idaho, and Washington, 1974-96

USGS Publications Warehouse

Maret, Terry R.; Dutton, DeAnn M.

1999-01-01

As part of the Northern Rockies Intermontane Basins study of the National Water-Quality Assessment Program, data collected between 1974 and 1996 were compiled to describe contaminants in tissue of riverine species. Tissue-contaminant data from 11 monitoring programs and studies representing 28 sites in the study area were summarized. Tissue-contaminant data for most streams generally were lacking. Many studies have focused on and around mining-affected areas on the Clark Fork and Coeur d'Alene Rivers and their major tributaries. DDT and PCBs and their metabolites and congeners were the synthetic organic contaminants most commonly detected in fish tissue. Fish collected from the Spokane River in Washington contained elevated concentrations of PCB arochlors, some of which exceeded guidelines for the protection of human health and predatory wildlife. Tissue samples of fish from the Flathead River watershed contained higher-than-expected concentrations of PCBs, which might have resulted from atmospheric transport. Trace element concentrations in fish and macroinvertebrates collected in and around mining areas were elevated compared with background concentrations. Some cadmium, copper, lead, and mercury concentrations in fish tissue were elevated compared with results from other studies, and some exceeded guidelines. Macroinvertebrates from the Coeur d'Alene River contained higher concentrations of cadmium, lead, and zinc than did macroinvertebrates from other river systems in mining-affected areas. A few sportfish fillet samples, most from the Spokane River in Washington, were collected to assess human health risk. Concentrations of PCBs in these fillets exceeded screening values for the protection of human health. At present, there is no coordinated, long-term fish tissue monitoring program for rivers in the study area, even though contaminants are present in fish at levels considered a threat to human health. Development of a coordinated, centralized national data base for contaminants in fish tissue is needed. The National Water-Quality Assessment Program can provide a framework for other agencies to evaluate tissue contaminants in the Northern Rockies Intermontane Basins study area. As of 1996, there are no fish consumption advisories or fishing restrictions as a result of elevated contaminants on any rivers within the study area.

Recent developments in human biomonitoring: non-invasive assessment of target tissue dose and effects of pneumotoxic metals.

PubMed

Mutti, A; Corradi, M

2006-01-01

Tobacco smoke and polluted environments substantially increase the lung burden of pneumotoxic chemicals, particularly pneumotoxic metallic elements. To achieve a better understanding of the early events between exposure to inhaled toxicants and the onset of adverse effects on the lung, the characterization of dose at the target organ would be extremely useful. Exhaled breath condensate (EBC), obtained by cooling exhaled air under conditions of spontaneous breathing, is a novel technique that could provide a non-invasive assessment of pulmonary pathobiology. Considering that EBC is water practically free of interfering solutes, it represents an ideal biological matrix for elemental characterization. Published data show that several toxic metals and trace elements are detectable in EBC, raising the possibility of using this medium to quantify the lung tissue dose of pneumotoxic substances. This novel approach may represent a significant advance over the analysis of alternative media (blood, serum, urine, hair), which are not as reliable (owing to interfering substances in the complex matrix) and reflect systemic rather than lung (target tissue) levels of both toxic metals and essential trace elements. Data obtained among workers occupationally exposed to either hard metals or chromium (VI) and in smokers with or without chronic obstructive pulmonary disease (COPD) are reviewed to show that--together with biomarkers of exposure--EBC also allows the simultaneous quantification of biomarkers of effect directly sampled from the epithelial lining fluid, thus providing novel insights on both kinetic and dynamic aspects of metal toxicology.

Cell block samples from malignant pleural effusion might be valid alternative samples for anaplastic lymphoma kinase detection in patients with advanced non-small-cell lung cancer.

PubMed

Zhou, Jianya; Yao, Hongtian; Zhao, Jing; Zhang, Shumeng; You, Qihan; Sun, Ke; Zou, Yinying; Zhou, Caicun; Zhou, Jianying

2015-06-01

To evaluate the clinical value of cell block samples from malignant pleural effusion (MPE) as alternative samples to tumour tissue for anaplastic lymphoma kinase (ALK) detection in patients with advanced non-small-cell lung cancer (NSCLC). Fifty-two matched samples were eligible for analysis. ALK status was detected by Ventana immunohistochemistry (IHC) (with the D5F3 clone), reverse transcription polymerase chain reaction (RT-PCR) and fluorescence in-situ hybridization (FISH) in MPE cell block samples, and by FISH in tumour tissue block samples. In total, ALK FISH results were obtained for 52 tumour tissue samples and 41 MPE cell block samples. Eight cases (15.4%) were ALK-positive in tumour tissue samples by FISH, and among matched MPE cell block samples, five were ALK-positive by FISH, seven were ALK-positive by RT-PCR, and eight were ALK-positive by Ventana IHC. The ALK status concordance rates between tumour tissue and MPE cell block samples were 78.9% by FISH, 98.1% by RT-PCR, and 100% by Ventana IHC. In MPE cell block samples, the sensitivity and specificity of Ventana IHC (100% and 100%) and RT-PCR (87.5% and 100%) were higher than those of FISH (62.5% and 100%). Malignant pleural effusion cell block samples had a diagnostic performance for ALK detection in advanced NSCLC that was comparable to that of tumour tissue samples. MPE cell block samples might be valid alternative samples for ALK detection when tissue is not available. Ventana IHC could be the most suitable method for ALK detection in MPE cell block samples. © 2014 John Wiley & Sons Ltd.

A workflow to preserve genome-quality tissue samples from plants in botanical gardens and arboreta.

PubMed

Gostel, Morgan R; Kelloff, Carol; Wallick, Kyle; Funk, Vicki A

2016-09-01

Internationally, gardens hold diverse living collections that can be preserved for genomic research. Workflows have been developed for genomic tissue sampling in other taxa (e.g., vertebrates), but are inadequate for plants. We outline a workflow for tissue sampling intended for two audiences: botanists interested in genomics research and garden staff who plan to voucher living collections. Standard herbarium methods are used to collect vouchers, label information and images are entered into a publicly accessible database, and leaf tissue is preserved in silica and liquid nitrogen. A five-step approach for genomic tissue sampling is presented for sampling from living collections according to current best practices. Collecting genome-quality samples from gardens is an economical and rapid way to make available for scientific research tissue from the diversity of plants on Earth. The Global Genome Initiative will facilitate and lead this endeavor through international partnerships.

Comparison of hard tissues that are useful for DNA analysis in forensic autopsy.

PubMed

Kaneko, Yu; Ohira, Hiroshi; Tsuda, Yukio; Yamada, Yoshihiro

2015-11-01

Forensic analysis of DNA from hard tissues can be important when investigating a variety of cases resulting from mass disaster or criminal cases. This study was conducted to evaluate the most suitable tissues, method and sample size for processing of hard tissues prior to DNA isolation. We also evaluated the elapsed time after death in relation to the quantity of DNA extracted. Samples of hard tissues (37 teeth, 42 skull, 42 rib, and 39 nails) from 42 individuals aged between 50 and 83 years were used. The samples were taken from remains following forensic autopsy (from 2 days to 2 years after death). To evaluate the integrity of the nuclear DNA isolated, the percentage of allele calls for short tandem repeat profiles were compared between the hard tissues. DNA typing results indicated that until 1 month after death, any of the four hard tissue samples could be used as an alternative to teeth, allowing analysis of all of the loci. However, in terms of the sampling site, collection method and sample size adjustment, the rib appeared to be the best choice in view of the ease of specimen preparation. Our data suggest that the rib could be an alternative hard tissue sample for DNA analysis of human remains. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Correlation between Infectivity and Disease Associated Prion Protein in the Nervous System and Selected Edible Tissues of Naturally Affected Scrapie Sheep

PubMed Central

Chianini, Francesca; Cosseddu, Gian Mario; Steele, Philip; Hamilton, Scott; Hawthorn, Jeremy; Síso, Sílvia; Pang, Yvonne; Finlayson, Jeanie; Eaton, Samantha L.; Reid, Hugh W.; Dagleish, Mark P.; Di Bari, Michele Angelo; D’Agostino, Claudia; Agrimi, Umberto; Terry, Linda; Nonno, Romolo

2015-01-01

The transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative disorders characterised by the accumulation of a pathological form of a host protein known as prion protein (PrP). The validation of abnormal PrP detection techniques is fundamental to allow the use of high-throughput laboratory based tests, avoiding the limitations of bioassays. We used scrapie, a prototype TSE, to examine the relationship between infectivity and laboratory based diagnostic tools. The data may help to optimise strategies to prevent exposure of humans to small ruminant TSE material via the food chain. Abnormal PrP distribution/accumulation was assessed by immunohistochemistry (IHC), Western blot (WB) and ELISA in samples from four animals. In addition, infectivity was detected using a sensitive bank vole bioassay with selected samples from two of the four sheep and protein misfolding cyclic amplification using bank vole brain as substrate (vPMCA) was also carried out in selected samples from one animal. Lymph nodes, oculomotor muscles, sciatic nerve and kidney were positive by IHC, WB and ELISA, although at levels 100–1000 fold lower than the brain, and contained detectable infectivity by bioassay. Tissues not infectious by bioassay were also negative by all laboratory tests including PMCA. Although discrepancies were observed in tissues with very low levels of abnormal PrP, there was an overall good correlation between IHC, WB, ELISA and bioassay results. Most importantly, there was a good correlation between the detection of abnormal PrP in tissues using laboratory tests and the levels of infectivity even when the titre was low. These findings provide useful information for risk modellers and represent a first step toward the validation of laboratory tests used to quantify prion infectivity, which would greatly aid TSE risk assessment policies. PMID:25807559

Characterization of T-cell subsets infiltrating post-burn hypertrophic scar tissues.

PubMed

Castagnoli, C; Trombotto, C; Ondei, S; Stella, M; Calcagni, M; Magliacani, G; Alasia, S T

1997-01-01

In this study, skin-infiltrating cells were characterized in both the active and remission phases of post-burn hypertrophic scar biopsies. Immunohistochemistry examination of active phase samples showed an abundant presence of Langerhans cells, T cells, macrophages, a low presence of natural killer cells and the lack of B lymphocytes. In active hypertrophic scars T lymphocytes infiltrate deep into the superficial dermis and are also observed in the epidermis: CD3+ cells were present at about 222 +/- 107 per 0.25 mm2. In particular the analysis of lymphocyte subpopulations showed that CD4+ T cells predominate in the dermis as well as in the epidermis of active hypertrophic scars whereas CD8+ cells were less well represented (CD4/CD8 ratio is 2.06). This distribution was also shown in remission phase samples and in normotrophic scar specimens, although the lymphocyte number was significantly lower. Approximately 70 per cent of T lymphocytes present in the tissue involved in active phase hypertrophic scar samples were activated (positive with anti-HLA-DR and IL-2 receptor antibodies) which is significantly higher than remission phase hypertrophic and normotrophic scars, in which positivity was 40 and 38 per cent, respectively. Upon activation, the lesional lymphocytes release several cytokines, locally and transiently, that interact with specific receptors in response to different stimulation. Central to the immune hypothesis of hypertrophic scars is that some of the T-cell lymphokines act on keratinocytes, fibroblasts and other cell types to induce changes characteristic of these scars. The presence and close proximity of activated T lymphocytes and antigen-presenting cells of various phenotypes in both the epidermis and dermis of hypertrophic tissues provides strong circumstantial evidence of a local immune response. However, the manner in which T cells achieve and maintain their activated state in hypertrophic tissues is not yet known, and both antigen-dependent and independent mechanisms may contribute.

Residual antibiotics in decontaminated human cardiovascular tissues intended for transplantation and risk of falsely negative microbiological analyses.

PubMed

Buzzi, Marina; Guarino, Anna; Gatto, Claudio; Manara, Sabrina; Dainese, Luca; Polvani, Gianluca; Tóthová, Jana D'Amato

2014-01-01

We investigated the presence of antibiotics in cryopreserved cardiovascular tissues and cryopreservation media, after tissue decontamination with antibiotic cocktails, and the impact of antibiotic residues on standard tissue bank microbiological analyses. Sixteen cardiovascular tissues were decontaminated with bank-prepared cocktails and cryopreserved by two different tissue banks according to their standard operating procedures. Before and after decontamination, samples underwent microbiological analysis by standard tissue bank methods. Cryopreserved samples were tested again with and without the removal of antibiotic residues using a RESEP tube, after thawing. Presence of antibiotics in tissue homogenates and processing liquids was determined by a modified agar diffusion test. All cryopreserved tissue homogenates and cryopreservation media induced important inhibition zones on both Staphylococcus aureus- and Pseudomonas aeruginosa-seeded plates, immediately after thawing and at the end of the sterility test. The RESEP tube treatment markedly reduced or totally eliminated the antimicrobial activity of tested tissues and media. Based on standard tissue bank analysis, 50% of tissues were found positive for bacteria and/or fungi, before decontamination and 2 out of 16 tested samples (13%) still contained microorganisms after decontamination. After thawing, none of the 16 cryopreserved samples resulted positive with direct inoculum method. When the same samples were tested after removal of antibiotic residues, 8 out of 16 (50%) were contaminated. Antibiotic residues present in tissue allografts and processing liquids after decontamination may mask microbial contamination during microbiological analysis performed with standard tissue bank methods, thus resulting in false negatives.

Residual Antibiotics in Decontaminated Human Cardiovascular Tissues Intended for Transplantation and Risk of Falsely Negative Microbiological Analyses

PubMed Central

Gatto, Claudio; Manara, Sabrina; Dainese, Luca; Polvani, Gianluca; Tóthová, Jana D'Amato

2014-01-01

We investigated the presence of antibiotics in cryopreserved cardiovascular tissues and cryopreservation media, after tissue decontamination with antibiotic cocktails, and the impact of antibiotic residues on standard tissue bank microbiological analyses. Sixteen cardiovascular tissues were decontaminated with bank-prepared cocktails and cryopreserved by two different tissue banks according to their standard operating procedures. Before and after decontamination, samples underwent microbiological analysis by standard tissue bank methods. Cryopreserved samples were tested again with and without the removal of antibiotic residues using a RESEP tube, after thawing. Presence of antibiotics in tissue homogenates and processing liquids was determined by a modified agar diffusion test. All cryopreserved tissue homogenates and cryopreservation media induced important inhibition zones on both Staphylococcus aureus- and Pseudomonas aeruginosa-seeded plates, immediately after thawing and at the end of the sterility test. The RESEP tube treatment markedly reduced or totally eliminated the antimicrobial activity of tested tissues and media. Based on standard tissue bank analysis, 50% of tissues were found positive for bacteria and/or fungi, before decontamination and 2 out of 16 tested samples (13%) still contained microorganisms after decontamination. After thawing, none of the 16 cryopreserved samples resulted positive with direct inoculum method. When the same samples were tested after removal of antibiotic residues, 8 out of 16 (50%) were contaminated. Antibiotic residues present in tissue allografts and processing liquids after decontamination may mask microbial contamination during microbiological analysis performed with standard tissue bank methods, thus resulting in false negatives. PMID:25397402

Advancing Optical Imaging for Breast Margin Assessment: An Analysis of Excisional Time, Cautery, and Patent Blue Dye on Underlying Sources of Contrast

PubMed Central

Bydlon, Torre M.; Barry, William T.; Kennedy, Stephanie A.; Brown, J. Quincy; Gallagher, Jennifer E.; Wilke, Lee G.; Geradts, Joseph; Ramanujam, Nimmi

2012-01-01

Breast conserving surgery (BCS) is a recommended treatment for breast cancer patients where the goal is to remove the tumor and a surrounding rim of normal tissue. Unfortunately, a high percentage of patients return for additional surgeries to remove all of the cancer. Post-operative pathology is the gold standard for evaluating BCS margins but is limited due to the amount of tissue that can be sampled. Frozen section analysis and touch-preparation cytology have been proposed to address the surgical needs but also have sampling limitations. These issues represent an unmet clinical need for guidance in resecting malignant tissue intra-operatively and for pathological sampling. We have developed a quantitative spectral imaging device to examine margins intra-operatively. The context in which this technology is applied (intra-operative or post-operative setting) is influenced by time after excision and surgical factors including cautery and the presence of patent blue dye (specifically Lymphazurin™, used for sentinel lymph node mapping). Optical endpoints of hemoglobin ([THb]), fat ([β-carotene]), and fibroglandular content via light scattering (<µs’>) measurements were quantified from diffuse reflectance spectra of lumpectomy and mastectomy specimens using a Monte Carlo model. A linear longitudinal mixed-effects model was used to fit the optical endpoints for the cautery and kinetics studies. Monte Carlo simulations and tissue mimicking phantoms were used for the patent blue dye experiments. [THb], [β-carotene], and <µs’> were affected by <3.3% error with <80 µM of patent blue dye. The percent change in [β-carotene], <µs’>, and [β-carotene]/<µs’> was <14% in 30 minutes, while percent change in [THb] was >40%. [β-carotene] and [β-carotene]/<µs’> were the only parameters not affected by cautery. This work demonstrates the importance of understanding the post-excision kinetics of ex-vivo tissue and the presence of cautery and patent blue dye for breast tumor margin assessment, to accurately interpret data and exploit underling sources of contrast. PMID:23251526

Impact assessment of repeated exposure of organotypic 3D bronchial and nasal tissue culture models to whole cigarette smoke.

PubMed

Kuehn, Diana; Majeed, Shoaib; Guedj, Emmanuel; Dulize, Remi; Baumer, Karine; Iskandar, Anita; Boue, Stephanie; Martin, Florian; Kostadinova, Radina; Mathis, Carole; Ivanov, Nikolai V; Frentzel, Stefan; Hoeng, Julia; Peitsch, Manuel C

2015-02-12

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

Correlation of MLH1 and MGMT methylation levels between peripheral blood leukocytes and colorectal tissue DNA samples in colorectal cancer patients.

PubMed

Li, Xia; Wang, Yibaina; Zhang, Zuoming; Yao, Xiaoping; Ge, Jie; Zhao, Yashuang

2013-11-01

CpG island methylation in the promoter regions of the DNA mismatch repair gene mutator L homologue 1 ( MLH1 ) and DNA repair gene O 6 -methylguanine-DNA methyltransferase ( MGMT ) genes has been shown to occur in the leukocytes of peripheral blood and colorectal tissue. However, it is unclear whether the methylation levels in the blood leukocytes and colorectal tissue are correlated. The present study analyzed and compared the levels of MGMT and MLH1 gene methylation in the leukocytes of peripheral blood and colorectal tissues obtained from patients with colorectal cancer (CRC). The methylation levels of MGMT and MLH1 were examined using methylation-sensitive high-resolution melting (MS-HRM) analysis. A total of 44 patients with CRC were selected based on the MLH1 and MGMT gene methylation levels in the leukocytes of the peripheral blood. Corresponding colorectal tumor and normal tissues were obtained from each patient and the DNA methylation levels were determined. The correlation coefficients were evaluated using Spearman's rank test. Agreement was determined by generalized κ-statistics. Spearman's rank correlation coefficients (r) for the methylation levels of the MGMT and MLH1 genes in the leukocytes of the peripheral blood and normal colorectal tissue were 0.475 and 0.362, respectively (P=0.001 and 0.016, respectively). The agreement of the MGMT and MLH1 gene methylation levels in the leukocytes of the peripheral blood and normal colorectal tissue were graded as fair and poor (κ=0.299 and 0.126, respectively). The methylation levels of MGMT and MLH1 were moderately and weakly correlated between the patient-matched leukocytes and the normal colorectal tissue, respectively. Blood-derived DNA methylation measurements may not always represent the levels of normal colorectal tissue methylation.

Fit for purpose frozen tissue collections by RNA integrity number-based quality control assurance at the Erasmus MC tissue bank.

PubMed

Kap, Marcel; Oomen, Monique; Arshad, Shazia; de Jong, Bas; Riegman, Peter

2014-04-01

About 5000 frozen tissue samples are collected each year by the Erasmus Medical Center tissue bank. Two percent of these samples are randomly selected annually for RNA isolation and RNA Integrity Number (RIN) measurement. A similar quality assessment was conducted during centralization of a 20-year-old tissue collection from the cancer institute, a 15-year-old liver sample archive (-80°C), and a 13-year-old clinical pathology frozen biopsy archive (Liquid Nitrogen). Samples were divided into either high-quality (RIN ≥6.5) or low-quality overall categories, or into four "fit-for-purpose" quality groups: RIN <5: not reliable for demanding downstream analysis; 5 ≤RIN <6: suitable for RT-qPCR; 6 ≤RIN <8: suitable for gene array analysis; and RIN ≥8: suitable for all downstream techniques. In general, low RIN values were correlated with fatty, fibrous, pancreatic, or necrotic tissue. When the percentage of samples with RIN ≥6.5 is higher than 90%, the tissue bank performance is adequate. The annual 2011 quality control assessment showed that 90.3% (n=93) of all samples had acceptable RIN values; 97.4% (n=39) of the cancer institute collection had RIN values above 6.5; and 88.6% (n=123) of samples from the liver sample archive collection had RIN values higher than 6.5. As the clinical pathology biopsy collection contained only 58.8% (n=24) acceptable samples, the procurement protocols used for these samples needed immediate evaluation. When the distribution of RIN values of the different collections were compared, no significant differences were found, despite differences in average storage time and temperature. According to the principle of "fit-for-purpose" distribution, the vast majority of samples are considered good enough for most downstream techniques. In conclusion, an annual tissue bank quality control procedure provides useful information on tissue sample quality and sheds light on where and if improvements need to be made.

Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing.

PubMed

Andreotti, Renato; Pérez de León, Adalberto A; Dowd, Scot E; Guerrero, Felix D; Bendele, Kylie G; Scoles, Glen A

2011-01-06

Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus. This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts are expected as our understanding of its microbiota expands. Increased awareness of the role R. microplus can play in the transmission of pathogenic bacteria will enhance our ability to mitigate its economic impact on animal agriculture globally. This recognition should be included as part of analyses to assess the risk for re-invasion of areas like the United States of America where R. microplus was eradicated.

[Tuberculosis lymphadenitis in slaughtered swine from the State of São Paulo, Brazil: microscopic histopathology and demonstration of mycobacteria].

PubMed

Balian, S C; Ribeiro, P; Vasconcellos, S A; Pinheiro, S R; Ferreira Neto, J S; Guerra, J L; Xavier, J G; Morais, Z M; Telles, M A

1997-08-01

As the occurrence of tuberculosis lymphadenitis in swine constitutes a public health risk, especially in immunosuppressed individuals, the distribution of tuberculoid lesions and the presence of microbacteria in lymph nodes and hepatic and muscular tissue in swine for slaughter, in the State of S. Paulo, SP (Brazil), in the period from 1993 to 1994, were studied. MATERIAL E METHOD: Tuberculous lesions in 60 carcasses of swine, slaughtered at abattoirs during the period 1993-1994, were studied. When macroscopic lesions were observed, a representative sampling of lymph nodes, hepatic and muscular tissues (masseter and diaphragm) were examined using histopathological and microbacterial isolation techniques. The macroscopic lesions were found predominantly in mesenteric lymph nodes. The MAI Complex (M.avium-intracellulare) was isolated mainly in the carcass group that showed lesions and was not found in the control group. Microorganisms of the MAI Complex were not isolated from hepatic and muscular tissues. No clear relationship between the type of mycobacteria isolated and the macroscopic lesions observed during the carcass inspection was found.

Pro-inflammatory cytokine levels in human apical periodontitis: Correlation with clinical and histological findings.

PubMed

Jakovljevic, Aleksandar; Knezevic, Aleksandra; Karalic, Danijela; Soldatovic, Ivan; Popovic, Branka; Milasin, Jelena; Andric, Miroslav

2015-08-01

This study aimed to compare the levels of tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) between apical periodontitis lesions with different clinical and histological features. Based on clinical data and history of disease, 100 human apical periodontitis lesions were categorised as either asymptomatic or symptomatic lesions. According to histological examination, lesions were divided into periapical granulomas and radicular cysts. Pulp tissues of 25 impacted wisdom teeth were used as controls. Homogenised tissue samples were centrifuged and supernatants were used for the determination of cytokine levels by enzyme-linked immunosorbent assay. Significantly higher levels of IL-1β and IL-6 were found in symptomatic lesions compared with asymptomatic lesions and control tissues (P < 0.001, P < 0.001, respectively). The concentration of IL-1β was significantly higher in radicular cysts compared with periapical granulomas (P = 0.003). Symptomatic lesions, as judged by high local production of IL-1β and IL-6, represent an immunologically active stage of the disease. © 2014 Australian Society of Endodontology.

Arsenic and mercury concentrations in major landscape components of an intensively cultivated watershed.

PubMed

Cooper, C M; Gillespie, W B

2001-01-01

To provide an understanding of arsenic (As) and mercury (Hg) concentrations in soil, sediment, water, and fish tissues, samples were collected from a Mississippi River alluvial floodplain located in northwest Mississippi. As concentrations increased approximately an order of magnitude from water (5.12 micrograms/l) to fish tissues (36.99 micrograms/kg) and an additional two orders of magnitude in soils, lake sediments, and wetland sediments (5728, 5614, and 6746 micrograms/kg), respectively. Average Hg concentrations in water, soils, lake sediments, and fish were 2.16 micrograms/l, 55.1, 14.5 and 125 micrograms/kg, respectively. As and Hg concentrations were within published ranges for uncontaminated soil, water, and sediments. As concentrations represented a low risk. Hg concentrations were also low but showed a greater tendency to concentrate in fish tissue. The dominant mode of entry of these materials into aquatic systems is through storm-generated runoff. Since both metals accompany sediments, agricultural conservation practices such as reduced tillage, buffer riparian strips, and bordering sediment ponds or drainage wetlands will minimize watershed input to aquatic systems.

Functional Integrative Levels in the Human Interactome Recapitulate Organ Organization

PubMed Central

Prieto, Carlos; Benkahla, Alia; De Las Rivas, Javier; Brun, Christine

2011-01-01

Interactome networks represent sets of possible physical interactions between proteins. They lack spatio-temporal information by construction. However, the specialized functions of the differentiated cell types which are assembled into tissues or organs depend on the combinatorial arrangements of proteins and their physical interactions. Is tissue-specificity, therefore, encoded within the interactome? In order to address this question, we combined protein-protein interactions, expression data, functional annotations and interactome topology. We first identified a subnetwork formed exclusively of proteins whose interactions were observed in all tested tissues. These are mainly involved in housekeeping functions and are located at the topological center of the interactome. This ‘Largest Common Interactome Network’ represents a ‘functional interactome core’. Interestingly, two types of tissue-specific interactions are distinguished when considering function and network topology: tissue-specific interactions involved in regulatory and developmental functions are central whereas tissue-specific interactions involved in organ physiological functions are peripheral. Overall, the functional organization of the human interactome reflects several integrative levels of functions with housekeeping and regulatory tissue-specific functions at the center and physiological tissue-specific functions at the periphery. This gradient of functions recapitulates the organization of organs, from cells to organs. Given that several gradients have already been identified across interactomes, we propose that gradients may represent a general principle of protein-protein interaction network organization. PMID:21799769

Small non-coding RNA profiling in human biofluids and surrogate tissues from healthy individuals: description of the diverse and most represented species.

PubMed

Ferrero, Giulio; Cordero, Francesca; Tarallo, Sonia; Arigoni, Maddalena; Riccardo, Federica; Gallo, Gaetano; Ronco, Guglielmo; Allasia, Marco; Kulkarni, Neha; Matullo, Giuseppe; Vineis, Paolo; Calogero, Raffaele A; Pardini, Barbara; Naccarati, Alessio

2018-01-09

The role of non-coding RNAs in different biological processes and diseases is continuously expanding. Next-generation sequencing together with the parallel improvement of bioinformatics analyses allows the accurate detection and quantification of an increasing number of RNA species. With the aim of exploring new potential biomarkers for disease classification, a clear overview of the expression levels of common/unique small RNA species among different biospecimens is necessary. However, except for miRNAs in plasma, there are no substantial indications about the pattern of expression of various small RNAs in multiple specimens among healthy humans. By analysing small RNA-sequencing data from 243 samples, we have identified and compared the most abundantly and uniformly expressed miRNAs and non-miRNA species of comparable size with the library preparation in four different specimens (plasma exosomes, stool, urine, and cervical scrapes). Eleven miRNAs were commonly detected among all different specimens while 231 miRNAs were globally unique across them. Classification analysis using these miRNAs provided an accuracy of 99.6% to recognize the sample types. piRNAs and tRNAs were the most represented non-miRNA small RNAs detected in all specimen types that were analysed, particularly in urine samples. With the present data, the most uniformly expressed small RNAs in each sample type were also identified. A signature of small RNAs for each specimen could represent a reference gene set in validation studies by RT-qPCR. Overall, the data reported hereby provide an insight of the constitution of the human miRNome and of other small non-coding RNAs in various specimens of healthy individuals.

Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast.

PubMed

Kao, Ya-Ting; Zhu, Xinxin; Xu, Fang; Min, Wei

2012-08-01

Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit. Herein we significantly improve the image contrast for deep scattering samples by harnessing reversibly switchable fluorescent proteins (RSFPs) which can be cycled between bright and dark states upon light illumination. Two distinct techniques, multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI), are demonstrated on tissue phantoms labeled with Dronpa protein. Such a focal switch approach can generate pseudo background-free images. Conceptually different from wave-based approaches that try to reduce light scattering in turbid samples, our work represents a molecule-based strategy that focused on imaging probes.

Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast

PubMed Central

Kao, Ya-Ting; Zhu, Xinxin; Xu, Fang; Min, Wei

2012-01-01

Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit. Herein we significantly improve the image contrast for deep scattering samples by harnessing reversibly switchable fluorescent proteins (RSFPs) which can be cycled between bright and dark states upon light illumination. Two distinct techniques, multiphoton deactivation and imaging (MPDI) and multiphoton activation and imaging (MPAI), are demonstrated on tissue phantoms labeled with Dronpa protein. Such a focal switch approach can generate pseudo background-free images. Conceptually different from wave-based approaches that try to reduce light scattering in turbid samples, our work represents a molecule-based strategy that focused on imaging probes. PMID:22876358

Organotins in fish muscle and liver from the Polish coast of the Baltic Sea: Is the total ban successful?

PubMed

Filipkowska, Anna; Złoch, Ilona; Wawrzyniak-Wydrowska, Brygida; Kowalewska, Grażyna

2016-10-15

Muscle and liver tissues of nine fish species were analyzed to assess butyltin and phenyltin contamination. The samples were collected from three basins located in the Southern Baltic Sea coastal zone that each represent different potential for organotin pollution. Maximum total concentrations of butyltin compounds (BTs) in the fish muscles and livers were 715 and 1132ng Sn g(-1) d.w., respectively, whereas triphenyltin (TPhT) was not detected. In the muscle samples, the predominant compound in the sum of butyltins was tributyltin (TBT), while in the liver samples, tributyltin degradation products were found in the majority. The results demonstrate that 6-7years after the implementation of the total ban on harmful organotin use in antifouling paints, butyltins remain present in fishes from the Polish coast of the Baltic Sea. According to the HELCOM recommendation, eight samples exceeded the good environmental status boundary for tributyltin in seafood. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines

PubMed Central

2010-01-01

Introduction Normal and neoplastic breast tissues are comprised of heterogeneous populations of epithelial cells exhibiting various degrees of maturation and differentiation. While cultured cell lines have been derived from both normal and malignant tissues, it remains unclear to what extent they retain similar levels of differentiation and heterogeneity as that found within breast tissues. Methods We used 12 reduction mammoplasty tissues, 15 primary breast cancer tissues, and 20 human breast epithelial cell lines (16 cancer lines, 4 normal lines) to perform flow cytometry for CD44, CD24, epithelial cell adhesion molecule (EpCAM), and CD49f expression, as well as immunohistochemistry, and in vivo tumor xenograft formation studies to extensively analyze the molecular and cellular characteristics of breast epithelial cell lineages. Results Human breast tissues contain four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) that differ on the basis of CD24, EpCAM and CD49f expression. Primary human breast cancer tissues also contain these four cellular states, but in altered proportions compared to normal tissues. In contrast, cultured cancer cell lines are enriched for rare basal and mesenchymal epithelial phenotypes, which are normally present in small numbers within human tissues. Similarly, cultured normal human mammary epithelial cell lines are enriched for rare basal and mesenchymal phenotypes that represent a minor fraction of cells within reduction mammoplasty tissues. Furthermore, although normal human mammary epithelial cell lines exhibit features of bi-potent progenitor cells they are unable to differentiate into mature luminal breast epithelial cells under standard culture conditions. Conclusions As a group breast cancer cell lines represent the heterogeneity of human breast tumors, but individually they exhibit increased lineage-restricted profiles that fall short of truly representing the intratumoral heterogeneity of individual breast tumors. Additionally, normal human mammary epithelial cell lines fail to retain much of the cellular diversity found in human breast tissues and are enriched for differentiation states that are a minority in breast tissues, although they do exhibit features of bi-potent basal progenitor cells. These findings suggest that collections of cell lines representing multiple cell types can be used to model the cellular heterogeneity of tissues. PMID:20964822

Human adipose-derived stem cells: definition, isolation, tissue-engineering applications.

PubMed

Nae, S; Bordeianu, I; Stăncioiu, A T; Antohi, N

2013-01-01

Recent researches have demonstrated that the most effective repair system of the body is represented by stem cells - unspecialized cells, capable of self-renewal through successive mitoses, which have also the ability to transform into different cell types through differentiation. The discovery of adult stem cells represented an important step in regenerative medicine because they no longer raises ethical or legal issues and are more accessible. Only in 2002, stem cells isolated from adipose tissue were described as multipotent stem cells. Adipose tissue stem cells benefits in tissue engineering and regenerative medicine are numerous. Development of adipose tissue engineering techniques offers a great potential in surpassing the existing limits faced by the classical approaches used in plastic and reconstructive surgery. Adipose tissue engineering clinical applications are wide and varied, including reconstructive, corrective and cosmetic procedures. Nowadays, adipose tissue engineering is a fast developing field, both in terms of fundamental researches and medical applications, addressing issues related to current clinical pathology or trauma management of soft tissue injuries in different body locations.

Viability and infectivity of Ichthyophonus sp. in post-mortem Pacific herring, Clupea pallasii

USGS Publications Warehouse

Kocan, Richard M.; Hart, Lucas M.; Lewandowski, Naomi; Hershberger, Paul

2014-01-01

Ichthyophonus-infected Pacific herring, Clupea pallasii, were allowed to decompose in ambient seawater then serially sampled for 29 days to evaluate parasite viability and infectivity for Pacific staghorn sculpin, Leptocottus armatus. Ichthyophonus sp. was viable in decomposing herring tissues for at least 29 days post-mortem and could be transmitted via ingestion to sculpin for up to 5 days. The parasite underwent morphologic changes during the first 48 hr following death of the host that were similar to those previously reported, but as host tissue decomposition progressed, several previously un-described forms of the parasite were observed. The significance of long-term survival and continued morphologic transformation in the post-mortem host is unknown, but it could represent a saprozoic phase of the parasite life cycle that has survival value for Ichthyophonus sp.

Textural analysis of optical coherence tomography skin images: quantitative differentiation between healthy and cancerous tissues

NASA Astrophysics Data System (ADS)

Adabi, Saba; Conforto, Silvia; Hosseinzadeh, Matin; Noe, Shahryar; Daveluy, Steven; Mehregan, Darius; Nasiriavanaki, Mohammadreza

2017-02-01

Optical Coherence Tomography (OCT) offers real-time high-resolution three-dimensional images of tissue microstructures. In this study, we used OCT skin images acquired from ten volunteers, neither of whom had any skin conditions addressing the features of their anatomic location. OCT segmented images are analyzed based on their optical properties (attenuation coefficient) and textural image features e.g., contrast, correlation, homogeneity, energy, entropy, etc. Utilizing the information and referring to their clinical insight, we aim to make a comprehensive computational model for the healthy skin. The derived parameters represent the OCT microstructural morphology and might provide biological information for generating an atlas of normal skin from different anatomic sites of human skin and may allow for identification of cell microstructural changes in cancer patients. We then compared the parameters of healthy samples with those of abnormal skin and classified them using a linear Support Vector Machines (SVM) with 82% accuracy.

Breast Angiosarcoma: Case Series and Expression of Vascular Endothelial Growth Factor

PubMed Central

Brar, Rondeep; West, Robert; Witten, Daniela; Raman, Bhargav; Jacobs, Charlotte; Ganjoo, Kristen

2009-01-01

Purpose Angiosarcoma of the breast is a rare, malignant tumor for which little is known regarding prognostic indicators and optimal therapeutic regimens. To address this issue, we performed a retrospective analysis of breast angiosarcoma cases seen at Stanford University along with immunohistochemical analysis for markers of angiogenesis. Methods Breast angiosarcoma cases seen between 1980 and 2008 were examined. Viable tissue blocks were analyzed for expression of vascular endothelial growth factor and its receptors. Results A total of 16 cases were identified. Data was collected regarding epidemiology, treatment, response rates, disease-free survival, and the use of various imaging modalities. Five tissue blocks remained viable for immunohistochemical analysis. Vascular endothelial growth factor-A was positively expressed in 3 of these samples. Conclusion Angiosarcoma of the breast is an aggressive malignancy with a propensity for both local recurrence and distant metastases. Angiogenesis inhibition may represent a novel therapeutic modality in this rare, vascular malignancy. PMID:20737044

Metals in Bone Tissue of Antillean Manatees from the Gulf of Mexico and Chetumal Bay, Mexico.

PubMed

Romero-Calderón, Ana G; Morales-Vela, Benjamin; Rosíles-Martínez, René; Olivera-Gómez, León D; Delgado-Estrella, Alberto

2016-01-01

Concentrations of seven metals (As, Cd, Cr, Cu, Pb, Ni, and Zn) were analyzed in 33 bone tissue samples of Antillean manatees (Trichechus manatus manatus) found dead in lagoons and rivers of Tabasco and Campeche in the Gulf of Mexico and Chetumal Bay in the Caribbean region. The concentrations of Cr, Cu, Pb, and Zn were significantly different between regions, with greater levels found in the Gulf of Mexico group than in the Mexican Caribbean group (p < 0.05). Pb concentrations differed significantly between adults and calves. No differences were observed between sexes. Metal concentrations detected in the manatee bones were higher than most of those reported for bones in other marine mammals around the world. Future studies are necessary to establish whether the metal concentrations represent a risk to the health of the species.

Tissue microarrays and digital image analysis.

PubMed

Ryan, Denise; Mulrane, Laoighse; Rexhepaj, Elton; Gallagher, William M

2011-01-01

Tissue microarrays (TMAs) have recently emerged as very valuable tools for high-throughput pathological assessment, especially in the cancer research arena. This important technology, however, has yet to fully penetrate into the area of toxicology. Here, we describe the creation of TMAs representative of samples produced from conventional toxicology studies within a large-scale, multi-institutional pan-European project, PredTox. PredTox, short for Predictive Toxicology, formed part of an EU FP6 Integrated Project, Innovative Medicines for Europe (InnoMed), and aimed to study pre-clinically 16 compounds of known liver and/or kidney toxicity. In more detail, TMAs were constructed from materials corresponding to the full face sections of liver and kidney from rats treated with different drug candidates by members of the consortium. We also describe the process of digital slide scanning of kidney and liver sections, in the context of creating an online resource of histopathological data.

Comparison of rapid diagnostic tests to detect Mycobacterium avium subsp. paratuberculosis disseminated infection in bovine liver.

PubMed

Zarei, Mehdi; Ghorbanpour, Masoud; Tajbakhsh, Samaneh; Mosavari, Nader

2017-08-01

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, a chronic enteritis in cattle and other domestic and wild ruminants. The presence of MAP in tissues other than intestines and associated lymph nodes, such as meat and liver, is a potential public health concern. In the present study, the relationship between the results of rapid diagnostic tests of the Johne's disease, such as serum ELISA, rectal scraping PCR, and acid-fast staining, and the presence of MAP in liver was evaluated. Blood, liver, and rectal scraping samples were collected from 200 slaughtered cattle with unknown Johne's disease status. ELISA was performed to determine the MAP antibody activity in the serum. Acid-fast staining was performed on rectal scraping samples, and PCR was performed on rectal scraping and liver samples. PCR-positive liver samples were used for mycobacterial culture. Overall, the results of this study demonstrated that MAP can be detected and cultured from liver of slaughtered cattle and rapid diagnostic tests of Johne's disease have limited value in detecting cattle with MAP infection in liver. These findings show that the presence of MAP in liver tissue may occur in cows with negative results for rapid diagnostic tests and vice versa. Hence, liver might represent another possible risk of human exposure to MAP. Given concerns about a potential zoonotic role for MAP, these results show the necessity to find new methods for detecting cattle with MAP disseminated infection.

Mitochondrial and glycolytic metabolic compartmentalization in diffuse large B-cell lymphoma.

PubMed

Gooptu, Mahasweta; Whitaker-Menezes, Diana; Sprandio, John; Domingo-Vidal, Marina; Lin, Zhao; Uppal, Guldeep; Gong, Jerald; Fratamico, Roberto; Leiby, Benjamin; Dulau-Florea, Alina; Caro, Jaime; Martinez-Outschoorn, Ubaldo

2017-06-01

Metabolic heterogeneity between neoplastic cells and surrounding stroma has been described in several epithelial malignancies; however, the metabolic phenotypes of neoplastic lymphocytes and neighboring stroma in diffuse large B-cell lymphoma (DLBCL) is unknown. We investigated the metabolic phenotypes of human DLBCL tumors by using immunohistochemical markers of glycolytic and mitochondrial oxidative phosphorylation (OXPHOS) metabolism. The lactate importer MCT4 is a marker of glycolysis, whereas the lactate importer MCT1 and TOMM20 are markers of OXPHOS metabolism. Staining patterns were assessed in 33 DLBCL samples as well as 18 control samples (non-neoplastic lymph nodes). TOMM20 and MCT1 were highly expressed in neoplastic lymphocytes, indicating an OXPHOS phenotype, whereas non-neoplastic lymphocytes in the control samples did not express these markers. Stromal cells in DLBCL samples strongly expressed MCT4, displaying a glycolytic phenotype, a feature not seen in stromal elements of non-neoplastic lymphatic tissue. Furthermore, the differential expression of lactate exporters (MCT4) on tumor-associated stroma and lactate importers (MCT1) on neoplastic lymphocytes support the hypothesis that neoplastic cells are metabolically linked to the stroma likely via mutually beneficial reprogramming. MCT4 is a marker of tumor-associated stroma in neoplastic tissue. Our findings suggest that disruption of neoplastic-stromal cell metabolic heterogeneity including MCT1 and MCT4 blockade should be studied to determine if it could represent a novel treatment target in DLBCL. Copyright © 2017 Elsevier Inc. All rights reserved.

Community structures and antagonistic activities of the bacteria associated with surface-sterilized pepper plants grown in different field soils.

PubMed

Kang, Sin Ae; Han, Jae Woo; Kim, Beom Seok

2016-12-01

Endophytic bacteria may act individually or in consortia in controlling certain plant diseases. In this study, pepper plants (Capsicum annuum L. cv. Nokkwang) were cultivated in glasshouse conditions using field soils collected from two different geographic locations, Deokso (DS) and Gwangyang (GY) in Korea. Community structure and antifungal activity of pepper endophytic bacteria were analyzed using culture-independent (PCR-DGGE) and culture-dependent (plating) methods, respectively. Dissimilarities were observed between DGGE profiles of DS and GY samples at all plant tissues. However, sequencing of the major DGGE bands revealed an enrichment of Firmicutes in the leaves of plants propagated in either soil. Similar results were observed with the culturable assays. Firmicutes dominated the isolates from both leaf samples, DS leaf (100 %) and GY leaf (83.3 %), although the genus compositions of DS leaf and GY leaf isolates were different. We assessed the antifungal activity of each isolate recovered to better understand the potential role that these endophytic bacteria may play. Of the 27 representative isolates from DS plant samples, 17 isolates (63.0 %) had antagonistic activity against at least one of the fungi tested. Seventeen isolates from GY plant samples (58.6 %) displayed antagonistic properties. The results show that the endophytic communities differ in the same plant species when propagated in different soils. Exploring the internal tissues of plants growing in diverse soil environments could be a way to find potential candidates for biocontrol agents.

Edema fluid accumulation within necrotic brain tissue as a cause of the mass effect of cerebral contusion in head trauma patients.

PubMed

Katayama, Y; Kawamata, T

2003-01-01

The early massive edema caused by severe cerebral contusion results in progressive intracranial pressure (ICP) elevation and clinical deterioration within 24-72 hours post-trauma. Surgical excision of the necrotic brain tissue represents the only therapy, which can provide satisfactory control of the elevated ICP and clinical deterioration. In order to elucidate the mechanisms underlying the early massive edema, we have carried out a series of detailed clinical studies. Diffusion magnetic resonance (MR) imaging and apparent diffusion co-efficient (ADC) mapping suggest that cells in the central area of contusion undergo shrinkage, disintegration and homogenization, whereas cellular swelling is predominant in the peripheral area during the period of 24-72 hours post-trauma. The ADC values in the central and peripheral areas are maximally dissociated during this period. A large amount of edema fluid accumulates within the necrotic brain tissue of the central area beginning at approximately 24 hours post-trauma. We have found that fluid-blood interface formation within the central area does not represent an uncommon finding in various neuroimaging examinations of cerebral contusions, indicating layering of red blood cells within the necrotic brain tissue accumulating voluminous edema fluid. Intravenous slow infusion of gadolinium-DTPA and delayed MR imaging revealed that the central area of contusion can be enhanced at 24-48 hours post-trauma. implying that water supply from the blood vessels is not completely interrupted. Necrotic brain tissue sampled from the central area of contusion during surgery demonstrates a very high osmolality. It appears that the capacitance for edema fluid accumulation increases in the central area, whereas cellular swelling in the peripheral area elevates the resistance for edema fluid propagation. Combination of these circumstances may facilitate edema fluid accumulation in the central area. We also suggest that the dissociation of ADC values and high osmolality within the necrotic brain tissue may generate an osmotic potential across the central and peripheral areas and contribute to the early massive edema caused by cerebral contusion.

A workflow to preserve genome-quality tissue samples from plants in botanical gardens and arboreta1

PubMed Central

Gostel, Morgan R.; Kelloff, Carol; Wallick, Kyle; Funk, Vicki A.

2016-01-01

Premise of the study: Internationally, gardens hold diverse living collections that can be preserved for genomic research. Workflows have been developed for genomic tissue sampling in other taxa (e.g., vertebrates), but are inadequate for plants. We outline a workflow for tissue sampling intended for two audiences: botanists interested in genomics research and garden staff who plan to voucher living collections. Methods and Results: Standard herbarium methods are used to collect vouchers, label information and images are entered into a publicly accessible database, and leaf tissue is preserved in silica and liquid nitrogen. A five-step approach for genomic tissue sampling is presented for sampling from living collections according to current best practices. Conclusions: Collecting genome-quality samples from gardens is an economical and rapid way to make available for scientific research tissue from the diversity of plants on Earth. The Global Genome Initiative will facilitate and lead this endeavor through international partnerships. PMID:27672517

Histopathology of Middle East respiratory syndrome coronovirus (MERS-CoV) infection - clinicopathological and ultrastructural study.

PubMed

Alsaad, Khaled O; Hajeer, Ali H; Al Balwi, Mohammed; Al Moaiqel, Mohammed; Al Oudah, Nourah; Al Ajlan, Abdulaziz; AlJohani, Sameera; Alsolamy, Sami; Gmati, Giamal E; Balkhy, Hanan; Al-Jahdali, Hamdan H; Baharoon, Salim A; Arabi, Yaseen M

2018-02-01

The pathogenesis, viral localization and histopathological features of Middle East respiratory syndrome - coronavirus (MERS-CoV) in humans are not described sufficiently. The aims of this study were to explore and define the spectrum of histological and ultrastructural pathological changes affecting various organs in a patient with MERS-CoV infection and represent a base of MERS-CoV histopathology. We analysed the post-mortem histopathological findings and investigated localisation of viral particles in the pulmonary and extrapulmonary tissue by transmission electron microscopic examination in a 33-year-old male patient of T cell lymphoma, who acquired MERS-CoV infection. Tissue needle biopsies were obtained from brain, heart, lung, liver, kidney and skeletal muscle. All samples were collected within 45 min from death to reduce tissue decomposition and artefact. Histopathological examination showed necrotising pneumonia, pulmonary diffuse alveolar damage, acute kidney injury, portal and lobular hepatitis and myositis with muscle atrophic changes. The brain and heart were histologically unremarkable. Ultrastructurally, viral particles were localised in the pneumocytes, pulmonary macrophages, renal proximal tubular epithelial cells and macrophages infiltrating the skeletal muscles. The results highlight the pulmonary and extrapulmonary pathological changes of MERS-CoV infection and provide the first evidence of the viral presence in human renal tissue, which suggests tissue trophism for MERS-CoV in kidney. © 2017 John Wiley & Sons Ltd.

Microarray analysis of port wine stains before and after pulsed dye laser treatment.

PubMed

Laquer, Vivian T; Hevezi, Peter A; Albrecht, Huguette; Chen, Tina S; Zlotnik, Albert; Kelly, Kristen M

2013-02-01

Neither the pathogenesis of port wine stain (PWS) birthmarks nor tissue effects of pulsed dye laser (PDL) treatment of these lesions is fully understood. There are few published reports utilizing gene expression analysis in human PWS skin. We aim to compare gene expression in PWS before and after PDL, using DNA microarrays that represent most, if not all, human genes to obtain comprehensive molecular profiles of PWS lesions and PDL-associated tissue effects. Five human subjects had PDL treatment of their PWS. One week later, three biopsies were taken from each subject: normal skin (N); untreated PWS (PWS); PWS post-PDL (PWS + PDL). Samples included two lower extremity lesions, two facial lesions, and one facial nodule. High-quality total RNA isolated from skin biopsies was processed and applied to Affymetrix Human gene 1.0ST microarrays for gene expression analysis. We performed a 16 pair-wise comparison identifying either up- or down-regulated genes between N versus PWS and PWS versus PWS + PDL for four of the donor samples. The PWS nodule (nPWS) was analyzed separately. There was significant variation in gene expression profiles between individuals. By doing pair-wise comparisons between samples taken from the same donor, we were able to identify genes that may participate in the formation of PWS lesions and PDL tissue effects. Genes associated with immune, epidermal, and lipid metabolism were up-regulated in PWS skin. The nPWS exhibited more profound differences in gene expression than the rest of the samples, with significant differential expression of genes associated with angiogenesis, tumorigenesis, and inflammation. In summary, gene expression profiles from N, PWS, and PWS + PDL demonstrated significant variation within samples from the same donor and between donors. By doing pair-wise comparisons between samples taken from the same donor and comparing these results between donors, we were able to identify genes that may participate in formation of PWS and PDL effects. Our preliminary results indicate changes in gene expression of angiogenesis-related genes, suggesting that dysregulation of angiogenic signals and/or components may contribute to PWS pathology. Copyright © 2012 Wiley Periodicals, Inc.

Intrinsic Cell Stress is Independent of Organization in Engineered Cell Sheets.

PubMed

van Loosdregt, Inge A E W; Dekker, Sylvia; Alford, Patrick W; Oomens, Cees W J; Loerakker, Sandra; Bouten, Carlijn V C

2018-06-01

Understanding cell contractility is of fundamental importance for cardiovascular tissue engineering, due to its major impact on the tissue's mechanical properties as well as the development of permanent dimensional changes, e.g., by contraction or dilatation of the tissue. Previous attempts to quantify contractile cellular stresses mostly used strongly aligned monolayers of cells, which might not represent the actual organization in engineered cardiovascular tissues such as heart valves. In the present study, therefore, we investigated whether differences in organization affect the magnitude of intrinsic stress generated by individual myofibroblasts, a frequently used cell source for in vitro engineered heart valves. Four different monolayer organizations were created via micro-contact printing of fibronectin lines on thin PDMS films, ranging from strongly anisotropic to isotropic. Thin film curvature, cell density, and actin stress fiber distribution were quantified, and subsequently, intrinsic stress and contractility of the monolayers were determined by incorporating these data into sample-specific finite element models. Our data indicate that the intrinsic stress exerted by the monolayers in each group correlates with cell density. Additionally, after normalizing for cell density and accounting for differences in alignment, no consistent differences in intrinsic contractility were found between the different monolayer organizations, suggesting that the intrinsic stress exerted by individual myofibroblasts is independent of the organization. Consequently, this study emphasizes the importance of choosing proper architectural properties for scaffolds in cardiovascular tissue engineering, as these directly affect the stresses in the tissue, which play a crucial role in both the functionality and remodeling of (engineered) cardiovascular tissues.

Validation and use of three complementary analytical methods (LC-FLS, LC-MS/MS and ICP-MS) to evaluate the pharmacokinetics, biodistribution and stability of motexafin gadolinium in plasma and tissues.

PubMed

Miles, Dale R; Mesfin, Mimi; Mody, Tarak D; Stiles, Mark; Lee, Jean; Fiene, John; Denis, Bernie; Boswell, Garry W

2006-05-01

Liquid chromatography-fluorescence (LC-FLS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and inductively coupled plasma-mass spectrometry (ICP-MS) methods were developed and validated for the evaluation of motexafin gadolinium (MGd, Xcytrin) pharmacokinetics and biodistribution in plasma and tissues. The LC-FLS method exhibited the greatest sensitivity (0.0057 microg mL(-1)), and was used for pharmacokinetic, biodistribution, and protein binding studies with small sample sizes or low MGd concentrations. The LC-MS/MS method, which exhibited a short run time and excellent selectivity, was used for routine clinical plasma sample analysis. The ICP-MS method, which measured total Gd, was used in conjunction with LC methods to assess MGd stability in plasma. All three methods were validated using human plasma. The LC-FLS method was also validated using plasma, liver and kidneys from mice and rats. All three methods were shown to be accurate, precise and robust for each matrix validated. For three mice, the mean (standard deviation) concentration of MGd in plasma/tissues taken 5 hr after dosing with 23 mg kg(-1) MGd was determined by LC-FLS as follows: plasma (0.025+/-0.002 microg mL(-1)), liver (2.89+/-0.45 microg g(-1)), and kidney (6.09+/-1.05 microg g(-1)). Plasma samples from a subset of patients with brain metastases from extracranial tumors were analyzed using both LC-MS/MS and ICP-MS methods. For a representative patient, > or = 90% of the total Gd in plasma was accounted for as MGd over the first hour post dosing. By 24 hr post dosing, 63% of total Gd was accounted for as MGd, indicating some metabolism of MGd.

Detection of novel polymorphisms in the ckit gene of canine patients with lymphoma, melanoma, haemangiosarcoma, and osteosarcoma.

PubMed

Gramer, Irina; Kessler, Martin; Geyer, Joachim

2016-06-01

Tyrosine kinase inhibitors (TKIs) that specifically target cKIT represent a therapeutic approach for non-resectable canine mast cell tumours (MCTs) grade II/III. The therapeutic benefit of TKIs has been investigated in other tumours based on clinical response rates and identification of gain-of-function mutations. In the present study, cKIT expression in 14 dogs with osteosarcoma, melanoma, haemangiosarcoma, lymphoma, and fibrosarcoma was analysed. Tissue samples were used for cKIT sequencing to (I) detect the cKIT transcript and to (II) identify gain-of-function mutations. The cKIT transcript was detected in ten patients. Four novel amino acid substitutions and five silent polymorphisms were identified. Furthermore, an insertion mutation (GNSK) was discovered in the tissue, but not in the blood sample of one dog. CKIT expression was identified in a variety of canine tumours and, therefore, TKIs might have a broader therapeutic indication apart from treatment of MCTs. Further investigations will be necessary to localize the cKIT protein in the respective tumours and to evaluate the functional consequence of the cKIT variants identified in the present study.

High-Throughput Sequencing of miRNAs Reveals a Tissue Signature in Gastric Cancer and Suggests Novel Potential Biomarkers

PubMed Central

Darnet, Sylvain; Moreira, Fabiano C; Hamoy, Igor G; Burbano, Rommel; Khayat, André; Cruz, Aline; Magalhães, Leandro; Silva, Artur; Santos, Sidney; Demachki, Samia; Assumpção, Monica; Assumpção, Paulo; Ribeiro-dos-Santos, Ândrea

2015-01-01

Gastric cancer has a high incidence and mortality rate worldwide; however, the use of biomarkers for its clinical diagnosis remains limited. The microRNAs (miRNAs) are biomarkers with the potential to identify the risk and prognosis as well as therapeutic targets. We performed the ultradeep miRnomes sequencing of gastric adenocarcinoma and gastric antrum without tumor samples. We observed that a small set of those samples were responsible for approximately 80% of the total miRNAs expression, which might represent a miRNA tissue signature. Additionally, we identified seven miRNAs exhibiting significant differences, and, of these, hsa-miR-135b and hsa-miR-29c were able to discriminate antrum without tumor from gastric cancer regardless of the histological type. These findings were validated by quantitative real-time polymerase chain reaction. The results revealed that hsa-miR-135b and hsa-miR-29c are potential gastric adenocarcinoma occurrence biomarkers with the ability to identify individuals at a higher risk of developing this cancer, and could even be used as therapeutic targets to allow individualized clinical management. PMID:26157332

Mapping of HABs Contaminated In Green Shells (Perna viridis) in Semarang Bay

NASA Astrophysics Data System (ADS)

A'in, Churun; Suryanti, Suryanti; Haeruddin, Haeruddin

2018-02-01

The existence of Harmful Algae Blooms (HABs) can adversely affect the water like a mass death of fish and oxygen depletion. Some types of HABs can be contaminated with seafood and contain biotoxins that are detrimental to the health of humans who consume them. Green mussels (Perna viridis) has the properties of filter feeders so vulnerable to contamination HABs. This research was conducted to produce spatially thematic maps contaminated HABs in P. viridis so providing information about risk prediction P. viridis when consumed by humans. Sampling was done purposively in three (3) stations that represent the Bay Semarang namely western boundary waters (Kendal), middle (Semarang) and the eastern boundary (Demak). Sampling done two (2) times, namely East season (June -July) and the second transitional season (September) 2016. Analysis of HABs done either in water or body tissues of P. viridis through the analysis of food habit. The results shows that P. viridis genus positive contaminated HABs phytoplankton Tricodesmium and Ceratium. Spatial distribution and abundance of Tricodesmium genus Fitoplankton is wider and taller than the HABs Phytoplankton genus Ceratium. Group HABs are found in the tissues of P. viridis no potential as biotoxin that does not cause adverse health risks.

Guidance of aortic ablation using optical coherence tomography.

PubMed

Patel, Nirlep A; Li, Xingde; Stamper, Debra L; Fujimoto, James G; Brezinski, Mark E

2003-04-01

There is a significant need for an imaging modality that is capable of providing guidance for intravascular procedures, as current technologies suffer from significant limitations. In particular, laser ablation of in-stent restenosis, revascularization of chronic total occlusions, and pulmonary vein ablation could benefit from guidance. Optical coherence tomography (OCT), a recently introduced technology, is similar to ultrasound except that it measures the back-reflection of infrared light instead of sound. This study examines the ability of OCT to guide vascular laser ablation. Aorta samples underwent laser ablation using an argon laser at varying power outputs and were monitored with OCT collecting images at 4 frames. Samples were compared to the corresponding histopathology. Arterial layers could be differentiated in the images sequences. This allowed correlation of changes in the OCT image with power and duration in addition to histopathology. OCT provides real-time guidance of arterial ablation. At 4 frames, OCT was successfully able to show the microstructural changes in the vessel wall during laser ablation. Since current ablation procedures often injure surrounding tissue, the ability to minimize collateral damage to the adjoining tissue represents a useful advantage of this system. This study suggests a possible role for OCT in the guidance of intravascular procedures.

Laser capture microdissection tailored to type 1 diabetes mellitus research.

PubMed

Szulawski, Robert; Nakazawa, Masato; McCall, Kelly D; James, Calvin B L; Schwartz, Frank L

2016-01-01

RNA isolation from pancreatic islets poses unique challenges. Here, we present a reproducible means of obtaining high-quality RNA from juvenile rodent islets in sufficient quantities for use in ex vivo expression studies. Tissue was extracted from female non-obese diabetic (NOD) toll-like receptor 3 (TLR3)(+/+) and (TLR3)(-/-) mice in the pre-diabetic stage. Samples were frozen in liquid nitrogen, sectioned, fixed in a highly alcoholic solution, and stained with an alcoholic cresyl violet (CV) solution. Rehydration of the fixed sections was minimized. Islets were identified visually and isolated with the Leica LMD6000 laser capture microdissection (LCM) system to yield samples highly enriched in islet RNA. Real time qPCR was performed on the islet cDNA using probes for CXC chemokine ligand 10 (CXCL10), an inflammatory marker that plays a critical role in the pathogenesis of type 1 diabetes mellitus (TIDM). This method represents an improvement over currently described LCM techniques for rodent pancreatic islets and makes feasible expression studies using small amounts of starting tissue without the need for RNA pre-amplification. This has immediate implications for ongoing TIDM studies using the NOD mouse.

Factors influencing tissue nitrate concentration in field-grown wild rocket (Diplotaxis tenuifolia) in southern England.

PubMed

Weightman, R M; Huckle, A J; Roques, S E; Ginsburg, D; Dyer, C J

2012-01-01

Wild rocket (Diplotaxis tenuifolia) is a leafy vegetable known for its high tissue nitrate concentration (TNC) which can exceed the limits set in the relevant European legislation designed to protect human health. The aim of this work was to understand the factors influencing TNC and to develop best practice guidelines to growers. Commercial crops of field-grown wild rocket were studied over two seasons. In 2010, ten separate crops were sampled representing a range of soil types and time periods during the summer. Two fields sampled using a 'W'- or 'X'-shaped sampling pattern demonstrated that 10 incremental samples bulked to make 1 kg of fresh material could be used to provide an adequate sample for determination of TNC in the wild rocket crop, as is the case for other leafy vegetables. Of eight commercial crops sampled in 2010 with an average nitrogen (N) fertiliser application of 104 kg N ha(-1), two exceeded the limit of 6000 mg NO3(-) kg(-1) set in the legislation. In 2011, six N response experiments were carried out, and only two sites showed a significant yield response to N fertiliser. The reason for the lack of response at the other sites was principally due to high levels of soil mineral N prior to drilling, meaning the crops' requirement for N was satisfied without additional fertiliser N. In the experimental situation at an N fertiliser application rate of 120 kg N ha(-1), 50% of crops would have exceeded the 6000 mg NO3(-) kg(-1) limit. In both seasons, low radiation levels in the 5 days prior to harvest were shown to increase TNC, although the relationship was also influenced by N supply. Strategies for optimising N nutrition of field-grown wild rocket are discussed.

Liquid Microjunction Surface Sampling Probe Electrospray Mass Spectrometry for Detection of Drugs and Metabolites in Thin Tissue Sections

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Berkel, Gary J; Kertesz, Vilmos; Koeplinger, Kenneth A.

2008-01-01

A self-aspirating, liquid micro-junction surface sampling probe/electrospray emitter mass spectrometry system was demonstrated for use in the direct analysis of spotted and dosed drugs and their metabolites in thin tissue sections. Proof-of-principle sampling and analysis directly from tissue without the need for sample preparation was demonstrated first by raster scanning a region on a section of rat liver onto which reserpine was spotted. The mass spectral signal from selected reaction monitoring was used to develop a chemical image of the spotted drug on the tissue. The probe was also used to selectively spot sample areas of sagittal whole mouse bodymore » tissue sections that had been dosed orally (90 mg/kg) with R,S-sulforaphane 3 hrs prior to sacrifice. Sulforaphane and its glutathione and N-acetyl cysteine conjugates were monitored with selected reaction monitoring and detected in the stomach and various other tissues from the dosed mouse. No signal for these species was observed in the tissue from a control mouse. The same dosed tissue section was used to illustrate the possibility of obtaining a line scan across the whole body section. In total these results illustrate the potential for rapid screening of the distribution of drugs and metabolites in tissue sections with the micro-liquid junction surface sampling probe/electrospray mass spectrometry approach.« less

The value of intraoperative ultrasonography during the resection of relapsed irradiated malignant gliomas in the brain.

PubMed

Mursch, Kay; Scholz, Martin; Brück, Wolfgang; Behnke-Mursch, Julianne

2017-01-01

The aim of this study was to investigate whether intraoperative ultrasonography (IOUS) helped the surgeon navigate towards the tumor as seen in preoperative magnetic resonance imaging and whether IOUS was able to distinguish between tumor margins and the surrounding tissue. Twenty-five patients suffering from high-grade gliomas who were previously treated by surgery and radiotherapy were included. Intraoperatively, two histopathologic samples were obtained a sample of unequivocal tumor tissue (according to anatomical landmarks and the surgeon's visual and tactile impressions) and a small tissue sample obtained using a navigated needle when the surgeon decided to stop the resection. This specimen was considered to be a boundary specimen, where no tumor tissue was apparent. The decision to take the second sample was not influenced by IOUS. The effect of IOUS was analyzed semi-quantitatively. All 25 samples of unequivocal tumor tissue were histopathologically classified as tumor tissue and were hyperechoic on IOUS. Of the boundary specimens, eight were hypoechoic. Only one harbored tumor tissue (P=0.150). Seventeen boundaries were moderately hyperechoic, and these samples contained all possible histological results (i.e., tumor, infiltration, or no tumor). During surgery performed on relapsed, irradiated, high-grade gliomas, IOUS provided a reliable method of navigating towards the core of the tumor. At borders, it did not reliably distinguish between remnants or tumor-free tissue, but hypoechoic areas seldom contained tumor tissue.

Comparison of three methods for the derivation of a biologic scaffold composed of adipose tissue extracellular matrix.

PubMed

Brown, Bryan N; Freund, John M; Han, Li; Rubin, J Peter; Reing, Janet E; Jeffries, Eric M; Wolf, Mathew T; Tottey, Stephen; Barnes, Christopher A; Ratner, Buddy D; Badylak, Stephen F

2011-04-01

Extracellular matrix (ECM)-based scaffold materials have been used successfully in both preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. Results of numerous studies have shown that ECM scaffolds are capable of supporting the growth and differentiation of multiple cell types in vitro and of acting as inductive templates for constructive tissue remodeling after implantation in vivo. Adipose tissue represents a potentially abundant source of ECM and may represent an ideal substrate for the growth and adipogenic differentiation of stem cells harvested from this tissue. Numerous studies have shown that the methods by which ECM scaffold materials are prepared have a dramatic effect upon both the biochemical and structural properties of the resultant ECM scaffold material as well as the ability of the material to support a positive tissue remodeling outcome after implantation. The objective of the present study was to characterize the adipose ECM material resulting from three methods of decellularization to determine the most effective method for the derivation of an adipose tissue ECM scaffold that was largely free of potentially immunogenic cellular content while retaining tissue-specific structural and functional components as well as the ability to support the growth and adipogenic differentiation of adipose-derived stem cells. The results show that each of the decellularization methods produced an adipose ECM scaffold that was distinct from both a structural and biochemical perspective, emphasizing the importance of the decellularization protocol used to produce adipose ECM scaffolds. Further, the results suggest that the adipose ECM scaffolds produced using the methods described herein are capable of supporting the maintenance and adipogenic differentiation of adipose-derived stem cells and may represent effective substrates for use in tissue engineering and regenerative medicine approaches to soft tissue reconstruction.

A probable risk factor of female breast cancer: study on benign and malignant breast tissue samples.

PubMed

Rehman, Sohaila; Husnain, Syed M

2014-01-01

The study reports enhanced Fe, Cu, and Zn contents in breast tissues, a probable risk factor of breast cancer in females. Forty-one formalin-fixed breast tissues were analyzed using atomic absorption spectrophotometry. Twenty malignant, six adjacent to malignant and 15 benign tissues samples were investigated. The malignant tissues samples were of grade 11 and type invasive ductal carcinoma. The quantitative comparison between the elemental levels measured in the two types of specimen (benign and malignant) tissues (removed after surgery) suggests significant elevation of these metals (Fe, Cu, and Zn) in the malignant tissue. The specimens were collected just after mastectomy of women aged 19 to 59 years from the hospitals of Islamabad and Rawalpindi, Pakistan. Most of the patients belong to urban areas of Pakistan. Findings of study depict that these elements have a promising role in the initiation and development of carcinoma as consistent pattern of elevation for Fe, Cu, and Zn was observed. The results showed the excessive accumulation of Fe (229 ± 121 mg/L) in malignant breast tissue samples of patients (p < 0.05) to that in benign tissues samples (49.1 ± 11.4 mg/L). Findings indicated that excess accumulation of iron in malignant tissues can be a risk factor of breast cancer. In order to validate our method of analysis, certified reference material muscle tissue lyophilized (IAEA) MA-M-2/TM was analyzed for metal studied. Determined concentrations were quite in good agreement with certified levels. Asymmetric concentration distribution for Fe, Cu, and Zn was observed in both malignant and benign tissue samples.

Imaging biological tissues with electrical conductivity contrast below 1 S m−1 by means of magnetoacoustic tomography with magnetic induction

PubMed Central

Hu, Gang; Li, Xu; He, Bin

2010-01-01

Magnetoacoustic tomography with magnetic induction (MAT-MI) is a recently introduced imaging modality for noninvasive electrical impedance imaging, with ultrasound imaging resolution and a contrast reflecting the electrical conductivity properties of tissues. However, previous MAT-MI systems can only image samples that are much more conductive than real human or animal tissues. To image real biological tissue samples, a large-current-carrying coil that can give stronger magnetic stimulations and stronger MAT-MI acoustic signals is employed in this study. The conductivity values of all the tissue samples employed in this study are also directly measured using a well calibrated four-electrode system. The experimental results demonstrated the feasibility to image biological tissues with electrical conductivity contrast below 1.0 S∕m using the MAT-MI technique with safe level of electromagnetic energy applied to tissue samples. PMID:20938494

Imaging biological tissues with electrical conductivity contrast below 1 S m-1 by means of magnetoacoustic tomography with magnetic induction

NASA Astrophysics Data System (ADS)

Hu, Gang; Li, Xu; He, Bin

2010-09-01

Magnetoacoustic tomography with magnetic induction (MAT-MI) is a recently introduced imaging modality for noninvasive electrical impedance imaging, with ultrasound imaging resolution and a contrast reflecting the electrical conductivity properties of tissues. However, previous MAT-MI systems can only image samples that are much more conductive than real human or animal tissues. To image real biological tissue samples, a large-current-carrying coil that can give stronger magnetic stimulations and stronger MAT-MI acoustic signals is employed in this study. The conductivity values of all the tissue samples employed in this study are also directly measured using a well calibrated four-electrode system. The experimental results demonstrated the feasibility to image biological tissues with electrical conductivity contrast below 1.0 S/m using the MAT-MI technique with safe level of electromagnetic energy applied to tissue samples.

Water-quality assessment of the Rio Grande Valley, Colorado, New Mexico and Texas; organic compounds and trace elements in bed sediment and fish tissue, 1992-93

USGS Publications Warehouse

Carter, L.F.; Anderholm, S.K.

1997-01-01

The occurrence and distribution of contaminants in aquatic systems are major components of the National Water-Quality Assessment (NAWQA) Program. Bed-sediment samples were collected at 18 sites in the Rio Grande Valley study unit between September 1992 and March 1993 to characterize the geographic distribution of organic compounds, including chlorinated insecticides, polychlorinated biphenyls (PCB's), and other chlorinated hydrocarbons, and also trace elements. Two-millimeter-size- fraction sediment was analyzed for organic compounds and less than 63-micron-size-fraction sediment was analyzed for trace elements. Concentrations of p,p'-DDE were detected in 33 percent of the bed-sediment samples. With the exception of DDT-related compounds, no other organochlorine insecticides or polychlorinated biphenyls were detected in samples of bed sediment. Whole-body fish samples were collected at 11 of the bed- sediment sites and analyzed for organic compounds. Organic compounds were reported more frequently in samples of fish, and more types of organic compounds were found in whole-body fish samples than in bed-sediment samples. Concentrations of p,p'-DDE were detected in 91 percent of whole-body fish samples. Polychlorinated biphenyls, cis-chlordane, trans-chlordane, trans- nonachlor, and hexachlorobenzene were other organic compounds detected in whole-body samples of fish from at least one site. Because of the extent of mineralized areas in the Rio Grande Basin arsenic, cadmium, copper, lead, mercury, selenium, and zinc concentrations in bed-sediment samples could represent natural conditions at most sites. However, a combination of natural conditions and human activities appears to be associated with elevated trace-element concentrations in the bed-sediment sample from the site Rio Grande near Creede, Colorado, because this sample exceeded the background trace-element concentrations calculated for this study. Fish-liver samples were collected at 12 of the bed-sediment sites and analyzed for trace elements. Certain trace elements were detected at higher concentrations in fish-liver samples than in bed-sediment samples from the same site. Both bed-sediment and fish-tissue samples are necessary for a complete environmental assessment of the occurrence and distribution of trace elements.

76 FR 71315 - Endangered and Threatened Species; Take of Anadromous Fish

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-17

... the fish and would then sample them for biological information (fin tissue and scale samples). They..., measured, weighed, tissue-sampled, and checked for external marks and coded-wire tags depending on the.... Then the researchers would remove and preserve fish body tissues, otoliths, and coded wire tags (from...

Age estimation based on aspartic acid racemization in human sclera.

PubMed

Klumb, Karolin; Matzenauer, Christian; Reckert, Alexandra; Lehmann, Klaus; Ritz-Timme, Stefanie

2016-01-01

Age estimation based on racemization of aspartic acid residues (AAR) in permanent proteins has been established in forensic medicine for years. While dentine is the tissue of choice for this molecular method of age estimation, teeth are not always available which leads to the need to identify other suitable tissues. We examined the suitability of total tissue samples of human sclera for the estimation of age at death. Sixty-five samples of scleral tissue were analyzed. The samples were hydrolyzed and after derivatization, the extent of aspartic acid racemization was determined by gas chromatography. The degree of AAR increased with age. In samples from younger individuals, the correlation of age and D-aspartic acid content was closer than in samples from older individuals. The age-dependent racemization in total tissue samples proves that permanent or at least long-living proteins are present in scleral tissue. The correlation of AAR in human sclera and age at death is close enough to serve as basis for age estimation. However, the precision of age estimation by this method is lower than that of age estimation based on the analysis of dentine which is due to molecular inhomogeneities of total tissue samples of sclera. Nevertheless, the approach may serve as a valuable alternative or addition in exceptional cases.

Comparative tissue transcriptomics highlights dynamic differences among tissues but conserved metabolic transcript prioritization in preparation for arousal from torpor.

PubMed

Bogren, Lori K; Grabek, Katharine R; Barsh, Gregory S; Martin, Sandra L

2017-07-01

During the hibernation season, 13-lined ground squirrels spend days to weeks in torpor with body temperatures near freezing then spontaneously rewarm. The molecular drivers of the drastic physiological changes that orchestrate and permit torpor are not well understood. Although transcription effectively ceases at the low body temperatures of torpor, previous work has demonstrated that some transcripts are protected from bulk degradation in brown adipose tissue (BAT), consistent with the importance of their protein products for metabolic heat generation during arousal from torpor. We examined the transcriptome of skeletal muscle, heart, and liver to determine the patterns of differentially expressed genes in these tissues, and whether, like BAT, a subset of these were relatively increased during torpor. EDGE-tags were quantified from five distinct physiological states representing the seasonal and torpor-arousal cycles of 13-lined ground squirrels. Supervised clustering on relative transcript abundances with Random Forest separated the two states bracketing prolonged torpor, entrance into and aroused from torpor, in all three tissues. Independent analyses identified 3347, 6784, and 2433 differentially expressed transcripts among all sampling points in heart, skeletal muscle, and liver, respectively. There were few differentially expressed genes in common across all three tissues; these were enriched in mitochondrial and apoptotic pathway components. Divisive clustering of these data revealed unique cohorts of transcripts that increased across the torpor bout in each tissue with patterns reflecting various combinations of cycling within and between seasons as well as between torpor and arousal. Transcripts that increased across the torpor bout were likewise tissue specific. These data shed new light on the biochemical pathways that alter in concert with hibernation phenotype and provide a rich resource for further hypothesis-based studies.

Three-Dimensional Coculture of Meniscal Cells and Mesenchymal Stem Cells in Collagen Type I Hydrogel on a Small Intestinal Matrix-A Pilot Study Toward Equine Meniscus Tissue Engineering.

PubMed

Kremer, Antje; Ribitsch, Iris; Reboredo, Jenny; Dürr, Julia; Egerbacher, Monika; Jenner, Florien; Walles, Heike

2017-05-01

Meniscal injuries are the most frequently encountered soft tissue injuries in the equine stifle joint. Due to the inherent limited repair potential of meniscal tissue, meniscal injuries do not only affect the meniscus itself but also lead to impaired joint homeostasis and secondary osteoarthritis. The presented study compares 3D coculture constructs of primary equine mesenchymal stem cells (MSC) and meniscus cells (MC) seeded on three different scaffolds-a cell-laden collagen type I hydrogel (Col I gel), a tissue-derived small intestinal matrix scaffold (SIS-muc) and a combination thereof-for their qualification to be applied for meniscus tissue engineering. To investigate cell attachment of primary MC and MSC on SIS-muc matrix SEM pictures were performed. For molecular analysis, lyophilized samples of coculture constructs with different cell ratios (100% MC, 100% MSC, and 50% MC and 50% MSC, 20% MC, and 80% MSC) were digested and analyzed for DNA and GAG content. Active matrix remodeling of 3D coculture models was indicated by matrix metalloproteinases detection. For comparison of tissue-engineered constructs with the histologic architecture of natural equine menisci, paired lateral and medial menisci of 15 horses representing different age groups were examined. A meniscus phenotype with promising similarity to native meniscus tissue in its GAG/DNA expression in addition to Col I, Col II, and Aggrecan production was achieved using a scaffold composed of Col I gel on SIS-muc combined with a coculture of MC and MSC. The results encourage further development of this scaffold-cell combination for meniscus tissue engineering.

Fluorescence suppression using wavelength modulated Raman spectroscopy in fiber-probe-based tissue analysis.

PubMed

Praveen, Bavishna B; Ashok, Praveen C; Mazilu, Michael; Riches, Andrew; Herrington, Simon; Dholakia, Kishan

2012-07-01

In the field of biomedical optics, Raman spectroscopy is a powerful tool for probing the chemical composition of biological samples. In particular, fiber Raman probes play a crucial role for in vivo and ex vivo tissue analysis. However, the high-fluorescence background typically contributed by the auto fluorescence from both a tissue sample and the fiber-probe interferes strongly with the relatively weak Raman signal. Here we demonstrate the implementation of wavelength-modulated Raman spectroscopy (WMRS) to suppress the fluorescence background while analyzing tissues using fiber Raman probes. We have observed a significant signal-to-noise ratio enhancement in the Raman bands of bone tissue, which have a relatively high fluorescence background. Implementation of WMRS in fiber-probe-based bone tissue study yielded usable Raman spectra in a relatively short acquisition time (∼30  s), notably without any special sample preparation stage. Finally, we have validated its capability to suppress fluorescence on other tissue samples such as adipose tissue derived from four different species.

Organochlorine compounds in streambed sediment and in biological tissue from streams and their relations to land use, central Arizona

USGS Publications Warehouse

Gebler, Joseph B.

2000-01-01

Streambed-sediment samples from 13 sites and biological-tissue samples from 11 sites in the Gila River Basin in central Arizona were analyzed for 32 organochlorine compounds in streambed sediment and 28 compounds in biological tissue during 1996 as part of the U.S. Geological Survey's National Water-Quality Assessment program. The objectives of the study were to determine the occurrence and distribution of organochlorine compounds and their relation to land use. Sampling sites were categorized on the basis of major land uses in the basin or the source of water in the stream. Because land uses were mixed or had changed over time, some land-use categories were combined. Sites were categorized as forest/rangeland (6), forest/urban (1), urban (4), or agricultural/urban (2). Thirteen organochlorine compounds were detected in streambed-sediment samples, and 10 were detected in tissue samples. The number of compounds found in streambed-sediment samples from individual sites ranged from 0 to 10, and the range for individual tissue samples was 0 to 7. Comparison of the number of detections in streambed-sediment samples to the number of detections in tissue samples from particular sites where both were sampled yielded five instances where more compounds were detected in streambed sediment, six instances where more compounds were detected in tissue, and five instances where the number of detections in streambed sediment and tissue were equal. The frequency of detection of particular compounds for sites where both streambed sediment and tissue were sampled resulted in five compounds being detected more frequently in streambed sediment, five more frequently in tissue, and three compounds that were equally frequent in streambed sediment and in tissue. Few contaminants were detected in samples from the forest/rangeland sites; greater numbers of compounds were detected at the urban sites and at the forest/urban site. The greatest number of compounds and the highest concentrations of many contaminants were detected at agriculture/urban sites. The compound detected most frequently in streambed-sediment and tissue samples was p,p'-DDE. Streambed-sediment guideline values for the protection of aquatic life for p,p'-DDE and total DDT were exceeded at both agricultural/urban sites, The streambed-sediment guideline value for the protection of aquatic life for total chlordane was exceeded at one agricultural/urban site, one urban site, and the forest/urban site. The streambed-sediment guideline value for the protection of aquatic life for total PCB’s was exceeded at one agricultural/urban site. Guideline values for the protection of fish-eating wildlife for total DDT and for toxaphene were exceeded only in samples from the two agricultural/urban sites. The guideline value for the protection of fish-eating wildlife for total PCB’s was equaled or exceeded in samples from two sites—one urban and one agricultural/urban site. Screening values established by the U.S. Environmental Protection Agency for the protection of human health for edible portions of fish were exceeded by total DDT and by toxaphene in fish-tissue samples from both agricultural/urban sites. The human-health criterion for total PCB’s was exceeded in two fish-tissue samples from an agricultural site and from an urban site. Tissue samples analyzed in this study were for whole fish, and thus, concentration data are not entirely comparable to the screening values of the U.S. Environmental Protection Agency. Because these exceedences were an order of magnitude above the criteria, however, it is possible that concentrations in the edible portions of fish from these locations could present a human- health risk. Analyses of samples of edible portions of fish from these locations would be needed to adequately assess the presence or absence of a human-health risk. The similarity of the results of this study to the results of other studies of organochlorine compounds in the environment suggests that there is a correlation between contaminants in sediment and biological-tissue samples and land uses. As with other studies of the occurrence and distribution of organochlorine contaminants in streambed sediments and biological tissue, this study shows that many organochlorine compounds continue to persist in the environment and thus could pose a threat to aquatic life, fish-eating wildlife, and possibly to humans who consume contaminated fish.

An efficient field and laboratory workflow for plant phylotranscriptomic projects1

PubMed Central

Yang, Ya; Moore, Michael J.; Brockington, Samuel F.; Timoneda, Alfonso; Feng, Tao; Marx, Hannah E.; Walker, Joseph F.; Smith, Stephen A.

2017-01-01

Premise of the study: We describe a field and laboratory workflow developed for plant phylotranscriptomic projects that involves cryogenic tissue collection in the field, RNA extraction and quality control, and library preparation. We also make recommendations for sample curation. Methods and Results: A total of 216 frozen tissue samples of Caryophyllales and other angiosperm taxa were collected from the field or botanical gardens. RNA was extracted, stranded mRNA libraries were prepared, and libraries were sequenced on Illumina HiSeq platforms. These included difficult mucilaginous tissues such as those of Cactaceae and Droseraceae. Conclusions: Our workflow is not only cost effective (ca. $270 per sample, as of August 2016, from tissue to reads) and time efficient (less than 50 h for 10–12 samples including all laboratory work and sample curation), but also has proven robust for extraction of difficult samples such as tissues containing high levels of secondary compounds. PMID:28337391

Evaluation of sample holders designed for long-lasting X-ray micro-tomographic scans of ex-vivo soft tissue samples

NASA Astrophysics Data System (ADS)

Dudak, J.; Zemlicka, J.; Krejci, F.; Karch, J.; Patzelt, M.; Zach, P.; Sykora, V.; Mrzilkova, J.

2016-03-01

X-ray microradiography and microtomography are imaging techniques with increasing applicability in the field of biomedical and preclinical research. Application of hybrid pixel detector Timepix enables to obtain very high contrast of low attenuating materials such as soft biological tissue. However X-ray imaging of ex-vivo soft tissue samples is a difficult task due to its structural instability. Ex-vivo biological tissue is prone to fast drying-out which is connected with undesired changes of sample size and shape producing later on artefacts within the tomographic reconstruction. In this work we present the optimization of our Timepix equipped micro-CT system aiming to maintain soft tissue sample in stable condition. Thanks to the suggested approach higher contrast of tomographic reconstructions can be achieved while also large samples that require detector scanning can be easily measured.

Regulatory role of tumor necrosis factor receptor-associated factor 6 in breast cancer by activating the protein kinase B/glycogen synthase kinase 3β signaling pathway.

PubMed

Shen, Hongyu; Li, Liangpeng; Yang, Sujin; Wang, Dandan; Zhou, Siying; Chen, Xiu; Tang, Jinhai

2017-08-01

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an endogenous adaptor of innate and adaptive immune responses, and serves a crucial role in tumor necrosis factor receptor and toll‑like/interleukin‑1 receptor signaling. Although studies have demonstrated that TRAF6 has oncogenic activity, its potential contributions to breast cancer in human remains largely uninvestigated. The present study examined the expression levels and function of TRAF6 in breast carcinoma (n=32) and adjacent healthy (n=25) tissue samples. Compared with adjacent healthy tissues, TRAF6 protein expression levels were significantly upregulated in breast cancer tissues. Reverse transcription‑quantitative polymerase chain reaction analysis revealed a significant upregulation of the cellular proliferative marker Ki‑67 and proliferation cell nuclear antigen expression levels in breast carcinoma specimens. Furthermore, protein expression levels of the accessory molecule, transforming growth factor β‑activated kinase 1 (TAK1), were significantly increased in breast cancer patients, as detected by western blot analysis. As determined by MTT assay, TRAF6 exerted profoundly proliferative effects in the MCF‑7 breast cancer cell line; however, these detrimental effects were ameliorated by TAK1 inhibition. Notably, protein kinase B (AKT)/glycogen synthase kinase (GSK)3β phosphorylation levels were markedly upregulated in breast cancer samples, compared with adjacent healthy tissues. In conclusion, an altered TRAF6‑TAK1 axis and its corresponding downstream AKT/GSK3β signaling molecules may contribute to breast cancer progression. Therefore, TRAF6 may represent a potential therapeutic target for the treatment of breast cancer.

Survival of the Apical Papilla and Its Resident Stem Cells in a Case of Advanced Pulpal Necrosis and Apical Periodontitis.

PubMed

Chrepa, Vanessa; Pitcher, Brandon; Henry, Michael A; Diogenes, Anibal

2017-04-01

Apical papilla represents a source of an enriched mesenchymal stem cell (MSC) population (stem cells of the apical papilla [SCAPs]) that modulates root development and may participate in regenerative endodontic procedures in immature teeth with pulp necrosis. The characteristics and phenotype of this tissue in the presence of inflammation are largely unknown. The purpose of this study was to characterize a human apical papilla sample that was isolated from an immature tooth with pulp necrosis and apical periodontitis. Inflamed periapical tissue that included part of the apical papilla (apical papilla clinical sample [CS]) was collected from an immature mandibular premolar previously diagnosed with pulp necrosis and apical periodontitis during an apexification procedure. Harvested cells from this tissue (SCAP CS) were compared with inflamed periapical progenitor cells (IPAPCs) and normal SCAP (SCAP-RP89) in flow cytometry and quantitative osteogenesis experiments. Part of the issue was further processed for immunohistochemistry and compared with apical papilla and coronal pulp sections from normal immature teeth as well as inflamed periapical tissues from mature teeth. Similar to SCAP-RP89, 96.6% of the SCAP CS coexpressed the MSC markers CD73, CD90, and CD105, whereas only 66.3% of IPAPCs coexpressed all markers. The SCAP CS showed a significantly greater mineralization potential than both SCAP-RP89 and IPAPCs. Finally, immunohistochemical analysis revealed moderate infiltration of cells expressing the inflammatory markers CD45/68 in the apical papilla CS and prominent CD24, CD105, and von Willebrand factor expression. Under inflammatory conditions, human apical papilla was found moderately inflamed with retained SCAP vitality and stemness and increased osteogenic and angiogenesis potential. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Human herpesvirus multiplex ddPCR detection in brain tissue from low- and high-grade astrocytoma cases and controls.

PubMed

Lin, Cheng-Te Major; Leibovitch, Emily C; Almira-Suarez, M Isabel; Jacobson, Steven

2016-01-01

Glioblastoma (GBM) is a fatal CNS malignancy, representing 50 % of all gliomas with approximately 12-18 months survival time after initial diagnosis. Recently, the human herpesvirus cytomegalovirus (CMV) has been suggested to have an oncogenic role, yet this association remains controversial. In addition, human herpesvirus 6 (HHV-6) and Epstein-Barr virus (EBV) have also been associated with low-grade gliomas, but few studies have examined HHV-6 and EBV in glioblastomas. Droplet digital PCR (ddPCR) is a highly precise diagnostic tool that enables the absolute quantification of target DNA. This study examines the association between multiple human herpesviruses and astrocytomas. This study analyzed 112 brain tissue specimens, including 45 glioblastoma, 12 astrocytoma grade III, 2 astrocytoma grade II, 4 astrocytoma grade I, and 49 controls. All brain tissue samples were de-identified and pathologically confirmed. Each tissue block was sectioned for DNA extraction and CMV, EBV, HHV-6A and HHV-6B, and a cellular housekeeping gene were amplified by ddPCR. Neither CMV nor HHV-6A were detected in any of the astrocytoma samples. However, HHV-6B (p = 0.147) and EBV (p = 0.049) had a higher positivity frequency in the GBM compared to the controls. The undetectable CMV DNA in the astrocytoma cohort does not support the observation of an increased prevalence of CMV DNA in GBM, as reported in other studies. EBV has a significantly higher positivity in the GBM cohort compared to the controls, while HHV-6B has a higher but not statistically significant positivity in the case cohort. Whether these viruses play an oncogenic role in GBM remains to be further investigated.

A Novel High Mechanical Property PLGA Composite Matrix Loaded with Nanodiamond-Phospholipid Compound for Bone Tissue Engineering.

PubMed

Zhang, Fan; Song, Qingxin; Huang, Xuan; Li, Fengning; Wang, Kun; Tang, Yixing; Hou, Canglong; Shen, Hongxing

2016-01-20

A potential bone tissue engineering material was produced from a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA), loaded with nanodiamond phospholipid compound (NDPC) via physical mixing. On the basis of hydrophobic effects and physical absorption, we modified the original hydrophilic surface of the nanodiamond (NDs) with phospholipids to be amphipathic, forming a typical core-shell structure. The ND-phospholipid weight ratio was optimized to generate sample NDPC50 (i.e., ND-phospholipid weight ratio of 100:50), and NDPC50 was able to be dispersed in a PLGA matrix at up to 20 wt %. Compared to a pure PLGA matrix, the introduction of 10 wt % of NDPC (i.e., sample NDPC50-PF10) resulted in a significant improvement in the material's mechanical and surface properties, including a decrease in the water contact angle from 80 to 55°, an approximately 100% increase in the Young's modulus, and an approximate 550% increase in hardness, thus closely resembling that of human cortical bone. As a novel matrix supporting human osteoblast (hFOB1.19) growth, NDPC50-PFs with different amounts of NDPC50 demonstrated no negative effects on cell proliferation and osteogenic differentiation. Furthermore, we focused on the behaviors of NDPC-PFs implanted into mice for 8 weeks and found that NDPC-PFs induced acceptable immune response and can reduce the rapid biodegradation of PLGA matrix. Our results represent the first in vivo research on ND (or NDPC) as nanofillers in a polymer matrix for bone tissue engineering. The high mechanical properties, good in vitro and in vivo biocompatibility, and increased mineralization capability suggest that biodegradable PLGA composite matrices loaded with NDPC may potentially be useful for a variety of biomedical applications, especially bone tissue engineering.

Fungal Endophytes of Alpinia officinarum Rhizomes: Insights on Diversity and Variation across Growth Years, Growth Sites, and the Inner Active Chemical Concentration

PubMed Central

Shubin, Li; Juan, Huang; RenChao, Zhou; ShiRu, Xu; YuanXiao, Jin

2014-01-01

In the present study, the terminal-restriction fragment length polymorphism (T-RFLP) technique, combined with the use of a clone library, was applied to assess the baseline diversity of fungal endophyte communities associated with rhizomes of Alpinia officinarum Hance, a medicinal plant with a long history of use. A total of 46 distinct T-RFLP fragment peaks were detected using HhaI or MspI mono-digestion-targeted, amplified fungal rDNA ITS sequences from A. officinarum rhizomes. Cloning and sequencing of representative sequences resulted in the detection of members of 10 fungal genera: Pestalotiopsis, Sebacina, Penicillium, Marasmius, Fusarium, Exserohilum, Mycoleptodiscus, Colletotrichum, Meyerozyma, and Scopulariopsis. The T-RFLP profiles revealed an influence of growth year of the host plant on fungal endophyte communities in rhizomes of this plant species; whereas, the geographic location where A. officinarum was grown contributed to only limited variation in the fungal endophyte communities of the host tissue. Furthermore, non-metric multidimensional scaling (NMDS) analysis across all of the rhizome samples showed that the fungal endophyte community assemblages in the rhizome samples could be grouped according to the presence of two types of active indicator chemicals: total volatile oils and galangin. Our present results, for the first time, address a diverse fungal endophyte community is able to internally colonize the rhizome tissue of A. officinarum. The diversity of the fungal endophytes found in the A. officinarum rhizome appeared to be closely correlated with the accumulation of active chemicals in the host plant tissue. The present study also provides the first systematic overview of the fungal endophyte communities in plant rhizome tissue using a culture-independent method. PMID:25536070

Fungal endophytes of Alpinia officinarum rhizomes: insights on diversity and variation across growth years, growth sites, and the inner active chemical concentration.

PubMed

Shubin, Li; Juan, Huang; RenChao, Zhou; ShiRu, Xu; YuanXiao, Jin

2014-01-01

In the present study, the terminal-restriction fragment length polymorphism (T-RFLP) technique, combined with the use of a clone library, was applied to assess the baseline diversity of fungal endophyte communities associated with rhizomes of Alpinia officinarum Hance, a medicinal plant with a long history of use. A total of 46 distinct T-RFLP fragment peaks were detected using HhaI or MspI mono-digestion-targeted, amplified fungal rDNA ITS sequences from A. officinarum rhizomes. Cloning and sequencing of representative sequences resulted in the detection of members of 10 fungal genera: Pestalotiopsis, Sebacina, Penicillium, Marasmius, Fusarium, Exserohilum, Mycoleptodiscus, Colletotrichum, Meyerozyma, and Scopulariopsis. The T-RFLP profiles revealed an influence of growth year of the host plant on fungal endophyte communities in rhizomes of this plant species; whereas, the geographic location where A. officinarum was grown contributed to only limited variation in the fungal endophyte communities of the host tissue. Furthermore, non-metric multidimensional scaling (NMDS) analysis across all of the rhizome samples showed that the fungal endophyte community assemblages in the rhizome samples could be grouped according to the presence of two types of active indicator chemicals: total volatile oils and galangin. Our present results, for the first time, address a diverse fungal endophyte community is able to internally colonize the rhizome tissue of A. officinarum. The diversity of the fungal endophytes found in the A. officinarum rhizome appeared to be closely correlated with the accumulation of active chemicals in the host plant tissue. The present study also provides the first systematic overview of the fungal endophyte communities in plant rhizome tissue using a culture-independent method.

Increased expression of HOXB2 and HOXB13 proteins is associated with HPV infection and cervical cancer progression.

PubMed

Gonzalez-Herrera, Al; Salgado-Bernabe, M; Velazquez-Velazquez, Ck; Salcedo-Vargas, M; Andrade-Manzano, A; Avila-Moreno, F; Pina-Sanchez, P

2015-01-01

Cervical cancer (CeCa) is the second most common cancer in women in developing countries, and human papilloma virus (HPV) is the primary etiological factor. Aberrant expression of HOX transcription factors has been observed in several types of cancer. To date, however, no reports exist on the expression of HOXB2 and HOXB13 proteins during neoplastic progression in CeCa and its correlation with HPV infection. Expression of HOXB2 and HOXB13 proteins was assessed in tissue microarrays from normal cervical epithelium, cervical intraepithelial neoplasias grade 1-3, and CeCa. HPV was detected by PCR and sequencing. Expression of HOX-positive cells was determined in each diagnostic group. Percentage of HOXB2- and HOXB13-positive cells gradually increased from means of 10.9% and 16.7%, respectively, in samples from healthy women, to 75.2% and 88.6% in those from CeCa patients. Frequency of HPV infection also increased from 13% in healthy tissue samples to 92.3% in CeCa. Both HOXB2 and HOXB13 proteins were preferentially expressed in HPV+ samples. The present study represents the first report on the expression of both HOXB2 and HOXB13 proteins through cervix tumorigenesis, providing evidence that increased expression of such proteins is a common event during progression to CeCa.

Assessment of tissue-specific cortisol activity with regard to degeneration of the suspensory ligaments in horses with pituitary pars intermedia dysfunction.

PubMed

Hofberger, Sina C; Gauff, Felicia; Thaller, Denise; Morgan, Ruth; Keen, John A; Licka, Theresia F

2018-02-01

OBJECTIVE To identify signs of tissue-specific cortisol activity in samples of suspensory ligament (SL) and neck skin tissue from horses with and without pituitary pars intermedia dysfunction (PPID). SAMPLE Suspensory ligament and neck skin tissue samples obtained from 26 euthanized horses with and without PPID. PROCEDURES Tissue samples were collected from 12 horses with and 14 horses without PPID (controls). Two control horses had received treatment with dexamethasone; data from those horses were not used in statistical analyses. The other 12 control horses were classified as old horses (≥ 14 years old) and young horses (≤ 9 years old). Standard histologic staining, staining for proteoglycan accumulation, and immunostaining of SL and neck skin tissue sections for glucocorticoid receptors, insulin, 11β hydroxysteroid dehydrogenase type 1, and 11β hydroxysteroid dehydrogenase type 2 were performed. Findings for horses with PPID were compared with findings for young and old horses without PPID. RESULTS Compared with findings for old and young control horses, there were significantly more cells stained for glucocorticoid receptors in SL samples and for 11 β hydroxysteroid dehydrogenase type 1 in SL and skin tissue samples from horses with PPID. Insulin could not be detected in any of the SL or skin tissue samples. Horses with PPID had evidence of SL degeneration with significantly increased proteoglycan accumulation. Neck skin tissue was found to be significantly thinner in PPID-affected horses than in young control horses. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that tissue-specific dysregulation of cortisol metabolism may contribute to the SL degeneration associated with PPID in horses.

Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.

PubMed

Bernsen, M R; Dijkman, H B; de Vries, E; Figdor, C G; Ruiter, D J; Adema, G J; van Muijen, G N

1998-10-01

Molecular analysis of small tissue samples has become increasingly important in biomedical studies. Using a laser dissection microscope and modified nucleic acid isolation protocols, we demonstrate that multiple mRNA as well as DNA sequences can be identified from a single-cell sample. In addition, we show that the specificity of procurement of tissue samples is not compromised by smear contamination resulting from scraping of the microtome knife during sectioning of lesions. The procedures described herein thus allow for efficient RT-PCR or PCR analysis of multiple nucleic acid sequences from small tissue samples obtained by laser-assisted microdissection.

Quality control of human tissues--experience from the Indiana University Cancer Center-Lilly Research Labs human tissue bank.

PubMed

Sandusky, George E; Teheny, Katie Heinz; Esterman, Mike; Hanson, Jeff; Williams, Stephen D

2007-01-01

The success of molecular research and its applications in both the clinical and basic research arenas is strongly dependent on the collection, handling, storage, and quality control of fresh human tissue samples. This tissue bank was set up to bank fresh surgically obtained human tissue using a Clinical Annotated Tissue Database (CATD) in order to capture the associated patient clinical data and demographics using a one way patient encryption scheme to protect patient identification. In this study, we determined that high quality of tissue samples is imperative for both genomic and proteomic molecular research. This paper also contains a brief compilation of the literature involved in the patient ethics, patient informed consent, patient de-identification, tissue collection, processing, and storage as well as basic molecular research generated from the tissue bank using good clinical practices. The current applicable rules, regulations, and guidelines for handling human tissues are briefly discussed. More than 6,610 cancer patients have been consented (97% of those that were contacted by the consenter) and 16,800 tissue specimens have been banked from these patients in 9 years. All samples collected in the bank were QC'd by a pathologist. Approximately 1,550 tissue samples have been requested for use in basic, clinical, and/or biomarker cancer research studies. Each tissue aliquot removed from the bank for a research study were evaluated by a second H&E, if the samples passed the QC, they were submitted for genomic and proteomic molecular analysis/study. Approximately 75% of samples evaluated were of high histologic quality and used for research studies. Since 2003, we changed the patient informed consent to allow the tissue bank to gather more patient clinical follow-up information. Ninety two percent of the patients (1,865 patients) signed the new informed consent form and agreed to be re-contacted for follow-up information on their disease state. In addition, eighty five percent of patients (1,584) agreed to be re-contacted to provide a biological fluid sample to be used for biomarker research.

Large-scale atlas of microarray data reveals the distinct expression landscape of different tissues in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Fei; Maslov, Sergei; Yoo, Shinjae

Here, transcriptome datasets from thousands of samples of the model plant Arabidopsis thaliana have been collectively generated by multiple individual labs. Although integration and meta-analysis of these samples has become routine in the plant research community, it is often hampered by the lack of metadata or differences in annotation styles by different labs. In this study, we carefully selected and integrated 6,057 Arabidopsis microarray expression samples from 304 experiments deposited to NCBI GEO. Metadata such as tissue type, growth condition, and developmental stage were manually curated for each sample. We then studied global expression landscape of the integrated dataset andmore » found that samples of the same tissue tend to be more similar to each other than to samples of other tissues, even in different growth conditions or developmental stages. Root has the most distinct transcriptome compared to aerial tissues, but the transcriptome of cultured root is more similar to those of aerial tissues as the former samples lost their cellular identity. Using a simple computational classification method, we showed that the tissue type of a sample can be successfully predicted based on its expression profile, opening the door for automatic metadata extraction and facilitating re-use of plant transcriptome data. As a proof of principle we applied our automated annotation pipeline to 708 RNA-seq samples from public repositories and verified accuracy of our predictions with samples’ metadata provided by authors.« less

Large-scale atlas of microarray data reveals the distinct expression landscape of different tissues in Arabidopsis

DOE PAGES

He, Fei; Maslov, Sergei; Yoo, Shinjae; ...

2016-05-25

Here, transcriptome datasets from thousands of samples of the model plant Arabidopsis thaliana have been collectively generated by multiple individual labs. Although integration and meta-analysis of these samples has become routine in the plant research community, it is often hampered by the lack of metadata or differences in annotation styles by different labs. In this study, we carefully selected and integrated 6,057 Arabidopsis microarray expression samples from 304 experiments deposited to NCBI GEO. Metadata such as tissue type, growth condition, and developmental stage were manually curated for each sample. We then studied global expression landscape of the integrated dataset andmore » found that samples of the same tissue tend to be more similar to each other than to samples of other tissues, even in different growth conditions or developmental stages. Root has the most distinct transcriptome compared to aerial tissues, but the transcriptome of cultured root is more similar to those of aerial tissues as the former samples lost their cellular identity. Using a simple computational classification method, we showed that the tissue type of a sample can be successfully predicted based on its expression profile, opening the door for automatic metadata extraction and facilitating re-use of plant transcriptome data. As a proof of principle we applied our automated annotation pipeline to 708 RNA-seq samples from public repositories and verified accuracy of our predictions with samples’ metadata provided by authors.« less

Molecular detection of hepatitis E virus in wild boar population in eastern Romania.

PubMed

Porea, D; Anita, A; Demange, A; Raileanu, C; Oslobanu Ludu, L; Anita, D; Savuta, G; Pavio, N

2018-04-01

In industrialized countries, Hepatitis E is a recognized zoonosis, with wild boar and swine representing the main reservoirs for zoonotic genotype HEV-3 in Europe. Data related to HEV infection in wild boar population in Romania are restricted to serological surveys. Therefore, our main goal was to determine the HEV prevalence in wild boar population and to characterize HEV strains circulating in Romania. Using TaqMan real-time RT-PCR assay, we analyzed the presence of RNA HEV in 45 liver samples and five spleen samples collected from 50 wild boars. Samples were collected during the 2013-2015 hunting seasons. Nine samples of 50 were tested positive for HEV RNA, resulting an overall prevalence of 18%. Phylogenetic analysis revealed that the isolates clustered in different HEV-3 monophyletic groups, depending on the sampling county. This is the first study signalling, based on molecular analysis, the presence of HEV in wild boar population from Romania. Also, in this study, we report the detection of HEV in splenic tissue from wild boar. © 2017 Blackwell Verlag GmbH.

Monitoring Cole-Cole parameters during haemodialysis (HD).

PubMed

Al-Surkhi, Omar I; Riu, P J; Vazquez, F F; Ibeas, J

2007-01-01

The investigation of the hydration process during the haemodialysis treatment sessions is very important for the development of methods for predicting the unbalanced fluid shifts and hypotension crisis hence improving the quality of the haemodialysis procedure. Bioimpedance measurements can give valuable information about the tissue under measurement, therefore characterizing the tissue. In this work we propose a non-invasive method based on local multifrequency bioimpedance measurements that allow us to determine the fluid distribution and variations during haemodialysis. Clinical measurements were done using 10 HD patients during 60 HD sessions. Bioimpedance data, ultrafiltration volume, blood volume and blood heamatocrit variations were recorded continuously during the HD sessions. Bioimpedance of the local tissue was measured with a 4-elctrode impedance system using surface electrodes with sampling rate of 1meas./4min. at 6 different frequencies. The measured impedances were fitted into Cole-Cole model and the Cole-Cole parameters were continuously determined for each measurement point during the HD session. The 4 Cole-Cole parameters (R 00, R 0, Fc,alpha) and their variations were evaluated. Impedance values at infinite and zero (R 00, R 0) frequencies were extrapolated from Cole-Cole mathematical model. These values are assumed to represent the impedance of total tissue fluid and the impedance of the extracellular space respectively.

Comparison of different numerical treatments for x-ray phase tomography of soft tissue from differential phase projections

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pelliccia, Daniele; Vaz, Raquel; Svalbe, Imants

X-ray imaging of soft tissue is made difficult by their low absorbance. The use of x-ray phase imaging and tomography can significantly enhance the detection of these tissues and several approaches have been proposed to this end. Methods such as analyzer-based imaging or grating interferometry produce differential phase projections that can be used to reconstruct the 3D distribution of the sample refractive index. We report on the quantitative comparison of three different methods to obtain x-ray phase tomography with filtered back-projection from differential phase projections in the presence of noise. The three procedures represent different numerical approaches to solve themore » same mathematical problem, namely phase retrieval and filtered back-projection. It is found that obtaining individual phase projections and subsequently applying a conventional filtered back-projection algorithm produces the best results for noisy experimental data, when compared with other procedures based on the Hilbert transform. The algorithms are tested on simulated phantom data with added noise and the predictions are confirmed by experimental data acquired using a grating interferometer. The experiment is performed on unstained adult zebrafish, an important model organism for biomedical studies. The method optimization described here allows resolution of weak soft tissue features, such as muscle fibers.« less

Sub-diffusive scattering parameter maps recovered using wide-field high-frequency structured light imaging.

PubMed

Kanick, Stephen Chad; McClatchy, David M; Krishnaswamy, Venkataramanan; Elliott, Jonathan T; Paulsen, Keith D; Pogue, Brian W

2014-10-01

This study investigates the hypothesis that structured light reflectance imaging with high spatial frequency patterns [Formula: see text] can be used to quantitatively map the anisotropic scattering phase function distribution [Formula: see text] in turbid media. Monte Carlo simulations were used in part to establish a semi-empirical model of demodulated reflectance ([Formula: see text]) in terms of dimensionless scattering [Formula: see text] and [Formula: see text], a metric of the first two moments of the [Formula: see text] distribution. Experiments completed in tissue-simulating phantoms showed that simultaneous analysis of [Formula: see text] spectra sampled at multiple [Formula: see text] in the frequency range [0.05-0.5] [Formula: see text] allowed accurate estimation of both [Formula: see text] in the relevant tissue range [0.4-1.8] [Formula: see text], and [Formula: see text] in the range [1.4-1.75]. Pilot measurements of a healthy volunteer exhibited [Formula: see text]-based contrast between scar tissue and surrounding normal skin, which was not as apparent in wide field diffuse imaging. These results represent the first wide-field maps to quantify sub-diffuse scattering parameters, which are sensitive to sub-microscopic tissue structures and composition, and therefore, offer potential for fast diagnostic imaging of ultrastructure on a size scale that is relevant to surgical applications.

A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies

PubMed Central

Zlobec, Inti; Suter, Guido; Perren, Aurel; Lugli, Alessandro

2014-01-01

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a ‘donor’ block into a ‘recipient’ block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 ‘recipient’ blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research. PMID:25285857

Concordance in diabetic foot ulceration: a cross-sectional study of agreement between wound swabbing and tissue sampling in infected ulcers.

PubMed

Nelson, E Andrea; Wright-Hughes, Alexandra; Brown, Sarah; Lipsky, Benjamin A; Backhouse, Michael; Bhogal, Moninder; Ndosi, Mwidimi; Reynolds, Catherine; Sykes, Gill; Dowson, Christopher; Edmonds, Michael; Vowden, Peter; Jude, Edward B; Dickie, Tom; Nixon, Jane

2016-11-01

There is inadequate evidence to advise clinicians on the relative merits of swabbing versus tissue sampling of infected diabetic foot ulcers (DFUs). To determine (1) concordance between culture results from wound swabs and tissue samples from the same ulcer; (2) whether or not differences in bacterial profiles from swabs and tissue samples are clinically relevant; (3) concordance between results from conventional culture versus polymerase chain reaction (PCR); and (4) prognosis for patients with an infected DFU at 12 months' follow-up. This was a cross-sectional, multicentre study involving patients with diabetes and a foot ulcer that was deemed to be infected by their clinician. Microbiology specimens for culture were taken contemporaneously by swab and by tissue sampling from the same wound. In a substudy, specimens were also processed by PCR. A virtual 'blinded' clinical review compared the appropriateness of patients' initial antibiotic regimens based on the results of swab and tissue specimens. Patients' case notes were reviewed at 12 months to assess prognosis. The main study recruited 400 patients, with 247 patients in the clinical review. There were 12 patients in the PCR study and 299 patients in the prognosis study. Patients' median age was 63 years (range 26-99 years), their diabetes duration was 15 years (range 2 weeks-57 years), and their index ulcer duration was 1.8 months (range 3 days-12 years). Half of the ulcers were neuropathic and the remainder were ischaemic/neuroischaemic. Tissue results reported more than one pathogen in significantly more specimens than swabs {86.1% vs. 70.1% of patients, 15.9% difference [95% confidence interval (CI) 11.8% to 20.1%], McNemar's p -value < 0.0001}. The two sampling techniques reported a difference in the identity of pathogens for 58% of patients. The number of pathogens differed in 50.4% of patients. In the clinical review study, clinicians agreed on the need for a change in therapy for 73.3% of patients (considering swab and tissue results separately), but significantly more tissue than swab samples required a change in therapy. Compared with traditional culture, the PCR technique reported additional pathogens for both swab and tissue samples in six (50%) patients and reported the same pathogens in four (33.3%) patients and different pathogens in two (16.7%) patients. The estimated healing rate was 44.5% (95% CI 38.9% to 50.1%). At 12 months post sampling, 45 (15.1%) patients had died, 52 (17.4%) patients had a lower-extremity ipsilateral amputation and 18 (6.0%) patients had revascularisation surgery. We did not investigate the potential impact of microbiological information on care. We cannot determine if the improved information yield from tissue sampling is attributable to sample collection, sample handling, processing or reporting. Tissue sampling reported both more pathogens and more organisms overall than swabbing. Both techniques missed some organisms, with tissue sampling missing fewer than swabbing. Results from tissue sampling more frequently led to a (virtual) recommended change in therapy. Long-term prognosis for patients with an infected foot ulcer was poor. Research is needed to determine the effect of sampling/processing techniques on clinical outcomes and antibiotic stewardship. The National Institute for Health Research Health Technology Assessment programme.

Extraction efficiency and implications for absolute quantitation of propranolol in mouse brain, liver and kidney thin tissue sections using droplet-based liquid microjunction surface sampling-HPLC ESI-MS/MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kertesz, Vilmos; Weiskittel, Taylor M.; Vavek, Marissa

Currently, absolute quantitation aspects of droplet-based surface sampling for thin tissue analysis using a fully automated autosampler/HPLC-ESI-MS/MS system are not fully evaluated. Knowledge of extraction efficiency and its reproducibility is required to judge the potential of the method for absolute quantitation of analytes from thin tissue sections. Methods: Adjacent thin tissue sections of propranolol dosed mouse brain (10- μm-thick), kidney (10- μm-thick) and liver (8-, 10-, 16- and 24- μm-thick) were obtained. Absolute concentration of propranolol was determined in tissue punches from serial sections using standard bulk tissue extraction protocols and subsequent HPLC separations and tandem mass spectrometric analysis. Thesemore » values were used to determine propranolol extraction efficiency from the tissues with the droplet-based surface sampling approach. Results: Extraction efficiency of propranolol using 10- μm-thick brain, kidney and liver thin tissues using droplet-based surface sampling varied between ~45-63%. Extraction efficiency decreased from ~65% to ~36% with liver thickness increasing from 8 μm to 24 μm. Randomly selecting half of the samples as standards, precision and accuracy of propranolol concentrations obtained for the other half of samples as quality control metrics were determined. Resulting precision ( ±15%) and accuracy ( ±3%) values, respectively, were within acceptable limits. In conclusion, comparative quantitation of adjacent mouse thin tissue sections of different organs and of various thicknesses by droplet-based surface sampling and by bulk extraction of tissue punches showed that extraction efficiency was incomplete using the former method, and that it depended on the organ and tissue thickness. However, once extraction efficiency was determined and applied, the droplet-based approach provided the required quantitation accuracy and precision for assay validations. Furthermore, this means that once the extraction efficiency was calibrated for a given tissue type and drug, the droplet-based approach provides a non-labor intensive and high-throughput means to acquire spatially resolved quantitative analysis of multiple samples of the same type.« less

Extraction efficiency and implications for absolute quantitation of propranolol in mouse brain, liver and kidney thin tissue sections using droplet-based liquid microjunction surface sampling-HPLC ESI-MS/MS

DOE PAGES

Kertesz, Vilmos; Weiskittel, Taylor M.; Vavek, Marissa; ...

2016-06-22

Currently, absolute quantitation aspects of droplet-based surface sampling for thin tissue analysis using a fully automated autosampler/HPLC-ESI-MS/MS system are not fully evaluated. Knowledge of extraction efficiency and its reproducibility is required to judge the potential of the method for absolute quantitation of analytes from thin tissue sections. Methods: Adjacent thin tissue sections of propranolol dosed mouse brain (10- μm-thick), kidney (10- μm-thick) and liver (8-, 10-, 16- and 24- μm-thick) were obtained. Absolute concentration of propranolol was determined in tissue punches from serial sections using standard bulk tissue extraction protocols and subsequent HPLC separations and tandem mass spectrometric analysis. Thesemore » values were used to determine propranolol extraction efficiency from the tissues with the droplet-based surface sampling approach. Results: Extraction efficiency of propranolol using 10- μm-thick brain, kidney and liver thin tissues using droplet-based surface sampling varied between ~45-63%. Extraction efficiency decreased from ~65% to ~36% with liver thickness increasing from 8 μm to 24 μm. Randomly selecting half of the samples as standards, precision and accuracy of propranolol concentrations obtained for the other half of samples as quality control metrics were determined. Resulting precision ( ±15%) and accuracy ( ±3%) values, respectively, were within acceptable limits. In conclusion, comparative quantitation of adjacent mouse thin tissue sections of different organs and of various thicknesses by droplet-based surface sampling and by bulk extraction of tissue punches showed that extraction efficiency was incomplete using the former method, and that it depended on the organ and tissue thickness. However, once extraction efficiency was determined and applied, the droplet-based approach provided the required quantitation accuracy and precision for assay validations. Furthermore, this means that once the extraction efficiency was calibrated for a given tissue type and drug, the droplet-based approach provides a non-labor intensive and high-throughput means to acquire spatially resolved quantitative analysis of multiple samples of the same type.« less

Population Pharmacokinetic Modeling of the Unbound Levofloxacin Concentrations in Rat Plasma and Prostate Tissue Measured by Microdialysis

PubMed Central

Hurtado, Felipe K.; Weber, Benjamin; Derendorf, Hartmut; Hochhaus, Guenther

2014-01-01

Levofloxacin is a broad-spectrum fluoroquinolone used in the treatment of both acute and chronic bacterial prostatitis. Currently, the treatment of bacterial prostatitis is still difficult, especially due to the poor distribution of many antimicrobials into the prostate, thus preventing the drug to reach effective interstitial concentrations at the infection site. Newer fluoroquinolones show a greater penetration into the prostate. In the present study, we compared the unbound levofloxacin prostate concentrations measured by microdialysis to those in plasma after a 7-mg/kg intravenous bolus dose to Wistar rats. Plasma and dialysate samples were analyzed using a validated high-pressure liquid chromatography-fluorescence method. Both noncompartmental analysis (NCA) and population-based compartmental modeling (NONMEM 6) were performed. Unbound prostate tissue concentrations represented 78% of unbound plasma levels over a period of 12 h by comparing the extent of exposure (unbound AUC0–∞) of 6.4 and 4.8 h·μg/ml in plasma and tissue, respectively. A three-compartment model with simultaneous passive diffusion and saturable distribution kinetics from the prostate to the central compartment gave the best results in terms of curve fitting, precision of parameter estimates, and model stability. The following parameter values were estimated by the population model: V1 (0.38 liter; where V1 represents the volume of the central compartment), CL (0.22 liter/h), k12 (2.27 h−1), k21 (1.44 h−1), k13 (0.69 h−1), Vmax (7.19 μg/h), kM (0.35 μg/ml), V3/fuprostate (0.05 liter; where fuprostate represents the fraction unbound in the prostate), and k31 (3.67 h−1). The interindividual variability values for V1, CL, Vmax, and kM were 21, 37, 42, and 76%, respectively. Our results suggest that levofloxacin is likely to be substrate for efflux transporters in the prostate. PMID:24217697

Menstrual Blood as a Potential Source of Endometrial Derived CD3+ T Cells

PubMed Central

Sabbaj, Steffanie; Hel, Zdenek; Richter, Holly E.; Mestecky, Jiri; Goepfert, Paul A.

2011-01-01

Studies of T cell-mediated immunity in the human female genital tract have been problematic due to difficulties associated with the collection of mucosal samples. Consequently, most studies rely on biopsies from the lower female genital tract or remnant tissue from hysterectomies. Availability of samples from healthy women is limited, as most studies are carried out in women with underlying pathologies. Menstruation is the cyclical sloughing off of endometrial tissue, and thus it should be a source of endometrial cells without the need for a biopsy. We isolated and phenotyped T cells from menstrual and peripheral blood and from endometrial biopsy-derived tissue from healthy women to determine the types of T cells present in this compartment. Our data demonstrated that T cells isolated from menstrual blood are a heterogeneous population of cells with markers reminiscent of blood and mucosal cells as well as unique phenotypes not represented in either compartment. T cells isolated from menstrual blood expressed increased levels of HLA-DR, αEβ7 and CXCR4 and reduced levels of CD62L relative to peripheral blood. Menstrual blood CD4+ T cells were enriched for cells expressing both CCR7 and CD45RA, markers identifying naïve T cells and were functional as determined by antigen-specific intracellular cytokine production assays. These data may open new avenues of investigation for cell mediated immune studies involving the female reproductive tract without the need for biopsies. PMID:22174921

Expression of cytokine signaling genes in morbidly obese patients with non-alcoholic steatohepatitis and hepatic fibrosis.

PubMed

Estep, J Michael; Baranova, Ancha; Hossain, Noreen; Elariny, Hazem; Ankrah, Kathy; Afendy, Arian; Chandhoke, Vikas; Younossi, Zobair M

2009-05-01

White adipose tissue (WAT) from visceral adiposity plays an important role in the pathogenesis of non-alcoholic steatohepatitis (NASH). Development of NASH and its progression to fibrosis is partially due to cytokines and adipokines produced by WAT. The aim of this study was to assess the association of hepatic fibrosis and NASH by evaluating the intrinsic differences in the inflammatory cytokine signaling in the visceral adipose tissue obtained from morbidly obese patients. We used targeted microarrays representing human genes involved in the inflammatory and fibrogenic reactions to profile visceral adipose samples of 15 well-matched NASH patients with and without fibrosis. Additionally, visceral adipose samples were subjected to real-time polymerase chain reaction profiling of 84 inflammations related genes. Eight genes (CCL2, CCL4, CCL18, CCR1, IL10RB, IL15RA, and LTB) were differentially expressed in NASH with fibrosis. Additionally, an overlapping but distinct list of the differentially expressed genes were found in NASH with type II diabetes (DM; IL8, BLR1, IL2RA, CD40LG, IL1RN, IL15RA, and CCL4) as compared to NASH without DM. Inflammatory cytokines are differentially expressed in the adipose tissue of NASH with fibrosis, as well in NASH with DM. These findings point at the interaction of adipose inflammatory cytokines, DM, hepatic fibrosis in NASH, and its progression to cirrhosis and end-stage liver disease.

Simulation-assisted design of microfluidic sample traps for optimal trapping and culture of non-adherent single cells, tissues, and spheroids.

PubMed

Rousset, Nassim; Monet, Frédéric; Gervais, Thomas

2017-03-21

This work focuses on modelling design and operation of "microfluidic sample traps" (MSTs). MSTs regroup a widely used class of microdevices that incorporate wells, recesses or chambers adjacent to a channel to individually trap, culture and/or release submicroliter 3D tissue samples ranging from simple cell aggregates and spheroids, to ex vivo tissue samples and other submillimetre-scale tissue models. Numerous MST designs employing various trapping mechanisms have been proposed in the literature, spurring the development of 3D tissue models for drug discovery and personalized medicine. Yet, there lacks a general framework to optimize trapping stability, trapping time, shear stress, and sample metabolism. Herein, the effects of hydrodynamics and diffusion-reaction on tissue viability and device operation are investigated using analytical and finite element methods with systematic parametric sweeps over independent design variables chosen to correspond to the four design degrees of freedom. Combining different results, we show that, for a spherical tissue of diameter d < 500 μm, the simplest, closest to optimal trap shape is a cube of dimensions w equal to twice the tissue diameter: w = 2d. Furthermore, to sustain tissues without perfusion, available medium volume per trap needs to be 100× the tissue volume to ensure optimal metabolism for at least 24 hours.

A novel semi-quantitative method for measuring tissue bleeding.

PubMed

Vukcevic, G; Volarevic, V; Raicevic, S; Tanaskovic, I; Milicic, B; Vulovic, T; Arsenijevic, S

2014-03-01

In this study, we describe a new semi-quantitative method for measuring the extent of bleeding in pathohistological tissue samples. To test our novel method, we recruited 120 female patients in their first trimester of pregnancy and divided them into three groups of 40. Group I was the control group, in which no dilation was applied. Group II was an experimental group, in which dilation was performed using classical mechanical dilators. Group III was also an experimental group, in which dilation was performed using a hydraulic dilator. Tissue samples were taken from the patients' cervical canals using a Novak's probe via energetic single-step curettage prior to any dilation in Group I and after dilation in Groups II and III. After the tissue samples were prepared, light microscopy was used to obtain microphotographs at 100x magnification. The surfaces affected by bleeding were measured in the microphotographs using the Autodesk AutoCAD 2009 program and its "polylines" function. The lines were used to mark the area around the entire sample (marked A) and to create "polyline" areas around each bleeding area on the sample (marked B). The percentage of the total area affected by bleeding was calculated using the formula: N = Bt x 100 / At where N is the percentage (%) of the tissue sample surface affected by bleeding, At (A total) is the sum of the surfaces of all of the tissue samples and Bt (B total) is the sum of all the surfaces affected by bleeding in all of the tissue samples. This novel semi-quantitative method utilizes the Autodesk AutoCAD 2009 program, which is simple to use and widely available, thereby offering a new, objective and precise approach to estimate the extent of bleeding in tissue samples.

Application of an object-oriented programming paradigm in three-dimensional computer modeling of mechanically active gastrointestinal tissues.

PubMed

Rashev, P Z; Mintchev, M P; Bowes, K L

2000-09-01

The aim of this study was to develop a novel three-dimensional (3-D) object-oriented modeling approach incorporating knowledge of the anatomy, electrophysiology, and mechanics of externally stimulated excitable gastrointestinal (GI) tissues and emphasizing the "stimulus-response" principle of extracting the modeling parameters. The modeling method used clusters of class hierarchies representing GI tissues from three perspectives: 1) anatomical; 2) electrophysiological; and 3) mechanical. We elaborated on the first four phases of the object-oriented system development life-cycle: 1) analysis; 2) design; 3) implementation; and 4) testing. Generalized cylinders were used for the implementation of 3-D tissue objects modeling the cecum, the descending colon, and the colonic circular smooth muscle tissue. The model was tested using external neural electrical tissue excitation of the descending colon with virtual implanted electrodes and the stimulating current density distributions over the modeled surfaces were calculated. Finally, the tissue deformations invoked by electrical stimulation were estimated and represented by a mesh-surface visualization technique.

Listeria monocytogenes - Danger for health safety vegetable production.

PubMed

Kljujev, Igor; Raicevic, Vera; Jovicic-Petrovic, Jelena; Vujovic, Bojana; Mirkovic, Milica; Rothballer, Michael

2018-04-22

The microbiologically contaminated vegetables represent a risk for consumers, especially vegetables without thermal processing. It is known that human pathogen bacteria, such as Listeria monocytogenes, could exist on fresh vegetables. The fresh vegetables could become Listeria-contaminated if they come in touch with contaminated soil, manure, irrigation water. The aim of this work was to investigate the presence of Listeria spp. and L. monocytogenes in different kind of vegetables grown in field and greenhouse condition as well as surface and endophytic colonization plant roots of different vegetables species by L. monocytogenes in laboratory conditions. The detection of Listeria spp. and L. monocytogenes in vegetable samples was done using ISO and PCR methods. The investigation of colonization vegetable roots and detection Listeria-cells inside plant root tissue was done using Fluorescence in situ hybridization (FISH) method in combination with confocal laser scanning microscopy (CLSM). The results showed that 25.58% vegetable samples were positive for Listeria spp. and only one sample (carrot) was positive for L. monocytogenes out of 43 samples in total collected from field and greenhouse. The strain L. monocytogenes EGD-E surface and endophytic colonized carrot root in highest degree while strain L. monocytogenes SV4B was the most represented at leafy vegetable plants, such at lettuce (1.68 × 10 6  cells/mm 3 absolutely dry root) and spinach (1.39 × 10 6  cells/mm 3 absolutely dry root) root surface. The cells of L. monocytogenes SV4B were visible as single cells in interior tissue of plant roots (celery and sweet corn roots) as well as in the interior of the plant root cell at sweet corn root. The cells of L. monocytogenes EGD-E bind to the surface of the plant root and they were less commonly found out on root hair. In the inner layers of the root, those bacterial cells were inhabited intercellular spaces mainly as single cells very close to the larval vessels of root. Our results suggest that L. monocytogenes is very good endophytic colonizer of vegetable plant roots. Copyright © 2018 Elsevier Ltd. All rights reserved.

Microscopic dual-energy CT (microDECT): a flexible tool for multichannel ex vivo 3D imaging of biological specimens.

PubMed

Handschuh, S; Beisser, C J; Ruthensteiner, B; Metscher, B D

2017-07-01

Dual-energy computed tomography (DECT) uses two different x-ray energy spectra in order to differentiate between tissues, materials or elements in a single sample or patient. DECT is becoming increasingly popular in clinical imaging and preclinical in vivo imaging of small animal models, but there have been only very few reports on ex vivo DECT of biological samples at microscopic resolutions. The present study has three main aims. First, we explore the potential of microscopic DECT (microDECT) for delivering isotropic multichannel 3D images of fixed biological samples with standard commercial laboratory-based microCT setups at spatial resolutions reaching below 10 μm. Second, we aim for retaining the maximum image resolution and quality during the material decomposition. Third, we want to test the suitability for microDECT imaging of different contrast agents currently used for ex vivo staining of biological samples. To address these aims, we used microCT scans of four different samples stained with x-ray dense contrast agents. MicroDECT scans were acquired with five different commercial microCT scanners from four companies. We present a detailed description of the microDECT workflow, including sample preparation, image acquisition, image processing and postreconstruction material decomposition, which may serve as practical guide for applying microDECT. The MATLAB script (The Mathworks Inc., Natick, MA, USA) used for material decomposition (including a graphical user interface) is provided as a supplement to this paper (https://github.com/microDECT/DECTDec). In general, the presented microDECT workflow yielded satisfactory results for all tested specimens. Original scan resolutions have been mostly retained in the separate material fractions after basis material decomposition. In addition to decomposition of mineralized tissues (inherent sample contrast) and stained soft tissues, we present a case of double labelling of different soft tissues with subsequent material decomposition. We conclude that, in contrast to in vivo DECT examinations, small ex vivo specimens offer some clear advantages regarding technical parameters of the microCT setup and the use of contrast agents. These include a higher flexibility in source peak voltages and x-ray filters, a lower degree of beam hardening due to small sample size, the lack of restriction to nontoxic contrast agents and the lack of a limit in exposure time and radiation dose. We argue that microDECT, because of its flexibility combined with already established contrast agents and the vast number of currently unexploited stains, will in future represent an important technique for various applications in biological research. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Functional similarity and molecular divergence of a novel reproductive transcriptome in two male-pregnant Syngnathus pipefish species

PubMed Central

Small, Clayton M; Harlin-Cognato, April D; Jones, Adam G

2013-01-01

Evolutionary studies have revealed that reproductive proteins in animals and plants often evolve more rapidly than the genome-wide average. The causes of this pattern, which may include relaxed purifying selection, sexual selection, sexual conflict, pathogen resistance, reinforcement, or gene duplication, remain elusive. Investigative expansions to additional taxa and reproductive tissues have the potential to shed new light on this unresolved problem. Here, we embark on such an expansion, in a comparison of the brood-pouch transcriptome between two male-pregnant species of the pipefish genus Syngnathus. Male brooding tissues in syngnathid fishes represent a novel, nonurogenital reproductive trait, heretofore mostly uncharacterized from a molecular perspective. We leveraged next-generation sequencing (Roche 454 pyrosequencing) to compare transcript abundance in the male brooding tissues of pregnant with nonpregnant samples from Gulf (S. scovelli) and dusky (S. floridae) pipefish. A core set of protein-coding genes, including multiple members of astacin metalloprotease and c-type lectin gene families, is consistent between species in both the direction and magnitude of expression bias. As predicted, coding DNA sequence analysis of these putative “male pregnancy proteins” suggests rapid evolution relative to nondifferentially expressed genes and reflects signatures of adaptation similar in magnitude to those reported from Drosophila male accessory gland proteins. Although the precise drivers of male pregnancy protein divergence remain unknown, we argue that the male pregnancy transcriptome in syngnathid fishes, a clade diverse with respect to brooding morphology and mating system, represents a unique and promising object of study for understanding the perplexing evolutionary nature of reproductive molecules. PMID:24324861

Demographics of Head and Neck Cancer Patients: A Single Institution Experience

PubMed Central

Kitanova, Martina; Dzhenkov, Deyan L; Ghenev, Peter; Sapundzhiev, Nikolay

2017-01-01

Introduction Head and neck cancer (HNC) comprises a diverse group of oncological entities, originating from various tissue types and organ localizations, situated in the topographical regions of the head and neck (H&N). This single institution retrospective study was aimed at establishing the HNC patient demographics and categorizing the individual incidence of H&N malignancies, regarding their organ of origin and main histopathological type. Materials and methods All histologically verified cases of HNC from a single tertiary referral center were reviewed in a descriptive retrospective manner. Data sampling period was 47 months. Results Male to female ratio of the registered HNC cases was 3.24:1. The mean age of diagnosis was 63.84 ± 12.65 years, median 65 years. The most common HNC locations include the larynx 30.37% (n = 188), lips and oral cavity 29.08% (n = 180), pharynx 20.03% (n = 124) and salivary glands 10.94% (n = 68), with other locations such as the external nose, nasal cavity and sinuses and auricle and external ear canal harboring a minority of the cases. The main histopathological groups include squamous cell carcinoma 76.74% (n = 475) and adenocarcinoma 6.14% (n = 38), with other malignant entries such as other epithelial malignancies, primary tonsillar, mucosa-associated lymphoid tissue or parenchymal lymphomas, connective tissue neoplasias, neuroendocrine and vascular malignancies diagnosed in a minority of cases. Conclusion Considered to be relatively rare, HNC represents a diverse group of oncological entities with individual and specific demographic characteristics. The reported single institution results appear representative of the national incidence and characteristics of HNC. PMID:28875091

Pharmacokinetics of enrofloxacin and ceftiofur in plasma, interstitial fluid, and gastrointestinal tract of calves after subcutaneous injection, and bactericidal impacts on representative enteric bacteria.

PubMed

Foster, D M; Jacob, M E; Warren, C D; Papich, M G

2016-02-01

This study's objectives were to determine intestinal antimicrobial concentrations in calves administered enrofloxacin or ceftiofur sodium subcutaneously, and their impact on representative enteric bacteria. Ultrafiltration devices were implanted in the ileum and colon of 12 steers, which received either enrofloxacin or ceftiofur sodium. Samples were collected over 48 h after drug administration for pharmacokinetic/pharmacodynamic analysis. Enterococcus faecalis or Salmonella enterica (5 × 10(5) CFU/mL of each) were exposed in vitro to peak and tail (48 h postadministration) concentrations of both drugs at each location for 24 h to determine inhibition of growth and change in MIC. Enrofloxacin had tissue penetration factors of 1.6 and 2.5 in the ileum and colon, while ciprofloxacin, an active metabolite of enrofloxacin, was less able to cross into the intestine (tissue penetration factors of 0.7 and 1.7). Ceftiofur was rapidly eliminated leading to tissue penetration factors of 0.39 and 0.25. All concentrations of enrofloxacin were bactericidal for S. enterica and significantly reduced E. faecalis. Peak ceftiofur concentration was bactericidal for S. enterica, and tail concentrations significantly reduced growth. E. faecalis experienced growth at all ceftiofur concentrations. The MICs for both organisms exposed to peak and tail concentrations of antimicrobials were unchanged at the end of the study. Enrofloxacin and ceftiofur achieved intestinal concentrations capable of reducing intestinal bacteria, yet the short exposure of ceftiofur in the intestine may select for resistant organisms. © 2015 John Wiley & Sons Ltd.

High-throughput simultaneous analysis of RNA, protein, and lipid biomarkers in heterogeneous tissue samples.

PubMed

Reiser, Vladimír; Smith, Ryan C; Xue, Jiyan; Kurtz, Marc M; Liu, Rong; Legrand, Cheryl; He, Xuanmin; Yu, Xiang; Wong, Peggy; Hinchcliffe, John S; Tanen, Michael R; Lazar, Gloria; Zieba, Renata; Ichetovkin, Marina; Chen, Zhu; O'Neill, Edward A; Tanaka, Wesley K; Marton, Matthew J; Liao, Jason; Morris, Mark; Hailman, Eric; Tokiwa, George Y; Plump, Andrew S

2011-11-01

With expanding biomarker discovery efforts and increasing costs of drug development, it is critical to maximize the value of mass-limited clinical samples. The main limitation of available methods is the inability to isolate and analyze, from a single sample, molecules requiring incompatible extraction methods. Thus, we developed a novel semiautomated method for tissue processing and tissue milling and division (TMAD). We used a SilverHawk atherectomy catheter to collect atherosclerotic plaques from patients requiring peripheral atherectomy. Tissue preservation by flash freezing was compared with immersion in RNAlater®, and tissue grinding by traditional mortar and pestle was compared with TMAD. Comparators were protein, RNA, and lipid yield and quality. Reproducibility of analyte yield from aliquots of the same tissue sample processed by TMAD was also measured. The quantity and quality of biomarkers extracted from tissue prepared by TMAD was at least as good as that extracted from tissue stored and prepared by traditional means. TMAD enabled parallel analysis of gene expression (quantitative reverse-transcription PCR, microarray), protein composition (ELISA), and lipid content (biochemical assay) from as little as 20 mg of tissue. The mean correlation was r = 0.97 in molecular composition (RNA, protein, or lipid) between aliquots of individual samples generated by TMAD. We also demonstrated that it is feasible to use TMAD in a large-scale clinical study setting. The TMAD methodology described here enables semiautomated, high-throughput sampling of small amounts of heterogeneous tissue specimens by multiple analytical techniques with generally improved quality of recovered biomolecules.

A qualitative signature for early diagnosis of hepatocellular carcinoma based on relative expression orderings.

PubMed

Ao, Lu; Zhang, Zimei; Guan, Qingzhou; Guo, Yating; Guo, You; Zhang, Jiahui; Lv, Xingwei; Huang, Haiyan; Zhang, Huarong; Wang, Xianlong; Guo, Zheng

2018-04-23

Currently, using biopsy specimens to confirm suspicious liver lesions of early hepatocellular carcinoma are not entirely reliable because of insufficient sampling amount and inaccurate sampling location. It is necessary to develop a signature to aid early hepatocellular carcinoma diagnosis using biopsy specimens even when the sampling location is inaccurate. Based on the within-sample relative expression orderings of gene pairs, we identified a simple qualitative signature to distinguish both hepatocellular carcinoma and adjacent non-tumour tissues from cirrhosis tissues of non-hepatocellular carcinoma patients. A signature consisting of 19 gene pairs was identified in the training data sets and validated in 2 large collections of samples from biopsy and surgical resection specimens. For biopsy specimens, 95.7% of 141 hepatocellular carcinoma tissues and all (100%) of 108 cirrhosis tissues of non-hepatocellular carcinoma patients were correctly classified. Especially, all (100%) of 60 hepatocellular carcinoma adjacent normal tissues and 77.5% of 80 hepatocellular carcinoma adjacent cirrhosis tissues were classified to hepatocellular carcinoma. For surgical resection specimens, 99.7% of 733 hepatocellular carcinoma specimens were correctly classified to hepatocellular carcinoma, while 96.1% of 254 hepatocellular carcinoma adjacent cirrhosis tissues and 95.9% of 538 hepatocellular carcinoma adjacent normal tissues were classified to hepatocellular carcinoma. In contrast, 17.0% of 47 cirrhosis from non-hepatocellular carcinoma patients waiting for liver transplantation were classified to hepatocellular carcinoma, indicating that some patients with long-lasting cirrhosis could have already gained hepatocellular carcinoma characteristics. The signature can distinguish both hepatocellular carcinoma tissues and tumour-adjacent tissues from cirrhosis tissues of non-hepatocellular carcinoma patients even using inaccurately sampled biopsy specimens, which can aid early diagnosis of hepatocellular carcinoma. © 2018 The Authors. Liver International Published by John Wiley & Sons Ltd.

Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type.

PubMed

Smither, Sophie J; Weller, Simon A; Phelps, Amanda; Eastaugh, Lin; Ngugi, Sarah; O'Brien, Lyn M; Steward, Jackie; Lonsdale, Steve G; Lever, Mark S

2015-10-01

Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-resource, outbreak scenarios. The EBOV inactivation efficacy of Buffer AVL (Qiagen) was tested against marmoset serum (EBOV concentration of 1 × 10(8) 50% tissue culture infective dose per milliliter [TCID50 · ml(-1)]) and murine blood (EBOV concentration of 1 × 10(7) TCID50 · ml(-1)) at 4:1 vol/vol buffer/sample ratios. Posttreatment cell culture and enzyme-linked immunosorbent assay (ELISA) analysis indicated that treatment with Buffer AVL did not inactivate EBOV in 67% of samples, indicating that Buffer AVL, which is designed for RNA extraction and not virus inactivation, cannot be guaranteed to inactivate EBOV in diagnostic samples. Murine blood samples treated with ethanol (4:1 [vol/vol] ethanol/sample) or heat (60°C for 15 min) also showed no viral inactivation in 67% or 100% of samples, respectively. However, combined Buffer AVL and ethanol or Buffer AVL and heat treatments showed total viral inactivation in 100% of samples tested. The Buffer AVL plus ethanol and Buffer AVL plus heat treatments were also shown not to affect the extraction of PCR quality RNA from EBOV-spiked murine blood samples. © Crown copyright 2015.

Evaluation of Two Enzyme-Linked Immunosorbent Assay Kits for Chikungunya Virus IgM Using Samples from Deceased Organ and Tissue Donors.

PubMed

Prince, Harry E; Altrich, Michelle L; Nowicki, Marek J

2016-10-01

The identification of nearly 3,500 cases of chikungunya virus (CHIKV) infection in U.S. residents returning in 2014 and 2015 from areas in which it is endemic has raised concerns within the transplant community that, should recently infected individuals become organ and/or tissue donors, CHIKV would be transmitted to transplant recipients. Thus, tests designed to detect recent CHIKV infection among U.S. organ and tissue donors may become necessary in the future. Accordingly, we evaluated 2 enzyme-linked immunosorbent assays (ELISAs) for CHIKV IgM readily available in the United States using 1,000 deidentified serum or plasma specimens collected from donors between November 2014 and March 2015. The Euroimmun indirect ELISA identified 38 reactive specimens; however, all 38 were negative for CHIKV IgG and IgM in immunofluorescence assays (IFAs) conducted at a reference laboratory and, thus, were falsely reactive in the Euroimmun CHIKV IgM assay. The InBios IgM-capture ELISA identified 26 reactive samples, and one was still reactive (index ≥ 1.00) when retested using the InBios kit with a background subtraction modification to identify false reactivity. This reactive specimen was CHIKV IgM negative but IgG positive by IFAs at two reference laboratories; plaque reduction neutralization testing (PRNT) demonstrated CHIKV-specific reactivity. The IgG and PRNT findings strongly suggest that the InBios CHIKV IgM-reactive result represents true reactivity, even though the IgM IFA result was negative. If testing organ/tissue donors for CHIKV IgM becomes necessary, the limitations of the currently available CHIKV IgM ELISAs and options for their optimization must be understood to avoid organ/tissue wastage due to falsely reactive results. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Molecular analysis of infectious bursal disease virus from bursal tissues collected on FTA filter paper.

PubMed

Moscoso, Hugo; Alvarado, Ivan; Hofacre, Charles L

2006-09-01

We investigated the feasibility of using FTA filter cards for the storage of bursas of Fabricius containing infectious bursal disease virus (IBDV) and for IBDV detection by reverse transcriptase (RT)-polymerase chain reaction (PCR), and characterization by restriction fragment length polymorphism (RFLP) or nucleotide sequencing. The FTA card is a cotton-based cellulose membrane containing lyophilized chemicals that lyses many types of bacteria and viruses. IBDV was inactivated upon contact with the FTA as shown by the inability of the virus to be propagated in embryonating chicken eggs. Viral RNA in minced bursas or stamped bursas could be amplified by RT-PCR (VP2 gene fragment, 248 base pairs) after storage on FTA for at least 15 days at room temperature or 8 mo at -20 C. Analytical sensitivity of the test was between 0.5-5 ng of RNA template or 5 x 10(1) mean tissue culture infective dose (TCID50)/FTA spot. Detection rate of IBDV in domestic clinical samples collected on FTA or collected by the non-FTA standard procedure was 36.7% and 41.7%, respectively, which represents 88% agreement. Detection of IBDV from FTA cards inoculated with bursal tissues in the laboratory or in the field was 36.7% and 37.1%, respectively. Detection of IBDV from FTA samples when the cards were inoculated with bursal tissues and sent through customs into the United States was 32.9%. Analysis of the amplified products showed that molecular characterization of IBDV by RFLP or nucleotide sequencing is feasible in bursas stored on FTA at 25 C for 1-3 mo or at -20 C for at least 8 mo. The use of FTA for the collection of bursal tissues and simultaneous inactivation of IBDV allows the movement of specimens within the United States and also from outside the United States in compliance with federal regulations and in a manner adequate for molecular characterization.

Standardization and validation of real time PCR assays for the diagnosis of histoplasmosis using three molecular targets in an animal model.

PubMed

López, Luisa F; Muñoz, César O; Cáceres, Diego H; Tobón, Ángela M; Loparev, Vladimir; Clay, Oliver; Chiller, Tom; Litvintseva, Anastasia; Gade, Lalitha; González, Ángel; Gómez, Beatriz L

2017-01-01

Histoplasmosis is considered one of the most important endemic and systemic mycoses worldwide. Until now few molecular techniques have been developed for its diagnosis. The aim of this study was to develop and evaluate three real time PCR (qPCR) protocols for different protein-coding genes (100-kDa, H and M antigens) using an animal model. Fresh and formalin-fixed and paraffin-embedded (FFPE) lung tissues from BALB/c mice inoculated i.n. with 2.5x106 Histoplasma capsulatum yeast or PBS were obtained at 1, 2, 3, 4, 8, 12 and 16 weeks post-infection. A collection of DNA from cultures representing different clades of H. capsulatum (30 strains) and other medically relevant pathogens (36 strains of related fungi and Mycobacterium tuberculosis) were used to analyze sensitivity and specificity. Analytical sensitivity and specificity were 100% when DNAs from the different strains were tested. The highest fungal burden occurred at first week post-infection and complete fungal clearance was observed after the third week; similar results were obtained when the presence of H. capsulatum yeast cells was demonstrated in histopathological analysis. In the first week post-infection, all fresh and FFPE lung tissues from H. capsulatum-infected animals were positive for the qPCR protocols tested except for the M antigen protocol, which gave variable results when fresh lung tissue samples were analyzed. In the second week, all qPCR protocols showed variable results for both fresh and FFPE tissues. Samples from the infected mice at the remaining times post-infection and uninfected mice (controls) were negative for all protocols. Good agreement was observed between CFUs, histopathological analysis and qPCR results for the 100-kDa and H antigen protocols. We successfully standardized and validated three qPCR assays for detecting H. capsulatum DNA in fresh and FFPE tissues, and conclude that the 100-kDa and H antigen molecular assays are promising tests for diagnosing this mycosis.

Plasma ctDNA RAS mutation analysis for the diagnosis and treatment monitoring of metastatic colorectal cancer patients

PubMed Central

Vidal, J; Muinelo, L; Dalmases, A; Jones, F; Edelstein, D; Iglesias, M; Orrillo, M; Abalo, A; Rodríguez, C; Brozos, E; Vidal, Y; Candamio, S; Vázquez, F; Ruiz, J; Guix, M; Visa, L; Sikri, V; Albanell, J; Bellosillo, B; López, R; Montagut, C

2017-01-01

Abstract Background RAS assessment is mandatory for therapy decision in metastatic colorectal cancer (mCRC) patients. This determination is based on tumor tissue, however, genotyping of circulating tumor (ct)DNA offers clear advantages as a minimally invasive method that represents tumor heterogeneity. Our study aims to evaluate the use of ctDNA as an alternative for determining baseline RAS status and subsequent monitoring of RAS mutations during therapy as a component of routine clinical practice. Patients and methods RAS mutational status in plasma was evaluated in mCRC patients by OncoBEAM™ RAS CRC assay. Concordance of results in plasma and tissue was retrospectively evaluated. RAS mutations were also prospectively monitored in longitudinal plasma samples from selected patients. Results Analysis of RAS in tissue and plasma samples from 115 mCRC patients showed a 93% overall agreement. Plasma/tissue RAS discrepancies were mainly explained by spatial and temporal tumor heterogeneity. Analysis of clinico-pathological features showed that the site of metastasis (i.e. peritoneal, lung), the histology of the tumor (i.e. mucinous) and administration of treatment previous to blood collection negatively impacted the detection of RAS in ctDNA. In patients with baseline mutant RAS tumors treated with chemotherapy/antiangiogenic, longitudinal analysis of RAS ctDNA mirrored response to treatment, being an early predictor of response. In patients RAS wt, longitudinal monitoring of RAS ctDNA revealed that OncoBEAM was useful to detect emergence of RAS mutations during anti-EGFR treatment. Conclusion The high overall agreement in RAS mutational assessment between plasma and tissue supports blood-based testing with OncoBEAM™ as a viable alternative for genotyping RAS of mCRC patients in routine clinical practice. Our study describes practical clinico-pathological specifications to optimize RAS ctDNA determination. Moreover, OncoBEAM™ is useful to monitor RAS in patients undergoing systemic therapy to detect resistance and evaluate the efficacy of particular treatments. PMID:28419195

Avian mercury exposure and toxicological risk across western North America: A synthesis

USGS Publications Warehouse

Ackerman, Joshua T.; Eagles-Smith, Collin A.; Herzog, Mark; Hartman, Christopher; Peterson, Sarah; Evers, David C.; Jackson, Allyson K.; Elliott, John E.; Vander Pol, Stacy S.; Bryan, Colleen E.

2016-01-01

Methylmercury contamination of the environment is an important issue globally, and birds are useful bioindicators for mercury monitoring programs. The available data on mercury contamination of birds in western North America were synthesized. Original data from multiple databases were obtained and a literature review was conducted to obtain additional mercury concentrations. In total, 29219 original bird mercury concentrations from 225 species were compiled, and an additional 1712 mean mercury concentrations, representing 19998 individuals and 176 species, from 200 publications were obtained. To make mercury data comparable across bird tissues, published equations of tissue mercury correlations were used to convert all mercury concentrations into blood-equivalent mercury concentrations. Blood-equivalent mercury concentrations differed among species, foraging guilds, habitat types, locations, and ecoregions. Piscivores and carnivores exhibited the greatest mercury concentrations, whereas herbivores and granivores exhibited the lowest mercury concentrations. Bird mercury concentrations were greatest in ocean and salt marsh habitats and lowest in terrestrial habitats. Bird mercury concentrations were above toxicity benchmarks in many areas throughout western North America, and multiple hotspots were identified. Additionally, published toxicity benchmarks established in multiple tissues were summarized and translated into a common blood-equivalent mercury concentration. Overall, 66% of birds sampled in western North American exceeded a blood-equivalent mercury concentration of 0.2 μg/g wet weight (ww; above background levels), which is the lowest-observed effect level, 28% exceeded 1.0 μg/g ww (moderate risk), 8% exceeded 3.0 μg/g ww (high risk), and 4% exceeded 4.0 μg/g ww (severe risk). Mercury monitoring programs should sample bird tissues, such as adult blood and eggs, that are most-easily translated into tissues with well-developed toxicity benchmarks and that are directly relevant to bird reproduction. Results indicate that mercury contamination of birds is prevalent in many areas throughout western North America, and large-scale ecological attributes are important factors influencing bird mercury concentrations.

Avian mercury exposure and toxicological risk across western North America: A synthesis

PubMed Central

Ackerman, Joshua T.; Eagles-Smith, Collin A.; Herzog, Mark P.; Hartman, C. Alex; Peterson, Sarah H.; Evers, David C.; Jackson, Allyson K.; Elliott, John E.; Vander Pol, Stacy S.; Bryan, Colleen E.

2017-01-01

Methylmercury contamination of the environment is an important issue globally and birds are useful bioindicators for mercury monitoring programs. The available data on mercury contamination of birds in western North America were synthesized. Original data from multiple databases were obtained and a literature review was conducted to obtain additional mercury concentrations. In total, 29219 original bird mercury concentrations from 225 species were compiled, and an additional 1712 mean mercury concentrations, representing 19998 individuals and 176 species, from 200 publications were obtained. To make mercury data comparable across bird tissues, published equations of tissue mercury correlations were used to convert all mercury concentrations into blood-equivalent mercury concentrations. Blood-equivalent mercury concentrations differed among species, foraging guilds, habitat types, locations, and ecoregions. Piscivores and carnivores exhibited the greatest mercury concentrations, whereas herbivores and granivores exhibited the lowest mercury concentrations. Bird mercury concentrations were greatest in ocean and salt marsh habitats and lowest in terrestrial habitats. Bird mercury concentrations were above toxicity benchmarks in many areas throughout western North America, and multiple hotspots were identified. Additionally, published toxicity benchmarks established in multiple tissues were summarized and translated into a common blood-equivalent mercury concentration. Overall, 66% of birds sampled in western North American exceeded a blood-equivalent mercury concentration of 0.2 μg/g wet weight (ww; above background levels), which is the lowest-observed effect level, 28% exceeded 1.0 μg/g ww (moderate risk), 8% exceeded 3.0 μg/g ww (high risk), and 4% exceeded 4.0 μg/g ww (severe risk). Mercury monitoring programs should sample bird tissues, such as adult blood and eggs, that are most-easily translated into tissues with well-developed toxicity benchmarks and that are directly relevant to bird reproduction. Results indicate that mercury contamination of birds is prevalent in many areas throughout western North America, and large-scale ecological attributes are important factors influencing bird mercury concentrations. PMID:27093907

Determination of diagnostic standards on saturated soil extracts for cut roses grown in greenhouses

PubMed Central

Cabrera, Raúl Iskander

2017-01-01

This work comprises the theoretical determination and validation of diagnostic standards for the analysis of saturated soil extracts for cut rose flower crops (Rosa spp.) growing in the Bogota Plateau, Colombia. The data included 684 plant tissue analyses and 684 corresponding analyses of saturated soil extracts, all collected between January 2009 and June 2013. The tissue and soil samples were selected from 13 rose farms, and from cultivars grafted on the 'Natal Briar' rootstock. These concurrent samples of soil and plant tissues represented 251 production units (locations) of approximately 10,000 m2 distributed across the study area. The standards were conceived as a tool to improve the nutritional balance in the leaf tissue of rose plants and thereby define the norms for expressing optimum productive potential relative to nutritional conditions in the soil. To this end, previously determined diagnostic standard for rose leaf tissues were employed to obtain rates of foliar nutritional balance at each analyzed location and as criteria for determining the diagnostic norms for saturated soil extracts. Implementing this methodology to foliar analysis, showed a higher significant correlation for diagnostic indices. A similar behavior was observed in saturated soil extracts analysis, becoming a powerful tool for integrated nutritional diagnosis. Leaf analyses determine the most limiting nutrients for high yield and analyses of saturated soil extracts facilitate the possibility of correcting the fertigation formulations applied to soils or substrates. Recommendations are proposed to improve the balance in soil-plant system with which the possibility of yield increase becomes more probable. The main recommendations to increase and improve rose crop flower yields would be: continuously check pH values of SSE, reduce the amounts of P, Fe, Zn and Cu in fertigation solutions and carefully analyze the situation of Mn in the soil-plant system. PMID:28542547

76 FR 24862 - Proposed Information Collection; Comment Request; Protocol for Access to Tissue Specimen Samples...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-05-03

... Collection; Comment Request; Protocol for Access to Tissue Specimen Samples From the National Marine Mammal Tissue Bank AGENCY: National Oceanic and Atmospheric Administration (NOAA), Commerce. ACTION: Notice... National Marine Mammal Tissue Bank (NMMTB) was established by the National Marine Fisheries Service (NMFS...

Specimen Sample Preservation for Cell and Tissue Cultures

NASA Technical Reports Server (NTRS)

Meeker, Gabrielle; Ronzana, Karolyn; Schibner, Karen; Evans, Robert

1996-01-01

The era of the International Space Station with its longer duration missions will pose unique challenges to microgravity life sciences research. The Space Station Biological Research Project (SSBRP) is responsible for addressing these challenges and defining the science requirements necessary to conduct life science research on-board the International Space Station. Space Station will support a wide range of cell and tissue culture experiments for durations of 1 to 30 days. Space Shuttle flights to bring experimental samples back to Earth for analyses will only occur every 90 days. Therefore, samples may have to be retained for periods up to 60 days. This presents a new challenge in fresh specimen sample storage for cell biology. Fresh specimen samples are defined as samples that are preserved by means other than fixation and cryopreservation. The challenge of long-term storage of fresh specimen samples includes the need to suspend or inhibit proliferation and metabolism pending return to Earth-based laboratories. With this challenge being unique to space research, there have not been any ground based studies performed to address this issue. It was decided hy SSBRP that experiment support studies to address the following issues were needed: Fixative Solution Management; Media Storage Conditions; Fresh Specimen Sample Storage of Mammalian Cell/Tissue Cultures; Fresh Specimen Sample Storage of Plant Cell/Tissue Cultures; Fresh Specimen Sample Storage of Aquatic Cell/Tissue Cultures; and Fresh Specimen Sample Storage of Microbial Cell/Tissue Cultures. The objective of these studies was to derive a set of conditions and recommendations that can be used in a long duration microgravity environment such as Space Station that will permit extended storage of cell and tissue culture specimens in a state consistent with zero or minimal growth, while at the same time maintaining their stability and viability.

A random sampling approach for robust estimation of tissue-to-plasma ratio from extremely sparse data.

PubMed

Chu, Hui-May; Ette, Ene I

2005-09-02

his study was performed to develop a new nonparametric approach for the estimation of robust tissue-to-plasma ratio from extremely sparsely sampled paired data (ie, one sample each from plasma and tissue per subject). Tissue-to-plasma ratio was estimated from paired/unpaired experimental data using independent time points approach, area under the curve (AUC) values calculated with the naïve data averaging approach, and AUC values calculated using sampling based approaches (eg, the pseudoprofile-based bootstrap [PpbB] approach and the random sampling approach [our proposed approach]). The random sampling approach involves the use of a 2-phase algorithm. The convergence of the sampling/resampling approaches was investigated, as well as the robustness of the estimates produced by different approaches. To evaluate the latter, new data sets were generated by introducing outlier(s) into the real data set. One to 2 concentration values were inflated by 10% to 40% from their original values to produce the outliers. Tissue-to-plasma ratios computed using the independent time points approach varied between 0 and 50 across time points. The ratio obtained from AUC values acquired using the naive data averaging approach was not associated with any measure of uncertainty or variability. Calculating the ratio without regard to pairing yielded poorer estimates. The random sampling and pseudoprofile-based bootstrap approaches yielded tissue-to-plasma ratios with uncertainty and variability. However, the random sampling approach, because of the 2-phase nature of its algorithm, yielded more robust estimates and required fewer replications. Therefore, a 2-phase random sampling approach is proposed for the robust estimation of tissue-to-plasma ratio from extremely sparsely sampled data.

In Vitro Tissue Differentiation using Dynamics of Tissue Mechanical Properties

NASA Astrophysics Data System (ADS)

Lin, Wei-Chiang; Phillips, Paul J.

2002-03-01

Dynamics of tissue mechanical properties of various human tissue types were studied at macroscopic as well as microscopic level in vitro. This study was conducted to enable the development of a feedback system based on dynamics of tissue mechanical properties for intraoperative guidance for tumor treatment (e.g., RF ablation of liver tumor) and noninvasive tumor localization. Human liver tissues, including normal, cancerous, and cirrhotic tissues, were obtained from patients receiving liver transplant or tumor resection at Vanderbilt University Medical Center with the approval of the Vanderbilt Institutional Review Board. Tissue samples, once resected from the patients, were snap-frozen using liquid nitrogen and stored at -70 oC. Measurements of the mechanical properties of these tissue samples were conducted at the University of Tennessee at Knoxville. Dynamics of tissue mechanical properties were measured from both native and thermally coagulated tissue samples at macroscopic and microscopic level. Preliminary results suggest the dynamics of mechanical properties of normal liver tissues are very different from those of cancerous liver tissues. The correlation between the dynamics of mechanical properties at macroscopic level and those at microscopic level is currently under investigation.

An efficient, reliable and inexpensive device for the rapid homogenization of multiple tissue samples by centrifugation.

PubMed

Ilyin, S E; Plata-Salamán, C R

2000-02-15

Homogenization of tissue samples is a common first step in the majority of current protocols for RNA, DNA, and protein isolation. This report describes a simple device for centrifugation-mediated homogenization of tissue samples. The method presented is applicable to RNA, DNA, and protein isolation, and we show examples where high quality total cell RNA, DNA, and protein were obtained from brain and other tissue samples. The advantages of the approach presented include: (1) a significant reduction in time investment relative to hand-driven or individual motorized-driven pestle homogenization; (2) easy construction of the device from inexpensive parts available in any laboratory; (3) high replicability in the processing; and (4) the capacity for the parallel processing of multiple tissue samples, thus allowing higher efficiency, reliability, and standardization.

The classification of secondary colorectal liver cancer in human biopsy samples using angular dispersive x-ray diffraction and multivariate analysis

NASA Astrophysics Data System (ADS)

Theodorakou, Chrysoula; Farquharson, Michael J.

2009-08-01

The motivation behind this study is to assess whether angular dispersive x-ray diffraction (ADXRD) data, processed using multivariate analysis techniques, can be used for classifying secondary colorectal liver cancer tissue and normal surrounding liver tissue in human liver biopsy samples. The ADXRD profiles from a total of 60 samples of normal liver tissue and colorectal liver metastases were measured using a synchrotron radiation source. The data were analysed for 56 samples using nonlinear peak-fitting software. Four peaks were fitted to all of the ADXRD profiles, and the amplitude, area, amplitude and area ratios for three of the four peaks were calculated and used for the statistical and multivariate analysis. The statistical analysis showed that there are significant differences between all the peak-fitting parameters and ratios between the normal and the diseased tissue groups. The technique of soft independent modelling of class analogy (SIMCA) was used to classify normal liver tissue and colorectal liver metastases resulting in 67% of the normal tissue samples and 60% of the secondary colorectal liver tissue samples being classified correctly. This study has shown that the ADXRD data of normal and secondary colorectal liver cancer are statistically different and x-ray diffraction data analysed using multivariate analysis have the potential to be used as a method of tissue classification.

Increased expression of insulin-like growth factor-1 receptor is correlated with worse survival in canine appendicular osteosarcoma.

PubMed

Maniscalco, Lorella; Iussich, Selina; Morello, Emanuela; Martano, Marina; Gattino, Francesca; Miretti, Silvia; Biolatti, Bartolomeo; Accornero, Paolo; Martignani, Eugenio; Sánchez-Céspedes, Raquel; Buracco, Paolo; De Maria, Raffaella

2015-08-01

Insulin-like growth factor 1 receptor (IGF-1R) is a cell membrane receptor widely expressed in tissues and involved in different cancers in humans. IGF-1R expression in human osteosarcoma has been associated with the development of tumour metastasis and with prognosis, and represents an attractive therapeutic target. The goal of this study was to investigate the expression of IGF-1R in canine osteosarcoma tissues and cell lines and assess its role and prognostic value. Samples from 34 dogs were examined by immunohistochemistry for IGF-1R expression. IGF-1R/AKT/MAPK signalling was evaluated by western blot and quantitative polymerase chain reaction in the cell lines. In addition, the in vitro inhibition of IGF-1R with pycropodophillin (PPP) was used to evaluate molecular and biological effects. Immunohistochemical data showed that IGF-1R was expressed in 71% of the analysed osteosarcoma samples and that dogs with higher levels of IGF-IR expression (47% of cases) had decreased survival (P < 0.05) when compared to dogs with lower IGF-IR expression. Molecular studies demonstrated that in canine osteosarcoma IGF-IR is activated by IGF-1 mostly in a paracrine or endocrine (rather than autocrine) manner, leading to activation of AKT/MAPK signalling. PPP caused p-IGF-1R dephosphorylation with partial blocking of p-MAPK and p-AKT, as well as apoptosis. It was concluded that IGF-1R is expressed and plays a role in canine osteosarcoma and that its expression is correlated with a poor prognosis. As in humans, IGF-1R may represent a good therapeutic target and a prognostic factor for canine osteosarcoma. Copyright © 2014 Elsevier Ltd. All rights reserved.

Downregulation of external death receptor genes FAS and DR5 in colorectal cancer samples positive for human papillomavirus infection.

PubMed

Karbasi, Ashraf; Borhani, Nasim; Daliri, Karim; Kazemi, Bahram; Manoochehri, Mehdi

2015-06-01

Human papillomaviruses (HPV) have frequently been detected in colorectal cancer tumor samples, and may play a role in the pathogenesis of colorectal cancer. This study was designed to investigate the presence of DNA and RNA for the high-risk HPV genotypes 16 and 18 in samples of colorectal cancer tumors and adjacent normal tissues. We also investigated the expression of proapoptotic genes in HPV-positive colorectal tumors compared to normal tissue samples. Samples of tumoral and adjacent normal tissues were fresh-frozen, and HPV DNA was identified by nested and semiquantitative PCR. Real time PCR was used to quantitatively compare the expression of HPV-18 E6 and nine proapoptotic genes in HPV-positive tumors and samples of adjacent normal tissue. HPV-16 DNA was found in 10.5% of the tumor samples, and HPV-18 DNA was found in 23.6% of the samples. Real time PCR results showed lower expression of the E6 gene in HPV-positive tumors than in adjacent normal tissue. The expression of two proapoptotic genes, FAS and DR5, was significantly lower in tumor samples than in adjacent normal tissues. HPV infection, especially HPV-18, may play a role in colorectal cancer tumorigenesis by downregulating death receptor genes and interfering with the extrinsic pathway of apoptosis. Copyright © 2015 Elsevier GmbH. All rights reserved.

Tissue-based standoff biosensors for detecting chemical warfare agents

DOEpatents

Greenbaum, Elias; Sanders, Charlene A.

2003-11-18

A tissue-based, deployable, standoff air quality sensor for detecting the presence of at least one chemical or biological warfare agent, includes: a cell containing entrapped photosynthetic tissue, the cell adapted for analyzing photosynthetic activity of the entrapped photosynthetic tissue; means for introducing an air sample into the cell and contacting the air sample with the entrapped photosynthetic tissue; a fluorometer in operable relationship with the cell for measuring photosynthetic activity of the entrapped photosynthetic tissue; and transmitting means for transmitting analytical data generated by the fluorometer relating to the presence of at least one chemical or biological warfare agent in the air sample, the sensor adapted for deployment into a selected area.

The Use of Carcasses for the Analysis of Cetacean Population Genetic Structure: A Comparative Study in Two Dolphin Species

PubMed Central

Bilgmann, Kerstin; Möller, Luciana M.; Harcourt, Robert G.; Kemper, Catherine M.; Beheregaray, Luciano B.

2011-01-01

Advances in molecular techniques have enabled the study of genetic diversity and population structure in many different contexts. Studies that assess the genetic structure of cetacean populations often use biopsy samples from free-ranging individuals and tissue samples from stranded animals or individuals that became entangled in fishery or aquaculture equipment. This leads to the question of how representative the location of a stranded or entangled animal is with respect to its natural range, and whether similar results would be obtained when comparing carcass samples with samples from free-ranging individuals in studies of population structure. Here we use tissue samples from carcasses of dolphins that stranded or died as a result of bycatch in South Australia to investigate spatial population structure in two species: coastal bottlenose (Tursiops sp.) and short-beaked common dolphins (Delphinus delphis). We compare these results with those previously obtained from biopsy sampled free-ranging dolphins in the same area to test whether carcass samples yield similar patterns of genetic variability and population structure. Data from dolphin carcasses were gathered using seven microsatellite markers and a fragment of the mitochondrial DNA control region. Analyses based on carcass samples alone failed to detect genetic structure in Tursiops sp., a species previously shown to exhibit restricted dispersal and moderate genetic differentiation across a small spatial scale in this region. However, genetic structure was correctly inferred in D. delphis, a species previously shown to have reduced genetic structure over a similar geographic area. We propose that in the absence of corroborating data, and when population structure is assessed over relatively small spatial scales, the sole use of carcasses may lead to an underestimate of genetic differentiation. This can lead to a failure in identifying management units for conservation. Therefore, this risk should be carefully assessed when planning population genetic studies of cetaceans. PMID:21655285

Pollution gets personal! A first population-based human biomonitoring study in Austria.

PubMed

Hohenblum, Philipp; Steinbichl, Philipp; Raffesberg, Wolfgang; Weiss, Stefan; Moche, Wolfgang; Vallant, Birgit; Scharf, Sigrid; Haluza, Daniela; Moshammer, Hanns; Kundi, Michael; Piegler, Brigitte; Wallner, Peter; Hutter, Hans-Peter

2012-02-01

Humans are exposed to a broad variety of man-made chemicals. Human biomonitoring (HBM) data reveal the individual body burden irrespective of sources and routes of uptake. A first population-based study was started in Austria in 2008 and was finished at the end of May 2011. This cross sectional study aims at documenting the extent, the distribution and the determinants of human exposure to industrial chemicals as well as proving the feasibility of a representative HBM study. Overall, 150 volunteers (50 families) were selected by stratified random sampling. Exposure to phthalates, trisphosphates, polybrominated diphenyl ethers (PBDE), bisphenol A (along with nonyl- and octyl phenol) and methyl mercury was assessed. Sixteen of 18 PBDE determined were detected above the limit of quantification (LOQ) in blood samples with #153 and #197 the most abundant species. Bisphenol A in urine was measured in a subsample of 25 with only 4 samples found above the LOQ. In 3 of 100 urine samples at least one of 8 trisphosphate compounds assessed was above the LOQ. These first analytical results of the human biomonitoring data show that the body burden of the Austrian population with respect to the assessed compounds is comparable to or even lower than in other European countries. Overall, the study revealed that in order to develop a feasible protocol for representative human biomonitoring studies procedures have to be optimized to allow for non-invasive sampling of body tissues in accordance with the main metabolic pathways. Procedures of participants' recruitment were, however, labor intensive and have to be improved. Copyright © 2011 Elsevier GmbH. All rights reserved.

Characterization of In Vitro Engineered Human Adipose Tissues: Relevant Adipokine Secretion and Impact of TNF-α

PubMed Central

Aubin, Kim; Safoine, Meryem; Proulx, Maryse; Audet-Casgrain, Marie-Alice; Côté, Jean-François; Têtu, Félix-André; Roy, Alphonse; Fradette, Julie

2015-01-01

Representative modelling of human adipose tissue functions is central to metabolic research. Tridimensional models able to recreate human adipogenesis in a physiological tissue-like context in vitro are still scarce. We describe the engineering of white adipose tissues reconstructed from their cultured adipose-derived stromal precursor cells. We hypothesize that these reconstructed tissues can recapitulate key functions of AT under basal and pro-inflammatory conditions. These tissues, featuring human adipocytes surrounded by stroma, were stable and metabolically active in long-term cultures (at least 11 weeks). Secretion of major adipokines and growth factors by the reconstructed tissues was determined and compared to media conditioned by human native fat explants. Interestingly, the secretory profiles of the reconstructed adipose tissues indicated an abundant production of leptin, PAI-1 and angiopoietin-1 proteins, while higher HGF levels were detected for the human fat explants. We next demonstrated the responsiveness of the tissues to the pro-inflammatory stimulus TNF-α, as reflected by modulation of MCP-1, NGF and HGF secretion, while VEGF and leptin protein expression did not vary. TNF-α exposure induced changes in gene expression for adipocyte metabolism-associated mRNAs such as SLC2A4, FASN and LIPE, as well as for genes implicated in NF-κB activation. Finally, this model was customized to feature adipocytes representative of progressive stages of differentiation, thereby allowing investigations using newly differentiated or more mature adipocytes. In conclusion, we produced tridimensional tissues engineered in vitro that are able to recapitulate key characteristics of subcutaneous white adipose tissue. These tissues are produced from human cells and their neo-synthesized matrix elements without exogenous or synthetic biomaterials. Therefore, they represent unique tools to investigate the effects of pharmacologically active products on human stromal cells, extracellular matrix and differentiated adipocytes, in addition to compounds modulating adipogenesis from precursor cells. PMID:26367137

Fluorescence spectroscopy using excitation and emission matrix for quantification of tissue native fluorophores and cancer diagnosis

NASA Astrophysics Data System (ADS)

Wu, Binlin; Gayen, S. K.; Xu, M.

2014-03-01

Native fluorescence spectrum of normal and cancerous human prostate tissues is studied to distinguish between normal and cancerous tissues, and cancerous tissues at different cancer grade. The tissue samples were obtained from Cooperative Human Tissue Network (CHTN) and National Disease Research Interchange(NDRI). An excitation and emission matrix (EEM) was generated for each tissue sample by acquiring native fluorescence spectrum of the sample using multiple excitation wavelengths. The non-negative matrix factorization algorithm was used to generate fluorescence EEMs that correspond to the fluorophores in biological tissues, including tryptophan, collagen, elastin, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and the background paraffin. We hypothesize that, as a consequence of metabolic changes associated with the development of cancer, the concentrations of NADH and FAD are different in normal and cancerous tissues, and also different for different cancer grades. We used the ratio of the abundances of FAD and NADH to distinguish between normal and cancerous tissues, and the tissue cancer grade. The FAD-to-NADH ratio was found to be the highest for normal tissue and decreased as the cancer grade increased.

Principles, Techniques, and Applications of Tissue Microfluidics

NASA Technical Reports Server (NTRS)

Wade, Lawrence A.; Kartalov, Emil P.; Shibata, Darryl; Taylor, Clive

2011-01-01

The principle of tissue microfluidics and its resultant techniques has been applied to cell analysis. Building microfluidics to suit a particular tissue sample would allow the rapid, reliable, inexpensive, highly parallelized, selective extraction of chosen regions of tissue for purposes of further biochemical analysis. Furthermore, the applicability of the techniques ranges beyond the described pathology application. For example, they would also allow the posing and successful answering of new sets of questions in many areas of fundamental research. The proposed integration of microfluidic techniques and tissue slice samples is called "tissue microfluidics" because it molds the microfluidic architectures in accordance with each particular structure of each specific tissue sample. Thus, microfluidics can be built around the tissues, following the tissue structure, or alternatively, the microfluidics can be adapted to the specific geometry of particular tissues. By contrast, the traditional approach is that microfluidic devices are structured in accordance with engineering considerations, while the biological components in applied devices are forced to comply with these engineering presets.

One Sample, One Shot - Evaluation of sample preparation protocols for the mass spectrometric proteome analysis of human bile fluid without extensive fractionation.

PubMed

Megger, Dominik A; Padden, Juliet; Rosowski, Kristin; Uszkoreit, Julian; Bracht, Thilo; Eisenacher, Martin; Gerges, Christian; Neuhaus, Horst; Schumacher, Brigitte; Schlaak, Jörg F; Sitek, Barbara

2017-02-10

The proteome analysis of bile fluid represents a promising strategy to identify biomarker candidates for various diseases of the hepatobiliary system. However, to obtain substantive results in biomarker discovery studies large patient cohorts necessarily need to be analyzed. Consequently, this would lead to an unmanageable number of samples to be analyzed if sample preparation protocols with extensive fractionation methods are applied. Hence, the performance of simple workflows allowing for "one sample, one shot" experiments have been evaluated in this study. In detail, sixteen different protocols implying modifications at the stages of desalting, delipidation, deglycosylation and tryptic digestion have been examined. Each method has been individually evaluated regarding various performance criteria and comparative analyses have been conducted to uncover possible complementarities. Here, the best performance in terms of proteome coverage has been assessed for a combination of acetone precipitation with in-gel digestion. Finally, a mapping of all obtained protein identifications with putative biomarkers for hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) revealed several proteins easily detectable in bile fluid. These results can build the basis for future studies with large and well-defined patient cohorts in a more disease-related context. Human bile fluid is a proximal body fluid and supposed to be a potential source of disease markers. However, due to its biochemical composition, the proteome analysis of bile fluid still represents a challenging task and is therefore mostly conducted using extensive fractionation procedures. This in turn leads to a high number of mass spectrometric measurements for one biological sample. Considering the fact that in order to overcome the biological variability a high number of biological samples needs to be analyzed in biomarker discovery studies, this leads to the dilemma of an unmanageable number of necessary MS-based analyses. Hence, easy sample preparation protocols are demanded representing a compromise between proteome coverage and simplicity. In the presented study, such protocols have been evaluated regarding various technical criteria (e.g. identification rates, missed cleavages, chromatographic separation) uncovering the strengths and weaknesses of various methods. Furthermore, a cumulative bile proteome list has been generated that extends the current bile proteome catalog by 248 proteins. Finally, a mapping with putative biomarkers for hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) derived from tissue-based studies, revealed several of these proteins being easily and reproducibly detectable in human bile. Therefore, the presented technical work represents a solid base for future disease-related studies. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of the Tissue and Stromal Cell Components of Micro-Superficial Enhanced Fluid Fat Injection (Micro-SEFFI) for Facial Aging Treatment.

PubMed

Rossi, Martina; Roda, Barbara; Zia, Silvia; Vigliotta, Ilaria; Zannini, Chiara; Alviano, Francesco; Bonsi, Laura; Zattoni, Andrea; Reschiglian, Pierluigi; Gennai, Alessandro

2018-06-14

New microfat preparations provide material suitable for use as a regenerative filler for different facial areas. To support the development of new robust techniques for regenerative purposes, the cellular content of the sample should be considered. To evaluate the stromal vascular fraction (SVF) cell components of micro-superficial enhanced fluid fat injection (SEFFI) samples via a technique to harvest re-injectable tissue with minimum manipulation. The results were compared to those obtained from SEFFI samples. Microscopy analysis was performed to visualize the tissue structure. Micro-SEFFI samples were also fractionated using Celector ®, an innovative non-invasive separation technique, to provide an initial evaluation of sample fluidity and composition. SVFs obtained from SEFFI and micro-SEFFI were studied. Adipose stromal cells (ASCs) were isolated and characterized by proliferation and differentiation capacity assays. Microscopic and quality analyses of micro-SEFFI samples by Celector® confirmed the high fluidity and sample cellular composition in terms of red blood cell contamination, the presence of cell aggregates and extracellular matrix fragments. ASCs were isolated from adipose tissue harvested using SEFFI and micro-SEFFI systems. These cells were demonstrated to have a good proliferation rate and differentiation potential towards mesenchymal lineages. Despite the small sizes and low cellularity observed in micro-SEFFI-derived tissue, we were able to isolate stem cells. This result partially explains the regenerative potential of autologous micro-SEFFI tissue grafts. In addition, using this novel Celector® technology, tissues used for aging treatment were characterized analytically, and the adipose tissue composition was evaluated with no need for extra sample processing.

Design and implementation of coded aperture coherent scatter spectral imaging of cancerous and healthy breast tissue samples

PubMed Central

Lakshmanan, Manu N.; Greenberg, Joel A.; Samei, Ehsan; Kapadia, Anuj J.

2016-01-01

Abstract. A scatter imaging technique for the differentiation of cancerous and healthy breast tissue in a heterogeneous sample is introduced in this work. Such a technique has potential utility in intraoperative margin assessment during lumpectomy procedures. In this work, we investigate the feasibility of the imaging method for tumor classification using Monte Carlo simulations and physical experiments. The coded aperture coherent scatter spectral imaging technique was used to reconstruct three-dimensional (3-D) images of breast tissue samples acquired through a single-position snapshot acquisition, without rotation as is required in coherent scatter computed tomography. We perform a quantitative assessment of the accuracy of the cancerous voxel classification using Monte Carlo simulations of the imaging system; describe our experimental implementation of coded aperture scatter imaging; show the reconstructed images of the breast tissue samples; and present segmentations of the 3-D images in order to identify the cancerous and healthy tissue in the samples. From the Monte Carlo simulations, we find that coded aperture scatter imaging is able to reconstruct images of the samples and identify the distribution of cancerous and healthy tissues (i.e., fibroglandular, adipose, or a mix of the two) inside them with a cancerous voxel identification sensitivity, specificity, and accuracy of 92.4%, 91.9%, and 92.0%, respectively. From the experimental results, we find that the technique is able to identify cancerous and healthy tissue samples and reconstruct differential coherent scatter cross sections that are highly correlated with those measured by other groups using x-ray diffraction. Coded aperture scatter imaging has the potential to provide scatter images that automatically differentiate cancerous and healthy tissue inside samples within a time on the order of a minute per slice. PMID:26962543

Design and implementation of coded aperture coherent scatter spectral imaging of cancerous and healthy breast tissue samples.

PubMed

Lakshmanan, Manu N; Greenberg, Joel A; Samei, Ehsan; Kapadia, Anuj J

2016-01-01

A scatter imaging technique for the differentiation of cancerous and healthy breast tissue in a heterogeneous sample is introduced in this work. Such a technique has potential utility in intraoperative margin assessment during lumpectomy procedures. In this work, we investigate the feasibility of the imaging method for tumor classification using Monte Carlo simulations and physical experiments. The coded aperture coherent scatter spectral imaging technique was used to reconstruct three-dimensional (3-D) images of breast tissue samples acquired through a single-position snapshot acquisition, without rotation as is required in coherent scatter computed tomography. We perform a quantitative assessment of the accuracy of the cancerous voxel classification using Monte Carlo simulations of the imaging system; describe our experimental implementation of coded aperture scatter imaging; show the reconstructed images of the breast tissue samples; and present segmentations of the 3-D images in order to identify the cancerous and healthy tissue in the samples. From the Monte Carlo simulations, we find that coded aperture scatter imaging is able to reconstruct images of the samples and identify the distribution of cancerous and healthy tissues (i.e., fibroglandular, adipose, or a mix of the two) inside them with a cancerous voxel identification sensitivity, specificity, and accuracy of 92.4%, 91.9%, and 92.0%, respectively. From the experimental results, we find that the technique is able to identify cancerous and healthy tissue samples and reconstruct differential coherent scatter cross sections that are highly correlated with those measured by other groups using x-ray diffraction. Coded aperture scatter imaging has the potential to provide scatter images that automatically differentiate cancerous and healthy tissue inside samples within a time on the order of a minute per slice.

Water-quality data for the Talkeetna River and four streams in National Parks, Cook Inlet basin, Alaska, 1998

USGS Publications Warehouse

Frenzel, Steven A.; Dorava, Joseph M.

1999-01-01

Five streams in the Cook Inlet Basin, Alaska, were sampled in 1998 to provide the National Park Service with baseline information on water quality. Four of these streams drain National Park Service land: Costello and Colorado Creeks in Denali National Park and Preserve, Johnson River in Lake Clark National Park and Preserve, and Kamishak River in Katmai National Park and Preserve. The fifth site was on the Talkeetna River, outside of national park boundaries. Samples of stream water, streambed sediments, and fish tissues were collected for chemical analyses. Biological and geomorphic information was also collected at each site. Nutrient concentrations in stream water were low and commonly were less than analytical detection limits. Analyses of fish tissues for 28 organochlorine compounds at Talkeetna River and Costello Creek produced just one detection. Hexachlorobenzene was detected at a concentration of 5.70 micrograms per kilogram in slimy sculpin from the Talkeetna River. Streambed sediment samples from the Talkeetna River had three organochlorine compounds at detectable levels; hexachlorobenzene was measured at 13 micrograms per kilogram and two other compounds were below the minimum reporting levels. At Colorado Creek, Johnson River, and Kamishak River, where fish samples were not collected, no organochlorine compounds were detected in streambed sediment samples. Several semivolatile organic compounds were detected at Colorado Creek and Costello Creek. Only one compound, dibenzothiophene, detected at Costello Creek at a concentration of 85 micrograms per kilogram was above the minimum reporting limit. No semivolatile organic compounds were detected at the Talkeetna, Kamishak, or Johnson Rivers. Trace elements were detected in both fish tissues and streambed sediments. Macroinvertebrate and fish samples contained few taxa at all sites. Total numbers of macroinvertebrate taxa ranged from 19 at the Johnson River to 38 at the Talkeetna River. Diptera were the most abundant and diverse order of macroinvertebrates at all sites. Total numbers of diptera taxa ranged from 8 at the Kamishak River to 19 at the Talkeetna River. Fish communities were represented by a maximum of nine taxa at the Talkeetna River and were absent at Colorado Creek. The Johnson River sampling site produced small numbers of juvenile Dolly Varden, and Costello Creek produced small numbers of both juvenile Dolly Varden and slimy sculpin.

The impact of hydration changes in fresh bio-tissue on THz spectroscopic measurements.

PubMed

Png, G M; Choi, J W; Ng, B W-H; Mickan, S P; Abbott, D; Zhang, X-C

2008-07-07

We present a study of how residual hydration in fresh rat tissue samples can vastly alter their extracted terahertz (THz) optical properties and influence their health assessment. Fresh (as opposed to preserved) tissue most closely mimics in vivo conditions, but high water content creates many challenges for tissue handling and THz measurement. Our THz measurements of fresh tissue over time highlight the effect of tissue hydration on tissue texture and dimension, the latter directly influencing the accuracy of calculated optical properties. We then introduce lyophilization (freeze drying) as a viable solution for overcoming hydration and freshness problems. Lyophilization removes large amounts of water while retaining sample freshness. In addition, lyophilized tissue samples are easy to handle and their textures and dimensions do not vary over time, allowing for consistent and stable THz measurements. A comparison of lyophilized and fresh tissue shows for the first time that freeze drying may be one way of overcoming tissue hydration issues while preserving tissue cellular structure. Finally, we compare THz measurements from fresh tissue against necrotic tissue to verify freshness over time. Indeed, THz measurements from fresh and necrotic tissues show marked differences.

Research on Navy-Related Combat Casualty Care Issues, Navy Operational-Related Injuries and Illnesses and Approaches to Enhance Navy/Marine Corps Personnel Combat Performance.

DTIC Science & Technology

1998-06-01

role of bone morphogenetic protein (BMP) receptors in bone regeneration in periodontal tissues . Tissue samples for these studies are in the accrual...34 Characterization of Bone Morphogenetic Protein Receptors in Oral Tissues " collection of clinical samples will proceed in preparation for assay. • Relative to the...transcribed. • Relative to the project * Characterization of Bone Morphogenetic Protein Receptors in Oral Tissues ", collection of clinical samples is

Characterisation of signal enhancements achieved when utilizing a photon diode in deep Raman spectroscopy of tissue

PubMed Central

Vardaki, Martha Z.; Matousek, Pavel; Stone, Nicholas

2016-01-01

We characterise the performance of a beam enhancing element (‘photon diode’) for use in deep Raman spectroscopy (DRS) of biological tissues. The optical component enhances the number of laser photons coupled into a tissue sample by returning escaping photons back into it at the illumination zone. The method is compatible with transmission Raman spectroscopy, a deep Raman spectroscopy concept, and its implementation leads to considerable enhancement of detected Raman photon rates. In the past, the enhancement concept was demonstrated with a variety of samples (pharmaceutical tablets, tissue, etc) but it was not systematically characterized with biological tissues. In this study, we investigate the enhancing properties of the photon diode in the transmission Raman geometry as a function of: a) the depth and b) the optical properties of tissue samples. Liquid tissue phantoms were employed to facilitate systematic variation of optical properties. These were chosen to mimic optical properties of human tissues, including breast and prostate. The obtained results evidence that a photon diode can enhance Raman signals of tissues by a maximum of × 2.4, although it can also decrease the signals created towards the back of samples that exhibit high scattering or absorption properties. PMID:27375932

Biased visualization of hypoperfused tissue by computed tomography due to short imaging duration: improved classification by image down-sampling and vascular models.

PubMed

Mikkelsen, Irene Klærke; Jones, P Simon; Ribe, Lars Riisgaard; Alawneh, Josef; Puig, Josep; Bekke, Susanne Lise; Tietze, Anna; Gillard, Jonathan H; Warburton, Elisabeth A; Pedraza, Salva; Baron, Jean-Claude; Østergaard, Leif; Mouridsen, Kim

2015-07-01

Lesion detection in acute stroke by computed-tomography perfusion (CTP) can be affected by incomplete bolus coverage in veins and hypoperfused tissue, so-called bolus truncation (BT), and low contrast-to-noise ratio (CNR). We examined the BT-frequency and hypothesized that image down-sampling and a vascular model (VM) for perfusion calculation would improve normo- and hypoperfused tissue classification. CTP datasets from 40 acute stroke patients were retrospectively analysed for BT. In 16 patients with hypoperfused tissue but no BT, repeated 2-by-2 image down-sampling and uniform filtering was performed, comparing CNR to perfusion-MRI levels and tissue classification to that of unprocessed data. By simulating reduced scan duration, the minimum scan-duration at which estimated lesion volumes came within 10% of their true volume was compared for VM and state-of-the-art algorithms. BT in veins and hypoperfused tissue was observed in 9/40 (22.5%) and 17/40 patients (42.5%), respectively. Down-sampling to 128 × 128 resolution yielded CNR comparable to MR data and improved tissue classification (p = 0.0069). VM reduced minimum scan duration, providing reliable maps of cerebral blood flow and mean transit time: 5 s (p = 0.03) and 7 s (p < 0.0001), respectively). BT is not uncommon in stroke CTP with 40-s scan duration. Applying image down-sampling and VM improve tissue classification. • Too-short imaging duration is common in clinical acute stroke CTP imaging. • The consequence is impaired identification of hypoperfused tissue in acute stroke patients. • The vascular model is less sensitive than current algorithms to imaging duration. • Noise reduction by image down-sampling improves identification of hypoperfused tissue by CTP.

High Definition Confocal Imaging Modalities for the Characterization of Tissue-Engineered Substitutes.

PubMed

Mayrand, Dominique; Fradette, Julie

2018-01-01

Optimal imaging methods are necessary in order to perform a detailed characterization of thick tissue samples from either native or engineered tissues. Tissue-engineered substitutes are featuring increasing complexity including multiple cell types and capillary-like networks. Therefore, technical approaches allowing the visualization of the inner structural organization and cellular composition of tissues are needed. This chapter describes an optical clearing technique which facilitates the detailed characterization of whole-mount samples from skin and adipose tissues (ex vivo tissues and in vitro tissue-engineered substitutes) when combined with spectral confocal microscopy and quantitative analysis on image renderings.

High prevalences and a wide genetic diversity of simian retroviruses in non-human primate bushmeat in rural areas of the Democratic Republic of Congo

PubMed Central

Steve, Ahuka-Mundeke; Ahidjo, Ayouba; Placide, Mbala-Kingebeni; Caroline, Foncelle; Mukulumanya, Mubonga; Simon-Pierre, Ndimbo-Kumugo; Octavie, Lunguya-Metila; Valentin, Mbenzo-Abokome; Jean-Jacques, Muyembe-Tamfum; Eric, Delaporte; Martine, Peeters

2017-01-01

Like the majority of emerging infectious diseases, HIV and HTLV are of zoonotic origin. Here we assess the risk of cross-species transmissions of their simian counterparts, SIV and STLV, from nonhuman primates (NHP) to humans in the Democratic Republic of Congo (DRC). A total of 331 samples, derived from NHP bushmeat, were collected as dried blood spots (DBS, n=283) or as tissue samples (n=36) at remote forest sites in northern and eastern DRC. SIV antibody prevalences in DBS were estimated with a novel high throughput immune assay with antigens representing the actual known diversity of HIV/SIV lineages. Antibody positive samples were confirmed by PCR and sequence analysis. Screening for STLV infection was done with universal primers in tax and new strains were further characterized in LTR. SIV and STLV infection in tissue samples was done by PCR only. Overall, 5% and 15.4% of NHP bushmeat was infected with SIV and STLV respectively. A new SIV lineage was identified in Allen's swamp monkeys (Allenopithecus nigroviridis). Three new STLV-1 subtypes were identified in Allen's swamp monkeys (Allenopithecus nigroviridis), blue monkeys (Cercopithecus mitis), red tailed guenons (Cercopithecus ascanius schmidti) and agile mangabeys (Cercocebus agilis). SIV and STLV prevalences varied according to species and geographic region. Our study illustrates clearly, even on a small sample size from a limited number of geograhic areas, that our knowledge on the genetic diversity and geographic distribution of simian retroviruses is still limited and that humans continue to be exposed to relative high proportions on infected NHP bushmeat. PMID:28050688

Spatial cluster analysis of nanoscopically mapped serotonin receptors for classification of fixed brain tissue

NASA Astrophysics Data System (ADS)

Sams, Michael; Silye, Rene; Göhring, Janett; Muresan, Leila; Schilcher, Kurt; Jacak, Jaroslaw

2014-01-01

We present a cluster spatial analysis method using nanoscopic dSTORM images to determine changes in protein cluster distributions within brain tissue. Such methods are suitable to investigate human brain tissue and will help to achieve a deeper understanding of brain disease along with aiding drug development. Human brain tissue samples are usually treated postmortem via standard fixation protocols, which are established in clinical laboratories. Therefore, our localization microscopy-based method was adapted to characterize protein density and protein cluster localization in samples fixed using different protocols followed by common fluorescent immunohistochemistry techniques. The localization microscopy allows nanoscopic mapping of serotonin 5-HT1A receptor groups within a two-dimensional image of a brain tissue slice. These nanoscopically mapped proteins can be confined to clusters by applying the proposed statistical spatial analysis. Selected features of such clusters were subsequently used to characterize and classify the tissue. Samples were obtained from different types of patients, fixed with different preparation methods, and finally stored in a human tissue bank. To verify the proposed method, samples of a cryopreserved healthy brain have been compared with epitope-retrieved and paraffin-fixed tissues. Furthermore, samples of healthy brain tissues were compared with data obtained from patients suffering from mental illnesses (e.g., major depressive disorder). Our work demonstrates the applicability of localization microscopy and image analysis methods for comparison and classification of human brain tissues at a nanoscopic level. Furthermore, the presented workflow marks a unique technological advance in the characterization of protein distributions in brain tissue sections.

Results of the Excreta Bioassay Quality Control Program for April 1, 2009 through March 31, 2010

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antonio, Cheryl L.

2012-07-19

A total of 58 urine samples and 10 fecal samples were submitted during the report period (April 1, 2009 through March 31, 2010) to General Engineering Laboratories, South Carolina by the Hanford Internal Dosimetry Program (IDP) to check the accuracy, precision, and detection levels of their analyses. Urine analyses for Sr, 238Pu, 239Pu, 241Am, 243Am 235U, 238U, elemental uranium and fecal analyses for 241Am, 238Pu and 239Pu were tested this year as well as four tissue samples for 238Pu, 239Pu, 241Am and 241Pu. The number of QC urine samples submitted during the report period represented 1.3% of the total samplesmore » submitted. In addition to the samples provided by IDP, GEL was also required to conduct their own QC program, and submit the results of analyses to IDP. About 33% of the analyses processed by GEL during the third year of this contract were quality control samples. GEL tested the performance of 21 radioisotopes, all of which met or exceeded the specifications in the Statement of Work within statistical uncertainty (Table 4).« less

Stable isotopic comparison between loggerhead sea turtle tissues.

PubMed

Vander Zanden, Hannah B; Tucker, Anton D; Bolten, Alan B; Reich, Kimberly J; Bjorndal, Karen A

2014-10-15

Stable isotope analysis has been used extensively to provide ecological information about diet and foraging location of many species. The difference in isotopic composition between animal tissue and its diet, or the diet-tissue discrimination factor, varies with tissue type. Therefore, direct comparisons between isotopic values of tissues are inaccurate without an appropriate conversion factor. We focus on the loggerhead sea turtle (Caretta caretta), for which a variety of tissues have been used to examine diet, habitat use, and migratory origin through stable isotope analysis. We calculated tissue-to-tissue conversions between two commonly sampled tissues. Epidermis and scute (the keratin covering on the carapace) were sampled from 33 adult loggerheads nesting at two beaches in Florida (Casey Key and Canaveral National Seashore). Carbon and nitrogen stable isotope ratios were measured in the epidermis and the youngest portion of the scute tissue, which reflect the isotopic composition of the diet and habitat over similar time periods of the order of several months. Significant linear relationships were observed between the δ(13)C and δ(15)N values of these two tissues, indicating they can be converted reliably. Whereas both epidermis and scute samples are commonly sampled from nesting sea turtles to study trophic ecology and habitat use, the data from these studies have not been comparable without reliable tissue-to-tissue conversions. The equations provided here allow isotopic datasets using the two tissues to be combined in previously published and subsequent studies of sea turtle foraging ecology and migratory movement. In addition, we recommend that future isotopic comparisons between tissues of any organism utilize linear regressions to calculate tissue-to-tissue conversions. Copyright © 2014 John Wiley & Sons, Ltd.

The components of somatostatin and ghrelin systems are altered in neuroendocrine lung carcinoids and associated to clinical-histological features.

PubMed

Herrera-Martínez, Aura D; Gahete, Manuel D; Sánchez-Sánchez, Rafael; Salas, Rosa Ortega; Serrano-Blanch, Raquel; Salvatierra, Ángel; Hofland, Leo J; Luque, Raúl M; Gálvez-Moreno, María A; Castaño, Justo P

2017-07-01

Lung carcinoids (LCs) are rare tumors that comprise 1-5% of lung malignancies but represent 20-30% of neuroendocrine tumors. Their incidence is progressively increasing and a better characterization of these tumors is required. Alterations in somatostatin (SST)/cortistatin (CORT) and ghrelin systems have been associated to development/progression of various endocrine-related cancers, wherein they may become useful diagnostic, prognostic and therapeutic biomarkers. We aimed to evaluate the expression levels of ghrelin and SST/CORT system components in LCs, as well as to explore their putative relationship with histological/clinical characteristics. An observational retrospective study was performed; 75 LC patients with clinical/histological characteristics were included. Samples from 46 patients were processed to isolate mRNA from tumor and adjacent non-tumor region, and the expression levels of SST/CORT and ghrelin systems components, determined by quantitative-PCR, were compared to those of 7 normal lung tissues. Patient cohort was characterized by mean age 53±15 years, 48% males, 34% with tobacco exposure; 71.4/28.6% typical/atypical carcinoids, 21.7% incidental tumors, 4.3% functioning tumors, 17.7% with metastasis. SST/CORT and ghrelin system components were expressed at variable levels in a high proportion of tumors, as well as in adjacent non-tumor tissues, while a lower proportion of normal lung samples also expressed these molecules. A gradation was observed from normal non-neoplastic lung tissues, non-tumor adjacent tissue and LCs, being SST, sst4, sst5, GHS-R1a and GHS-R1b overexpressed in tumor tissue compared to normal tissue. Importantly, several SST/CORT and ghrelin system components displayed significant correlations with relevant clinical parameters, such as necrosis, peritumoral and vascular invasion, or metastasis. Altogether, these data reveal a prominent, widespread expression of key SST/CORT/ghrelin system components in LCs, where they display clinical-histological correlations, which could provide novel, valuable markers for NET patient management. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous Multiparameter Cellular Energy Metabolism Profiling of Small Populations of Cells.

PubMed

Kelbauskas, Laimonas; Ashili, Shashaanka P; Lee, Kristen B; Zhu, Haixin; Tian, Yanqing; Meldrum, Deirdre R

2018-03-12

Functional and genomic heterogeneity of individual cells are central players in a broad spectrum of normal and disease states. Our knowledge about the role of cellular heterogeneity in tissue and organism function remains limited due to analytical challenges one encounters when performing single cell studies in the context of cell-cell interactions. Information based on bulk samples represents ensemble averages over populations of cells, while data generated from isolated single cells do not account for intercellular interactions. We describe a new technology and demonstrate two important advantages over existing technologies: first, it enables multiparameter energy metabolism profiling of small cell populations (<100 cells)-a sample size that is at least an order of magnitude smaller than other, commercially available technologies; second, it can perform simultaneous real-time measurements of oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and mitochondrial membrane potential (MMP)-a capability not offered by any other commercially available technology. Our results revealed substantial diversity in response kinetics of the three analytes in dysplastic human epithelial esophageal cells and suggest the existence of varying cellular energy metabolism profiles and their kinetics among small populations of cells. The technology represents a powerful analytical tool for multiparameter studies of cellular function.

Gene expression analysis of head and neck squamous cell carcinoma survival and recurrence

PubMed Central

Zhi, Xu; Lamperska, Katarzyna; Golusinski, Paweł; Schork, Nicholas J.; Luczewski, Lukasz; Kolenda, Tomasz; Golusinski, Wojciech; Masternak, Michal M.

2015-01-01

The squamous cell carcinomas represent about 90 % of all head and neck cancers, ranking the sixth most common human cancer. Approximately 450,000 of new cases of head and neck squamous cell carcinoma (HNSCC) are diagnosed every year. Unfortunately, because of diagnosis at the advanced stages and early metastasis to the lymph nodes, the HNSCC is associated with very high death rate. Identification of signature biomarkers and molecularly targeted therapies could provide more effective and specific cancer treatment, prevent recurrence, and increase survival rate. We used paired tumor and adjacent normal tissue samples to screen with RT² Profiler™ PCR Array Human Cancer PathwayFinderTM. Total of 20 up-regulated genes and two down-regulated genes were screened out. Out of 22 genes, 12 genes were subsequently validated to be significantly altered in the HNSCC; the samples were from all 41 patients. Five year survival and recurrence selected genes that could represent the biomarkers of survival and recurrence of the disease. We believe that comprehensive understanding of the unique genetic characteristics of HNSCC could provide novel diagnostic biomarkers and meet the requirement for molecular-targeted therapy for the HNSCC. PMID:25575813

A stochastic model for soft tissue failure using acoustic emission data.

PubMed

Sánchez-Molina, D; Martínez-González, E; Velázquez-Ameijide, J; Llumà, J; Rebollo Soria, M C; Arregui-Dalmases, C

2015-11-01

The strength of soft tissues is due mainly to collagen fibers. In most collagenous tissues, the arrangement of the fibers is random, but has preferred directions. The random arrangement makes it difficult to make deterministic predictions about the starting process of fiber breaking under tension. When subjected to tensile stress the fibers are progressively straighten out and then start to be stretched. At the beginning of fiber breaking, some of the fibers reach their maximum tensile strength and break down while some others remain unstressed (this latter fibers will assume then bigger stress until they eventually arrive to their failure point). In this study, a sample of human esophagi was subjected to a tensile breaking of fibers, up to the complete failure of the specimen. An experimental setup using Acoustic Emission to detect the elastic energy released is used during the test to detect the location of the emissions and the number of micro-failures per time unit. The data were statistically analyzed in order to be compared to a stochastic model which relates the level of stress in the tissue and the probability of breaking given the number of previously broken fibers (i.e. the deterioration in the tissue). The probability of a fiber breaking as the stretch increases in the tissue can be represented by a non-homogeneous Markov process which is the basis of the stochastic model proposed. This paper shows that a two-parameter model can account for the fiber breaking and the expected distribution for ultimate stress is a Fréchet distribution. Copyright © 2015 Elsevier Ltd. All rights reserved.

Global transcriptional profiling reveals similarities and differences between human stem cell-derived cardiomyocyte clusters and heart tissue.

PubMed

Synnergren, Jane; Améen, Caroline; Jansson, Andreas; Sartipy, Peter

2012-02-27

It is now well documented that human embryonic stem cells (hESCs) can differentiate into functional cardiomyocytes. These cells constitute a promising source of material for use in drug development, toxicity testing, and regenerative medicine. To assess their utility as replacement or complement to existing models, extensive phenotypic characterization of the cells is required. In the present study, we used microarrays and analyzed the global transcription of hESC-derived cardiomyocyte clusters (CMCs) and determined similarities as well as differences compared with reference samples from fetal and adult heart tissue. In addition, we performed a focused analysis of the expression of cardiac ion channels and genes involved in the Ca(2+)-handling machinery, which in previous studies have been shown to be immature in stem cell-derived cardiomyocytes. Our results show that hESC-derived CMCs, on a global level, have a highly similar gene expression profile compared with human heart tissue, and their transcriptional phenotype was more similar to fetal than to adult heart. Despite the high similarity to heart tissue, a number of significantly differentially expressed genes were identified, providing some clues toward understanding the molecular difference between in vivo sourced tissue and stem cell derivatives generated in vitro. Interestingly, some of the cardiac-related ion channels and Ca(2+)-handling genes showed differential expression between the CMCs and heart tissues. These genes may represent candidates for future genetic engineering to create hESC-derived CMCs that better mimic the phenotype of the cardiomyocytes present in the adult human heart.

Variability in isotope discrimination factors in coral reef fishes: implications for diet and food web reconstruction.

PubMed

Wyatt, Alex S J; Waite, Anya M; Humphries, Stuart

2010-10-27

Interpretation of stable isotope ratios of carbon and nitrogen (δ(13)C and δ(15)N) is generally based on the assumption that with each trophic level there is a constant enrichment in the heavier isotope, leading to diet-tissue discrimination factors of 3.4‰ for (15)N (ΔN) and ∼0.5‰ for (13)C (ΔC). Diet-tissue discrimination factors determined from paired tissue and gut samples taken from 152 individuals from 26 fish species at Ningaloo Reef, Western Australia demonstrate a large amount of variability around constant values. While caution is necessary in using gut contents to represent diet due to the potential for high temporal variability, there were significant effects of trophic position and season that may also lead to variability in ΔN under natural conditions. Nitrogen enrichment increased significantly at higher trophic levels (higher tissue δ(15)N), with significantly higher ΔN in carnivorous species. Changes in diet led to significant changes in ΔN, but not tissue δ(15)N, between seasons for several species: Acanthurus triostegus, Chromis viridis, Parupeneus signatus and Pomacentrus moluccensis. These results confirm that the use of meta-analysis averages for ΔN is likely to be inappropriate for accurately determining diets and trophic relationships using tissue stable isotope ratios. Where feasible, discrimination factors should be directly quantified for each species and trophic link in question, acknowledging the potential for significant variation away from meta-analysis averages and, perhaps, controlled laboratory diets and conditions.

Genotyping of Endosperms to Determine Seed Dormancy Genes Regulating Germination Through Embryonic, Endospermic, or Maternal Tissues in Rice

PubMed Central

Gu, Xing-You; Zhang, Jinfeng; Ye, Heng; Zhang, Lihua; Feng, Jiuhuan

2014-01-01

Seed dormancy is imposed by one or more of the embryo, endosperm, and maternal tissues that belong to two generations and represent two ploidy levels. Many quantitative trait loci (QTL) have been identified for seed dormancy as measured by gross effects on reduced germination rate or delayed germination in crop or model plants. This research developed an endosperm genotype−based genetic approach to determine specific tissues through which a mapped QTL regulates germination using rice as a model. This approach involves testing germination velocity for partially after-ripened seeds harvested from single plants heterozygous for a tested QTL and genotyping endosperms from individual germinated and nongerminated seeds with a codominant DNA marker located on the QTL peak region. Information collected about the QTL includes genotypic frequencies in germinated and/or nongerminated subpopulations; allelic frequency distributions during a germination period; endosperm or embryo genotypic differences in germination velocity; and genotypic frequencies for gametes involved in the double fertilization to form the sampled seeds. Using this approach, the seed dormancy loci SD12, SD1-2, and SD7-1 were determined to regulate germination through the embryo, endosperm, and maternal tissues, respectively; SD12 and SD1-2 acted additively on germination velocity in the offspring tissues; and SD12 also was associated with the preferential fertilization of male gametes in rice. This new genetic approach can be used to characterize mapped genes/QTL for tissue-specific functions in endospermic seeds and for marker-assisted selection of QTL alleles before or immediately after germination in crop breeding. PMID:25480961

Evaluation of two-dimensional electrophoresis and liquid chromatography – tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samples

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schad, Martina; Lipton, Mary S.; Giavalisco, Patrick

2005-07-14

Laser microdissection (LM) allows the collection of homogeneous tissue- and cell specific plant samples. The employment of this technique with subsequent protein analysis has thus far not been reported for plant tissues, probably due to the difficulties associated with defining a reasonable cellular morphology and, in parallel, allowing efficient protein extraction from tissue samples. The relatively large sample amount needed for successful proteome analysis is an additional issue that complicates protein profiling on a tissue- or even cell-specific level. In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies, there ismore » as yet no amplification procedure for proteins available. In the current study, we compared different tissue preparation techniques prior to LM/laser pressure catapulting (LMPC) with respect to their suitability for protein retrieval. Cryosectioning was identified as the best compromise between tissue morphology and effective protein extraction. After collection of vascular bundles from Arabidopsis thaliana stem tissue by LMPC, proteins were extracted and subjected to protein analysis, either by classical two-dimensional gel electrophoresis (2-DE), or by high-efficiency liquid chromatography (LC) in conjunction with tandem mass spectrometry (MS/MS). Our results demonstrate that both methods can be used with LMPC collected plant material. But because of the significantly lower sample amount required for LC-MS/MS than for 2-DE, the combination of LMPC and LC-MS/MS has a higher potential to promote comprehensive proteome analysis of specific plant tissues.« less

Cold-Temperature Plastic Resin Embedding of Liver for DNA- and RNA-Based Genotyping

PubMed Central

Finkelstein, Sydney D.; Dhir, Rajiv; Rabinovitz, Mordechai; Bischeglia, Michelle; Swalsky, Patricia A.; DeFlavia, Petrina; Woods, Jeffrey; Bakker, Anke; Becich, Michael

1999-01-01

The standard practice of tissue fixation in 10% formalin followed by embedding in paraffin wax preserves cellular morphology at the expense of availability and quality of DNA and RNA. The negative effect on cellular constituents results from a combination of extensive cross-linking and strand scission of DNA, RNA, and proteins induced by formaldehyde as well as RNA loss secondary to ubiquitous RNase activity and negative effects of high temperature exposure during paraffin melting, microscopic section collection, and tissue adherence to glass slides. An effective strategy to correlate cellular phenotype with molecular genotype involves microdissection of tissue sections based on specific histopathological features followed by genotyping of minute representative samples for specific underlying molecular alterations. Currently, this approach is limited to short-length polymerase chain reaction amplification (<250 bp) of DNA, due to the negative effects of standard tissue fixation and processing. To overcome this obstacle and permit both cellular morphology and nucleic acid content to be preserved to the fullest extent, we instituted a system of cold-temperature plastic resin embedding based on the use of the water-miscible methyl methacrylate polymer known as Immunobed (Polysciences, Warminster, PA). The system is simple, easy to adapt to clinical practice, and cost-effective. Immunobed tissue sections demonstrate a cellular appearance equivalent or even superior to that of standard tissue sections. Moreover, thin sectioning (0.5–1.0 μm thickness) renders ultrastructural evaluation feasible on plastic-embedded blocks. Tissue microdissection is readily performed, yielding high levels of long DNA and RNA for genomic and transcription-based correlative molecular analysis. We recommend the use of Immunobed or similar products for use in molecular anatomical pathology. PMID:11272904

Quantitative colorimetry of atherosclerotic plaque using the L*a*b* color space during angioscopy for the detection of lipid cores underneath thin fibrous caps.

PubMed

Ishibashi, Fumiyuki; Yokoyama, Shinya; Miyahara, Kengo; Dabreo, Alexandra; Weiss, Eric R; Iafrati, Mark; Takano, Masamichi; Okamatsu, Kentaro; Mizuno, Kyoichi; Waxman, Sergio

2007-12-01

Yellow plaques seen during angioscopy are thought to represent lipid cores underneath thin fibrous caps (LCTCs) and may be indicative of vulnerable sites. However, plaque color assessment during angioscopy has been criticized because of its qualitative nature. The purpose of the present study was to test the ability of a quantitative colorimetric system to measure yellow color intensity of atherosclerotic plaques during angioscopy and to characterize the color of LCTCs. Using angioscopy and a quantitative colorimetry system based on the L*a*b* color space [L* describes brightness (-100 to +100), b* describes blue to yellow (-100 to +100)], the optimal conditions for measuring plaque color were determined in three flat standard color samples and five artificial plaque models in cylinder porcine carotid arteries. In 88 human tissue samples, the colorimetric characteristics of LCTCs were then evaluated. In in-vitro samples and ex-vivo plaque models, brightness L* between 40 and 80 was determined to be optimal for acquiring b* values, and the variables unique to angioscopy in color perception did not impact b* values after adjusting for brightness L* by manipulating light or distance. In ex-vivo human tissue samples, b* value >/=23 (35.91 +/- 8.13) with L* between 40 and 80 was associated with LCTCs (fibrous caps <100 mum). Atherosclerotic plaque color can be consistently measured during angioscopy with quantitative colorimetry. High yellow color intensity, determined by this system, was associated with LCTCs. Quantitative colorimetry during angioscopy may be used for detection of LCTCs, which may be markers of vulnerability.

High enantiomeric excess of the flavor relevant 4-alkyl-branched Fatty acids in milk fat and subcutaneous adipose tissue of sheep and goat.

PubMed

Kaffarnik, Stefanie; Heid, Carolina; Kayademir, Yasemin; Eibler, Dorothee; Vetter, Walter

2015-01-21

Volatile 4-alkyl-branched fatty acids are characteristic flavor compounds of sheep and goat. Due to the methyl branch, the carbon C-4 represents a stereogenic center with the possible presence of one or both enantiomers in the respective samples. In this study, we used enantioselective gas chromatography to study the enantiomeric composition of 4-methyloctanoic acid (4-Me-8:0) and 4-ethyloctanoic acid (4-Et-8:0) in milk and dairy products from sheep and goat as well as in goat subcutaneous tissue. Different columns coated with modified cyclodextrins were tested to resolve racemic 4-alkyl-branched fatty acid methyl ester standards. The best enantiomer resolution was obtained on 25% octakis(2,3,6-tri-O-ethyl)-γ-cyclodextrin (γ-TECD) diluted in OV-1701. For analysis of the food samples, the lipids were extracted and fatty acids in the extracts were converted into fatty acid methyl esters. Non-aqueous reversed-phase high-performance liquid chromatography was used to fractionate the samples in order to gain one solution enriched in 4-Me-8:0 methyl ester and one solution enriched with 4-Et-8:0 methyl ester. Subsequent analysis by enantioselective gas chromatography with mass spectrometry allowed only the detection of one enantiomer of 4-Me-8:0 and 4-Et-8:0 in the samples. By means of a non-racemic standard of 4-Me-8:0, it was found that the predominant enantiomer was (R)-4-Me-8:0.

The Use of an Electronic System for Soft Tissue Balancing in Primary Total Knee Arthroplasties: Clinical and Radiological Evaluation.

PubMed

D'Angelo, Fabio; Puricelli, Marco; Binda, Tommaso; Surace, Michele Francesco; Floridi, Chiara; Cherubino, Paolo

2015-05-01

The eLibra® Dynamic Knee Balancing System (Synvasive Technology, Zimmer, Warsaw, IN) is an instrument designed to address the flexion stability during a TKA. It provides an objective measurement of the soft-tissue forces in the two compartments before the final cuts are made, allowing to obtain patient-specific rotational orientation of the femoral component. Between March 2010 and March 2012, the eLibra® system was used during the implantation of 75 TKAs in 75 patients at the author's institution. Preoperative and postoperative clinical assessment were evaluated using the Knee Society Score (KSS) and the Visual Analogical Scale (VAS). Radiographic evaluation was performed with weight-bearing radiographs in antero-posterior and lateral views in order to study the presence of radiolucencies. In a sample of 20 patients, representative of the population studied, the rotation of the femoral component was measured by two independent observers using the C-arm Cone Beam CT scan (XperCT/Allura FD20 angiography system; Philips, Best, Netherlands). At a mean follow-up of 42.3 months (29-54 months), three patients died from causes not related to the surgery. We had one case of aseptic loosening three years after surgery. None of the patients reported complications peri- or postoperatively. Clinical evaluation showed an improvement in KSS scoring, from preoperative means of 48.35 and 47.53 points for clinical and functional aspects, respectively, to postoperative means of 88.03 and 91.2 points, respectively (p<0.001 for both aspects). The current study demonstrates that the use of the eLibra® device is simple and reproducible. It could help surgeons objectively quantify ligament balance and perform soft tissue-guided resection in a reproducible way, resulting in better post-operative stability and reduced complications. The use of the postoperative cone beam computed tomography (CBCT), in a representative sample of patients, revealed a specific and optimal orientation of the femoral component with a mean of 2.18° of external rotation.

Determination of naturally occurring formaldehyde levels in sap and wood tissue of maple trees using gas chromatgraphy/mass spectrometry.

PubMed

Lagacé, Luc; Gaudy, Réjean; Perez-Locas, Carolina; Sadiki, Mustapha

2012-01-01

The occurrence of formaldehyde in sap and wood tissue of treated and untreated maple sugar trees was investigated using GC/MS. Samples were collected at different periods of the 2009 season and at different locations in Quebec, Canada. The natural concentration of formaldehyde found in untreated samples varied according to periods and locations and ranged from below the LOQ to 1.82 mg/kg for sap samples and from 2.39 to 8.92 mg/kg of fresh tissue for wood samples. Late season samples tended to have higher concentrations of formaldehyde. Samples of sap and wood tissue from tapholes treated with solutions of formaldehyde showed increased concentrations of formaldehyde for many days after treatment and were clearly distinct from untreated samples. These results will be useful to elaborate new inspection procedures for sugarbushes to control the illegal use of formaldehyde.

Remote biopsy darting and marking of polar bears

USGS Publications Warehouse

Pagano, Anthony M.; Peacock, Elizabeth; McKinney, Melissa A.

2014-01-01

Remote biopsy darting of polar bears (Ursus maritimus) is less invasive and time intensive than physical capture and is therefore useful when capture is challenging or unsafe. We worked with two manufacturers to develop a combination biopsy and marking dart for use on polar bears. We had an 80% success rate of collecting a tissue sample with a single biopsy dart and collected tissue samples from 143 polar bears on land, in water, and on sea ice. Dye marks ensured that 96% of the bears were not resampled during the same sampling period, and we recovered 96% of the darts fired. Biopsy heads with 5 mm diameters collected an average of 0.12 g of fur, tissue, and subcutaneous adipose tissue, while biopsy heads with 7 mm diameters collected an average of 0.32 g. Tissue samples were 99.3% successful (142 of 143 samples) in providing a genetic and sex identification of individuals. We had a 64% success rate collecting adipose tissue and we successfully examined fatty acid signatures in all adipose samples. Adipose lipid content values were lower compared to values from immobilized or harvested polar bears, indicating that our method was not suitable for quantifying adipose lipid content.

Biobanking of fresh frozen tissue from clinical surgical specimens: transport logistics, sample selection, and histologic characterization.

PubMed

Botling, Johan; Micke, Patrick

2011-01-01

Access to high-quality fresh frozen tissue is critical for translational cancer research and molecular -diagnostics. Here we describe a workflow for the collection of frozen solid tissue samples derived from fresh human patient specimens after surgery. The routines have been in operation at Uppsala University Hospital since 2001. We have integrated cryosection and histopathologic examination of each biobank sample into the biobank manual. In this way, even small, macroscopically ill-defined lesions can be -procured without a diagnostic hazard due to the removal of uncharacterized tissue from a clinical -specimen. Also, knowledge of the histomorphology of the frozen tissue sample - tumor cell content, stromal components, and presence of necrosis - is pivotal before entering a biobank case into costly molecular profiling studies.

Model-based recovery of histological parameters from multispectral images of the colon

NASA Astrophysics Data System (ADS)

Hidovic-Rowe, Dzena; Claridge, Ela

2005-04-01

Colon cancer alters the macroarchitecture of the colon tissue. Common changes include angiogenesis and the distortion of the tissue collagen matrix. Such changes affect the colon colouration. This paper presents the principles of a novel optical imaging method capable of extracting parameters depicting histological quantities of the colon. The method is based on a computational, physics-based model of light interaction with tissue. The colon structure is represented by three layers: mucosa, submucosa and muscle layer. Optical properties of the layers are defined by molar concentration and absorption coefficients of haemoglobins; the size and density of collagen fibres; the thickness of the layer and the refractive indexes of collagen and the medium. Using the entire histologically plausible ranges for these parameters, a cross-reference is created computationally between the histological quantities and the associated spectra. The output of the model was compared to experimental data acquired in vivo from 57 histologically confirmed normal and abnormal tissue samples and histological parameters were extracted. The model produced spectra which match well the measured data, with the corresponding spectral parameters being well within histologically plausible ranges. Parameters extracted for the abnormal spectra showed the increase in blood volume fraction and changes in collagen pattern characteristic of the colon cancer. The spectra extracted from multi-spectral images of ex-vivo colon including adenocarcinoma show the characteristic features associated with normal and abnormal colon tissue. These findings suggest that it should be possible to compute histological quantities for the colon from the multi-spectral images.

Genome-wide combination profiling of DNA copy number and methylation for deciphering biomarkers in non-small cell lung cancer patients.

PubMed

Son, Ji Woong; Jeong, Kang Jin; Jean, Woo-Sean; Park, Soon Young; Jheon, Sanghoon; Cho, Hyun Min; Park, Chang Gyo; Lee, Hoi Young; Kang, Jaeku

2011-12-01

Early detection of lung cancer provides the highest potential for saving lives. To date, no routine screening method enabling early detection is available, which is a key factor in the disease's high mortality rate. Copy number changes and DNA methylation alterations are good indicators of carcinogenesis and cancer prognosis. In this study, we attempted to combine profiles of DNA copy number and methylation patterns in 20 paired cancerous and noncancerous tissue samples from non-small cell lung cancer (NSCLC) patients, and we detected several clinically important genes with genetic and epigenetic relationships. Using array comparative genomic hybridization (aCGH), statistically significant differences were observed across the histological subtypes for gains at 1p31.1, 3q26.1, and 3q26.31-3q29 as well as for losses at 1p21.1, 2q33.3, 2q37.3, 3p12.3, 4q35.2, and 13q34 in squamous cell carcinoma (SQ) patients, and losses at 12q24.33 were measured in adenocarcinoma (AD) patients (p < 0.05). In an analysis of DNA methylation at 1505 autosomal CpG loci that are associated with 807 cancer-related genes, we identified six and nine loci with higher and lower DNA methylation levels, respectively, in tumor tissue compared to non-tumor lung tissues from AD patients. In addition, three loci with higher and seven loci with lower DNA methylation levels were identified in tumor tissue from SQ patients compared to non-tumor lung tissue. Subsequently, we searched for regions exhibiting concomitant hypermethylation and genomic loss in both ADs and SQs. One clone representing 7p15.2 (which includes candidate genes such as HOXA9 and HOXA11) and one target ID representing HOXA9_E252_R were detected. Quantitative real-time PCR identified the potential candidate gene HOXA9 as being down-regulated in the majority of NSCLC patients. Moreover, following HOXA9 over-expression, the invasion of representative cell lines, A549 and HCC95, were significantly inhibited. Taken together, our results show that the combined profiling analysis technique is a useful tool for identifying biomarkers in lung cancer and that HOXA9 might be a potential candidate gene for the pathogenesis and diagnosis of NSCLC patients. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

A pilot to examine the logistical and feasibility issues in testing deceased tissue donors for vCJD using tonsil as the analyte.

PubMed

Warwick, Ruth M; Armitage, W John; Chandrasekar, Akila; Mallinson, Gary; Poniatowski, Stefan; Clarkson, Anthony

2012-03-01

Transplanted tissues have transmitted transmissible spongiform encephalopathies and in the UK there have been more cases of variant Creutzfeldt-Jakob disease (vCJD) than elsewhere in the world. A pilot study was undertaken to look at the feasibility of testing for vCJD in deceased donors using tonsillar tissue. This pilot showed that obtaining consent for removal and testing tonsil tissue was feasible. Donor eligibility for inclusion in the pilot was limited to tissue donors from the National Health Service Blood and Transplant, Tissue Services and to donors shared with the Corneal Transplant Service Eye Banks. Obtaining tonsillar tissue in the immediate post-mortem period was limited by the presence of rigor mortis. Tonsillar tissue was suitable for routine analysis for the presence of prion associated with vCJD in deceased tissue donors. Production and processing of tissue was straightforward and a low assay background was obtained from most samples. Since palatine and lingual tonsil tissue can be obtained in pairs it was possible, in the majority of cases, to set aside an intact sample for confirmatory testing if required. In one instance a sample was reactive by Western blot. However, the pattern of reactivity was not typical for that obtained from vCJD patients. Unfortunately the sample was not of sufficient quality for the confirmatory test to provide a conclusive result.

DNA Yield From Tissue Samples in Surgical Pathology and Minimum Tissue Requirements for Molecular Testing.

PubMed

Austin, Melissa C; Smith, Christina; Pritchard, Colin C; Tait, Jonathan F

2016-02-01

Complex molecular assays are increasingly used to direct therapy and provide diagnostic and prognostic information but can require relatively large amounts of DNA. To provide data to pathologists to help them assess tissue adequacy and provide prospective guidance on the amount of tissue that should be procured. We used slide-based measurements to establish a relationship between processed tissue volume and DNA yield by A260 from 366 formalin-fixed, paraffin-embedded tissue samples submitted for the 3 most common molecular assays performed in our laboratory (EGFR, KRAS, and BRAF). We determined the average DNA yield per unit of tissue volume, and we used the distribution of DNA yields to calculate the minimum volume of tissue that should yield sufficient DNA 99% of the time. All samples with a volume greater than 8 mm(3) yielded at least 1 μg of DNA, and more than 80% of samples producing less than 1 μg were extracted from less than 4 mm(3) of tissue. Nine square millimeters of tissue should produce more than 1 μg of DNA 99% of the time. We conclude that 2 tissue cores, each 1 cm long and obtained with an 18-gauge needle, will almost always provide enough DNA for complex multigene assays, and our methodology may be readily extrapolated to individual institutional practice.

Arterial Suture in Acute Radiation Sickness - USSR

DTIC Science & Technology

1960-06-30

was surrounded, in the area of the suture, by abundantly developed cicatrical tissue and was tightly adhered to the surrounding tissue. When there...later, the vascular cicatrix represents solid cicatrical tissue, and, on the side of the vascular lumen, is covered with endothelim, Silk threads were

The transcriptome and miRNome profiling of glioblastoma tissues and peritumoral regions highlights molecular pathways shared by tumors and surrounding areas and reveals differences between short-term and long-term survivors

PubMed Central

Fazi, Barbara; Felsani, Armando; Grassi, Luigi; Moles, Anna; D'Andrea, Daniel; Toschi, Nicola; Sicari, Daria; De Bonis, Pasquale; Anile, Carmelo; Guerrisi, Maria Giovanna; Luca, Emilia; Farace, Maria Giulia; Maira, Giulio

2015-01-01

Glioblastoma multiforme (GBM) is the most common and deadliest primary brain tumor, driving patients to death within 15 months after diagnosis (short term survivors, ST), with the exception of a small fraction of patients (long term survivors, LT) surviving longer than 36 months. Here we present deep sequencing data showing that peritumoral (P) areas differ from healthy white matter, but share with their respective frankly tumoral (C) samples, a number of mRNAs and microRNAs representative of extracellular matrix remodeling, TGFβ and signaling, of the involvement of cell types different from tumor cells but contributing to tumor growth, such as microglia or reactive astrocytes. Moreover, we provide evidence about RNAs differentially expressed in ST vs LT samples, suggesting the contribution of TGF-β signaling in this distinction too. We also show that the edited form of miR-376c-3p is reduced in C vs P samples and in ST tumors compared to LT ones. As a whole, our study provides new insights into the still puzzling distinction between ST and LT tumors, and sheds new light onto that “grey” zone represented by the area surrounding the tumor, which we show to be characterized by the expression of several molecules shared with the proper tumor mass. PMID:26188123

Impact of Dermoscopy and Reflectance Confocal Microscopy on the Histopathologic Diagnosis of Lentigo Maligna/Lentigo Maligna Melanoma.

PubMed

Mataca, Ema; Migaldi, Mario; Cesinaro, Anna M

2018-06-15

Equivocal pigmented lesions of the head are usually biopsied to avoid inappropriate treatment. Clinical approach has evolved from simple visual examination to sophisticated techniques for selecting the biopsy sites. This study aimed to retrospectively evaluate the efficiency of dermoscopy (DE) and reflectance confocal microscopy (RCM) in sampling a histopathologically representative focus of lentigo maligna/lentigo maligna melanoma. Punch biopsies and surgical excisions of 72 patients, 37 men and 35 women (median age 70.6 years, range 39-90 years), affected by lentigo maligna/lentigo maligna melanoma of the head, sent from a single dermatology clinic, were reviewed for the presence of 5 histopathologic criteria: atypical junctional melanocytes, increased junctional melanocytes, follicular colonization, pagetoid spread and melanocytic junctional nests, plus other minor features. Forty-two patients were biopsied under DE and 30 under RCM guidance. Accuracy of the 2 techniques in sampling a representative tissue overlapped in most cases, although RCM selected sites to biopsy with more histopathologic criteria, in particular pagetoid spread and melanocytic nests. Interestingly, with RCM, inflammation and melanophages were observed more in biopsy than in excision. False positive cases were not registered. Compared with the sampling at naked eye, our results show that DE and RCM help selecting the most appropriate areas for biopsies, thus allowing not only more robust histopathologic diagnoses, but also a more accurate microstaging of tumor.

The transcriptome and miRNome profiling of glioblastoma tissues and peritumoral regions highlights molecular pathways shared by tumors and surrounding areas and reveals differences between short-term and long-term survivors.

PubMed

Fazi, Barbara; Felsani, Armando; Grassi, Luigi; Moles, Anna; D'Andrea, Daniel; Toschi, Nicola; Sicari, Daria; De Bonis, Pasquale; Anile, Carmelo; Guerrisi, Maria Giovanna; Luca, Emilia; Farace, Maria Giulia; Maira, Giulio; Ciafré, Silvia Anna; Mangiola, Annunziato

2015-09-08

Glioblastoma multiforme (GBM) is the most common and deadliest primary brain tumor, driving patients to death within 15 months after diagnosis (short term survivors, ST), with the exception of a small fraction of patients (long term survivors, LT) surviving longer than 36 months. Here we present deep sequencing data showing that peritumoral (P) areas differ from healthy white matter, but share with their respective frankly tumoral (C) samples, a number of mRNAs and microRNAs representative of extracellular matrix remodeling, TGFβ and signaling, of the involvement of cell types different from tumor cells but contributing to tumor growth, such as microglia or reactive astrocytes. Moreover, we provide evidence about RNAs differentially expressed in ST vs LT samples, suggesting the contribution of TGF-β signaling in this distinction too. We also show that the edited form of miR-376c-3p is reduced in C vs P samples and in ST tumors compared to LT ones. As a whole, our study provides new insights into the still puzzling distinction between ST and LT tumors, and sheds new light onto that "grey" zone represented by the area surrounding the tumor, which we show to be characterized by the expression of several molecules shared with the proper tumor mass.

X-ray Absorption Near Edge Structure Spectroscopy to Resolve the in Vivo Chemistry of the Redox-Active Indazolium trans-[Tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019)

PubMed Central

2013-01-01

Indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (1, KP1019) and its analogue sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (2, KP1339) are promising redox-active anticancer drug candidates that were investigated with X-ray absorption near edge structure spectroscopy. The analysis was based on the concept of the coordination charge and ruthenium model compounds representing possible coordinations and oxidation states in vivo. 1 was investigated in citrate saline buffer (pH 3.5) and in carbonate buffer (pH 7.4) at 37 °C for different time intervals. Interaction studies on 1 with glutathione in saline buffer and apo-transferrin in carbonate buffer were undertaken, and the coordination of 1 and 2 in tumor tissues was studied too. The most likely coordinations and oxidation states of the compound under the above mentioned conditions were assigned. Microprobe X-ray fluorescence of tumor thin sections showed the strong penetration of ruthenium into the tumor tissue, with the highest concentrations near blood vessels and in the edge regions of the tissue samples. PMID:23282017

Understanding diet and modeling changes in the omega-3 and omega-6 fatty acid composition of U.S. garrison foods for active duty personnel.

PubMed

Marriott, Bernadette P; Yu, Karina; Majchrzak-Hong, Sharon; Johnson, Jeremiah; Hibbeln, Joseph R

2014-11-01

Research indicates that dietary omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) are important in reducing the risk of mental illness. We used the DoD Survey of Health Related Behaviors among Active Duty Military Personnel (HRBS) to assess current military dietary patterns and meal locations. We used the Lands Equation to model PUFAs in a sample Garrison diet and the nutritional impact of substitution of foods higher in omega-3 PUFAs and lower in omega-6 PUFAs on tissue composition. The military diet was very poor quality compared to 2010 Healthy People Guidelines. A representative Garrison diet does not meet our estimated healthy n-3 HUFA intake at 3.5 g/d, corresponding with a tissue composition of 60% n-3 in HUFA (i.e., 40% n-6 in HUFA). Substitution of n-3 rich eggs, poultry, pork and other food commodities, combined with use on low linoleic acid oils, may contribute significantly to attaining healthier n-6/n-3 proportions in the tissue. Reprint & Copyright © 2014 Association of Military Surgeons of the U.S.

The Japanese Society of Pathology Guidelines on the handling of pathological tissue samples for genomic research: Standard operating procedures based on empirical analyses.

PubMed

Kanai, Yae; Nishihara, Hiroshi; Miyagi, Yohei; Tsuruyama, Tatsuhiro; Taguchi, Kenichi; Katoh, Hiroto; Takeuchi, Tomoyo; Gotoh, Masahiro; Kuramoto, Junko; Arai, Eri; Ojima, Hidenori; Shibuya, Ayako; Yoshida, Teruhiko; Akahane, Toshiaki; Kasajima, Rika; Morita, Kei-Ichi; Inazawa, Johji; Sasaki, Takeshi; Fukayama, Masashi; Oda, Yoshinao

2018-02-01

Genome research using appropriately collected pathological tissue samples is expected to yield breakthroughs in the development of biomarkers and identification of therapeutic targets for diseases such as cancers. In this connection, the Japanese Society of Pathology (JSP) has developed "The JSP Guidelines on the Handling of Pathological Tissue Samples for Genomic Research" based on an abundance of data from empirical analyses of tissue samples collected and stored under various conditions. Tissue samples should be collected from appropriate sites within surgically resected specimens, without disturbing the features on which pathological diagnosis is based, while avoiding bleeding or necrotic foci. They should be collected as soon as possible after resection: at the latest within about 3 h of storage at 4°C. Preferably, snap-frozen samples should be stored in liquid nitrogen (about -180°C) until use. When intending to use genomic DNA extracted from formalin-fixed paraffin-embedded tissue, 10% neutral buffered formalin should be used. Insufficient fixation and overfixation must both be avoided. We hope that pathologists, clinicians, clinical laboratory technicians and biobank operators will come to master the handling of pathological tissue samples based on the standard operating procedures in these Guidelines to yield results that will assist in the realization of genomic medicine. © 2018 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

Mercury contamination in Khramulia (Capoeta capoeta) from the Cheshme Kile and Zarrin Gol Rivers in Iran and human health risk assessment.

PubMed

Malvandi, Hassan; Sari, Abbas Esmaili; Aliabadian, Mansour

2014-10-01

Total mercury concentrations were determined in muscle tissue of Khramulia (Capoeta capoeta) captured in the Cheshme Kile and Zarrin Gol Rivers, Iran. In Cheshme Kile River, 49 fish samples were collected. The mean total mercury concentration in the muscles of C. capoeta from this area was 249 ng g(-1) dw. In Zarrin Gol River, where 62 fish samples were collected, the total mercury in muscles averaged 164 ng g(-1) dw. A significant difference was found between means of mercury in the rivers (p < 0.001). All samples from the two rivers had mean mercury concentrations below the maximum allowable limits for mercury set by the Food and Agriculture Organization, World Health Organization, Standardization Administration of China and Environmental Protection Agency. The results of this study indicate that the values of hazard target quotient and estimated weekly intake are low and represent a negligible risk for human health.

FANTOM5 CAGE profiles of human and mouse samples.

PubMed

Noguchi, Shuhei; Arakawa, Takahiro; Fukuda, Shiro; Furuno, Masaaki; Hasegawa, Akira; Hori, Fumi; Ishikawa-Kato, Sachi; Kaida, Kaoru; Kaiho, Ai; Kanamori-Katayama, Mutsumi; Kawashima, Tsugumi; Kojima, Miki; Kubosaki, Atsutaka; Manabe, Ri-Ichiroh; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakazato, Kenichi; Ninomiya, Noriko; Nishiyori-Sueki, Hiromi; Noma, Shohei; Saijyo, Eri; Saka, Akiko; Sakai, Mizuho; Simon, Christophe; Suzuki, Naoko; Tagami, Michihira; Watanabe, Shoko; Yoshida, Shigehiro; Arner, Peter; Axton, Richard A; Babina, Magda; Baillie, J Kenneth; Barnett, Timothy C; Beckhouse, Anthony G; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James; Brombacher, Frank; Carlisle, Ailsa J; Clevers, Hans C; Davis, Carrie A; Detmar, Michael; Dohi, Taeko; Edge, Albert S B; Edinger, Matthias; Ehrlund, Anna; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Eslami, Afsaneh; Fagiolini, Michela; Fairbairn, Lynsey; Farach-Carson, Mary C; Faulkner, Geoffrey J; Ferrai, Carmelo; Fisher, Malcolm E; Forrester, Lesley M; Fujita, Rie; Furusawa, Jun-Ichi; Geijtenbeek, Teunis B; Gingeras, Thomas; Goldowitz, Daniel; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas J; Hamaguchi, Masahide; Hara, Mitsuko; Hasegawa, Yuki; Herlyn, Meenhard; Heutink, Peter; Hitchens, Kelly J; Hume, David A; Ikawa, Tomokatsu; Ishizu, Yuri; Kai, Chieko; Kawamoto, Hiroshi; Kawamura, Yuki I; Kempfle, Judith S; Kenna, Tony J; Kere, Juha; Khachigian, Levon M; Kitamura, Toshio; Klein, Sarah; Klinken, S Peter; Knox, Alan J; Kojima, Soichi; Koseki, Haruhiko; Koyasu, Shigeo; Lee, Weonju; Lennartsson, Andreas; Mackay-Sim, Alan; Mejhert, Niklas; Mizuno, Yosuke; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Morris, Kelly J; Motohashi, Hozumi; Mummery, Christine L; Nakachi, Yutaka; Nakahara, Fumio; Nakamura, Toshiyuki; Nakamura, Yukio; Nozaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A; Passier, Robert; Patrikakis, Margaret; Pombo, Ana; Pradhan-Bhatt, Swati; Qin, Xian-Yang; Rehli, Michael; Rizzu, Patrizia; Roy, Sugata; Sajantila, Antti; Sakaguchi, Shimon; Sato, Hiroki; Satoh, Hironori; Savvi, Suzana; Saxena, Alka; Schmidl, Christian; Schneider, Claudio; Schulze-Tanzil, Gundula G; Schwegmann, Anita; Sheng, Guojun; Shin, Jay W; Sugiyama, Daisuke; Sugiyama, Takaaki; Summers, Kim M; Takahashi, Naoko; Takai, Jun; Tanaka, Hiroshi; Tatsukawa, Hideki; Tomoiu, Andru; Toyoda, Hiroo; van de Wetering, Marc; van den Berg, Linda M; Verardo, Roberto; Vijayan, Dipti; Wells, Christine A; Winteringham, Louise N; Wolvetang, Ernst; Yamaguchi, Yoko; Yamamoto, Masayuki; Yanagi-Mizuochi, Chiyo; Yoneda, Misako; Yonekura, Yohei; Zhang, Peter G; Zucchelli, Silvia; Abugessaisa, Imad; Arner, Erik; Harshbarger, Jayson; Kondo, Atsushi; Lassmann, Timo; Lizio, Marina; Sahin, Serkan; Sengstag, Thierry; Severin, Jessica; Shimoji, Hisashi; Suzuki, Masanori; Suzuki, Harukazu; Kawai, Jun; Kondo, Naoto; Itoh, Masayoshi; Daub, Carsten O; Kasukawa, Takeya; Kawaji, Hideya; Carninci, Piero; Forrest, Alistair R R; Hayashizaki, Yoshihide

2017-08-29

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

FANTOM5 CAGE profiles of human and mouse samples

PubMed Central

Noguchi, Shuhei; Arakawa, Takahiro; Fukuda, Shiro; Furuno, Masaaki; Hasegawa, Akira; Hori, Fumi; Ishikawa-Kato, Sachi; Kaida, Kaoru; Kaiho, Ai; Kanamori-Katayama, Mutsumi; Kawashima, Tsugumi; Kojima, Miki; Kubosaki, Atsutaka; Manabe, Ri-ichiroh; Murata, Mitsuyoshi; Nagao-Sato, Sayaka; Nakazato, Kenichi; Ninomiya, Noriko; Nishiyori-Sueki, Hiromi; Noma, Shohei; Saijyo, Eri; Saka, Akiko; Sakai, Mizuho; Simon, Christophe; Suzuki, Naoko; Tagami, Michihira; Watanabe, Shoko; Yoshida, Shigehiro; Arner, Peter; Axton, Richard A.; Babina, Magda; Baillie, J. Kenneth; Barnett, Timothy C.; Beckhouse, Anthony G.; Blumenthal, Antje; Bodega, Beatrice; Bonetti, Alessandro; Briggs, James; Brombacher, Frank; Carlisle, Ailsa J.; Clevers, Hans C.; Davis, Carrie A.; Detmar, Michael; Dohi, Taeko; Edge, Albert S.B.; Edinger, Matthias; Ehrlund, Anna; Ekwall, Karl; Endoh, Mitsuhiro; Enomoto, Hideki; Eslami, Afsaneh; Fagiolini, Michela; Fairbairn, Lynsey; Farach-Carson, Mary C.; Faulkner, Geoffrey J.; Ferrai, Carmelo; Fisher, Malcolm E.; Forrester, Lesley M.; Fujita, Rie; Furusawa, Jun-ichi; Geijtenbeek, Teunis B.; Gingeras, Thomas; Goldowitz, Daniel; Guhl, Sven; Guler, Reto; Gustincich, Stefano; Ha, Thomas J.; Hamaguchi, Masahide; Hara, Mitsuko; Hasegawa, Yuki; Herlyn, Meenhard; Heutink, Peter; Hitchens, Kelly J.; Hume, David A.; Ikawa, Tomokatsu; Ishizu, Yuri; Kai, Chieko; Kawamoto, Hiroshi; Kawamura, Yuki I.; Kempfle, Judith S.; Kenna, Tony J.; Kere, Juha; Khachigian, Levon M.; Kitamura, Toshio; Klein, Sarah; Klinken, S. Peter; Knox, Alan J.; Kojima, Soichi; Koseki, Haruhiko; Koyasu, Shigeo; Lee, Weonju; Lennartsson, Andreas; Mackay-sim, Alan; Mejhert, Niklas; Mizuno, Yosuke; Morikawa, Hiromasa; Morimoto, Mitsuru; Moro, Kazuyo; Morris, Kelly J.; Motohashi, Hozumi; Mummery, Christine L.; Nakachi, Yutaka; Nakahara, Fumio; Nakamura, Toshiyuki; Nakamura, Yukio; Nozaki, Tadasuke; Ogishima, Soichi; Ohkura, Naganari; Ohno, Hiroshi; Ohshima, Mitsuhiro; Okada-Hatakeyama, Mariko; Okazaki, Yasushi; Orlando, Valerio; Ovchinnikov, Dmitry A.; Passier, Robert; Patrikakis, Margaret; Pombo, Ana; Pradhan-Bhatt, Swati; Qin, Xian-Yang; Rehli, Michael; Rizzu, Patrizia; Roy, Sugata; Sajantila, Antti; Sakaguchi, Shimon; Sato, Hiroki; Satoh, Hironori; Savvi, Suzana; Saxena, Alka; Schmidl, Christian; Schneider, Claudio; Schulze-Tanzil, Gundula G.; Schwegmann, Anita; Sheng, Guojun; Shin, Jay W.; Sugiyama, Daisuke; Sugiyama, Takaaki; Summers, Kim M.; Takahashi, Naoko; Takai, Jun; Tanaka, Hiroshi; Tatsukawa, Hideki; Tomoiu, Andru; Toyoda, Hiroo; van de Wetering, Marc; van den Berg, Linda M.; Verardo, Roberto; Vijayan, Dipti; Wells, Christine A.; Winteringham, Louise N.; Wolvetang, Ernst; Yamaguchi, Yoko; Yamamoto, Masayuki; Yanagi-Mizuochi, Chiyo; Yoneda, Misako; Yonekura, Yohei; Zhang, Peter G.; Zucchelli, Silvia; Abugessaisa, Imad; Arner, Erik; Harshbarger, Jayson; Kondo, Atsushi; Lassmann, Timo; Lizio, Marina; Sahin, Serkan; Sengstag, Thierry; Severin, Jessica; Shimoji, Hisashi; Suzuki, Masanori; Suzuki, Harukazu; Kawai, Jun; Kondo, Naoto; Itoh, Masayoshi; Daub, Carsten O.; Kasukawa, Takeya; Kawaji, Hideya; Carninci, Piero; Forrest, Alistair R.R.; Hayashizaki, Yoshihide

2017-01-01

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities. PMID:28850106

Connective Tissue Disorders and Cardiovascular Complications: The indomitable role of Transforming Growth Factor-beta signaling

PubMed Central

Wheeler, Jason B.; Ikonomidis, John S.; Jones, Jeffrey A.

2015-01-01

Marfan Syndrome (MFS) and Loeys-Dietz Syndrome (LDS) represent heritable connective tissue disorders that cosegregate with a similar pattern of cardiovascular defects (thoracic aortic aneurysm, mitral valve prolapse/regurgitation, and aortic dilatation with regurgitation). This pattern of cardiovascular defects appears to be expressed along a spectrum of severity in many heritable connective tissue disorders and raises suspicion of a relationship between the normal development of connective tissues and the cardiovascular system. Given the evidence of increased transforming growth factor-beta (TGF-β) signaling in MFS and LDS, this signaling pathway may represent the common link in this relationship. To further explore this hypothetical link, this chapter will review the TGF-β signaling pathway, heritable connective tissue syndromes related to TGF-β receptor (TGFBR) mutations, and discuss the pathogenic contribution of TGF-β to these syndromes with a primary focus on the cardiovascular system. PMID:24443024

Importance of tissue preparation methods in FTIR micro-spectroscopical analysis of biological tissues: 'traps for new users'.

PubMed

Zohdi, Vladislava; Whelan, Donna R; Wood, Bayden R; Pearson, James T; Bambery, Keith R; Black, M Jane

2015-01-01

Fourier Transform Infrared (FTIR) micro-spectroscopy is an emerging technique for the biochemical analysis of tissues and cellular materials. It provides objective information on the holistic biochemistry of a cell or tissue sample and has been applied in many areas of medical research. However, it has become apparent that how the tissue is handled prior to FTIR micro-spectroscopic imaging requires special consideration, particularly with regards to methods for preservation of the samples. We have performed FTIR micro-spectroscopy on rodent heart and liver tissue sections (two spectroscopically very different biological tissues) that were prepared by desiccation drying, ethanol substitution and formalin fixation and have compared the resulting spectra with that of fully hydrated freshly excised tissues. We have systematically examined the spectra for any biochemical changes to the native state of the tissue caused by the three methods of preparation and have detected changes in infrared (IR) absorption band intensities and peak positions. In particular, the position and profile of the amide I, key in assigning protein secondary structure, changes depending on preparation method and the lipid absorptions lose intensity drastically when these tissues are hydrated with ethanol. Indeed, we demonstrate that preserving samples through desiccation drying, ethanol substitution or formalin fixation significantly alters the biochemical information detected using spectroscopic methods when compared to spectra of fresh hydrated tissue. It is therefore imperative to consider tissue preparative effects when preparing, measuring, and analyzing samples using FTIR spectroscopy.

Control of the rate of cell enlargement: Excision, wall relaxation, and growth-induced water potentials.

PubMed

Boyer, J S; Cavalieri, A J; Schulze, E D

1985-04-01

A new guillotine thermocouple psychrometer was used to make continuous measurements of water potential before and after the excision of elongating and mature regions of darkgrown soybean (Glycine max L. Merr.) stems. Transpiration could not occur, but growth took place during the measurement if the tissue was intact. Tests showed that the instrument measured the average water potential of the sampled tissue and responded rapidly to changes in water potential. By measuring tissue osmotic potential (Ψ s ), turgor pressure (Ψ p ) could be calculated. In the intact plant, Ψ s and Ψ p were essentially constant for the entire 22 h measurement, but Ψ s was lower and Ψ p higher in the elongating region than in the mature region. This caused the water potential in the elongating region to be lower than in the mature region. The mature tissue equilibrated with the water potential of the xylem. Therefore, the difference in water potential between mature and elongating tissue represented a difference between the xylem and the elongating region, reflecting a water potential gradient from the xylem to the epidermis that was involved in supplying water for elongation. When mature tissue was excised with the guillotine, Ψ s and Ψ p did not change. However, when elongating tissue was excised, water was absorbed from the xylem, whose water potential decreased. This collapsed the gradient and prevented further water uptake. Tissue Ψ p then decreased rapidly (5 min) by about 0.1 MPa in the elongating tissue. The Ψ p decreased because the cell walls relaxed as extension, caused by Ψ p , continued briefly without water uptake. The Ψ p decreased until the minimum for wall extension (Y) was reached, whereupon elongation ceased. This was followed by a slow further decrease in Y but no additional elongation. In elongating tissue excised with mature tissue attached, there was almost no effect on water potential or Ψ p for several hours. Nevertheless, growth was reduced immediately and continued at a decreasing rate. In this case, the mature tissue supplied water to the elongating tissue and the cell walls did not relax. Based on these measurements, a theory is presented for simultaneously evaluating the effects of water supply and water demand associated with growth. Because wall relaxation measured with the psychrometer provided a new method for determining Y and wall extensibility, all the factors required by the theory could be evaluated for the first time in a single sample. The analysis showed that water uptake and wall extension co-limited elongation in soybean stems under our conditions. This co-limitation explains why elongation responded immediately to a decrease in the water potential of the xylem and why excision with attached mature tissue caused an immediate decrease in growth rate without an immediate change in Ψ p.

Trace levels of Fukushima disaster radionuclides in East Pacific albacore.

PubMed

Neville, Delvan R; Phillips, A Jason; Brodeur, Richard D; Higley, Kathryn A

2014-05-06

The Fukushima Daiichi power station released several radionuclides into the Pacific following the March 2011 earthquake and tsunami. A total of 26 Pacific albacore (Thunnus alalunga) caught off the Pacific Northwest U.S. coast between 2008 and 2012 were analyzed for (137)Cs and Fukushima-attributed (134)Cs. Both 2011 (2 of 2) and several 2012 (10 of 17) edible tissue samples exhibited increased activity concentrations of (137)Cs (234-824 mBq/kg of wet weight) and (134)Cs (18.2-356 mBq/kg of wet weight). The remaining 2012 samples and all pre-Fukushima (2008-2009) samples possessed lower (137)Cs activity concentrations (103-272 mBq/kg of wet weight) with no detectable (134)Cs activity. Age, as indicated by fork length, was a strong predictor for both the presence and concentration of (134)Cs (p < 0.001). Notably, many migration-aged fish did not exhibit any (134)Cs, suggesting that they had not recently migrated near Japan. None of the tested samples would represent a significant change in annual radiation dose if consumed by humans.

Brominated flame retardants in polar bears (Ursus maritimus) from Alaska, the Canadian Arctic, East Greenland, and Svalbard.

PubMed

Muir, Derek C G; Backus, Sean; Derocher, Andrew E; Dietz, Rune; Evans, Thomas J; Gabrielsen, Geir W; Nagy, John; Norstrom, Ross J; Sonne, Christian; Stirling, Ian; Taylor, Mitch K; Letcher, Robert J

2006-01-15

Polybrominated diphenyl ethers (PBDEs) were determined in adipose tissue of adult and subadult female polar bears sampled between 1999 and 2002 from sub-populations in Arctic Canada, eastern Greenland, and Svalbard, and in males and females collected from 1994 to 2002 in northwestern Alaska. Only 4 congeners (BDE47, 99, 100, and 153) were consistently identified in all samples. BDE47 was the major PBDE congener representing from 65% to 82% of the sum (sigma) PBDEs. Age was not a significant covariate for individual PBDEs or sigmaPBDE. Higher proportions of BDE 99, 100, and 153 were generally found in samples from the Canadian Arctic than from Svalbard or the Bering-Chukchi Sea area of Alaska. Geometric mean sigmaPBDE concentrations were highest for female polar bear fat samples collected from Svalbard (50 ng/g lipid weight (lw)) and East Greenland (70 ng/g lw). Significantly lower sigmaPBDE concentrations were found in fat of bears from Canada and Alaska (means ranging from 7.6 to 22 ng/g lw).

Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing

PubMed Central

2011-01-01

Background Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. Results Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus. Conclusions This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts are expected as our understanding of its microbiota expands. Increased awareness of the role R. microplus can play in the transmission of pathogenic bacteria will enhance our ability to mitigate its economic impact on animal agriculture globally. This recognition should be included as part of analyses to assess the risk for re-invasion of areas like the United States of America where R. microplus was eradicated. PMID:21211038

Iophenoxic acid derivatives as markers of oral baits to wildlife. New tools for their detection in tissues of a game species and safety considerations for human exposure.

PubMed

Sage, Mickael; Fourel, Isabelle; Lahoreau, Jennifer; Siat, Vivien; Berny, Philippe; Rossi, Sophie

2013-05-01

The bait-marker iophenoxic acid (IPA) and its derivatives are increasingly used for evaluating and optimizing the cost-effectiveness of baiting campaigns on wildlife, particularly on game species such as the wild boar. We aimed to determine whether concentrations of the three main IPA derivatives ethyl, methyl and propyl-IPA measured on thoracic liquid extracts (TLE) of hunted wild boars may be representative of two exposure doses, 40 and 200 mg, from 20 to 217 days after ingestion. Then we developed a method of detection of the three IPA derivatives by LC/ESI-MS-MS in muscle and liver to evaluate the suitability of these two other tissues for monitoring the marked bait consumption and for measuring available residues in the meat of marked animals. Three semi-captive wild boars received 40 mg of each IPA derivative, three received 200 mg, and three, as controls, did not receive IPA. Blood serum was sampled 20, 197 or 217 days after IPA exposure according to animals and to the derivative. Wild boars were shot by gun after the different times of serum sampling times, and TLE, muscle and liver were sampled. Our results suggest that TLE is not a relevant tissue for quantitatively expressing IPA exposure. Due to interference, no analytical method was validated on TLE containing digestive material. On the other hand, quantifications in the muscle and particularly in the liver could discriminate wild boars that had ingested the two IPA doses from 20 days until 7 months after exposure, especially for the two long term markers ethyl and propyl-IPA. So IPA quantifications in the liver sampled on hunted animals appear to be a reliable tool for monitoring bait consumption in the field at a large scale. Nevertheless, whatever the ingested dose, ethyl- and propyl-IPA concentrations measured in the muscle and the liver of tested animals until 217 days after exposure, remained higher than 0.01 mg/kg, the Maximal Residue Limit (MRL) is recommended for molecules for which no toxicological data are available. Based on the range of IPA residues available in these two tissues, implications for humans consuming marked animals are discussed.

Improved quantitative recovery of Listeria monocytogenes from stainless steel surfaces using a one-ply composite tissue.

PubMed

Vorst, Keith L; Todd, Ewen C D; Rysert, Elliot T

2004-10-01

Four sampling devices, a sterile environmental sponge (ES), a sterile cotton-tipped swab (CS), a sterile calcium alginate fiber-tipped swab (CAS), and a one-ply composite tissue (CT), were evaluated for quantitative recovery of Listeria monocytogenes from a food-grade stainless steel surface. Sterile 304-grade stainless steel plates (6 by 6 cm) were inoculated with approximately 106 CFU/cm2 L. monocytogenes strain Scott A and dried for 1 h. The ES and CT sampling devices were rehydrated in phosphate buffer solution. After plate swabbing, ES and CT were placed in 40 ml of phosphate buffer solution, stomached for 1 min and hand massaged for 30 s. Each CS and CAS device was rehydrated in 0.1% peptone before swabbing. After swabbing, CS and CAS were vortexed in 0.1% peptone for 1 min. Samples were spiral plated on modified Oxford agar with modified Oxford agar Rodac Contact plates used to recover any remaining cells from the stainless steel surface. Potential inhibition from CT was examined in both phosphate buffer solution and in a modified disc-diffusion assay. Recovery was 2.70, 1.34, and 0.62 log greater using CT compared with ES, CS, and CAS, respectively, with these differences statistically significant (P < 0.001) for ES and CT and for CAS, CS, and CT (P < 0.05). Rodac plates were typically overgrown following ES, positive after CS and CAS, and negative after CT sampling. CT was noninhibitory in both phosphate buffer solution and the modified disc-diffusion assay. Using scanning electron microscopy, Listeria cells were observed on stainless steel plates sampled with each sampling device except CT. The CT device, which is inexpensive and easy to use, represents a major improvement over other methods in quantifying L. monocytogenes on stainless steel surfaces and is likely applicable to enrichment of environmental samples.

A comparison of sample preparation strategies for biological tissues and subsequent trace element analysis using LA-ICP-MS.

PubMed

Bonta, Maximilian; Török, Szilvia; Hegedus, Balazs; Döme, Balazs; Limbeck, Andreas

2017-03-01

Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is one of the most commonly applied methods for lateral trace element distribution analysis in medical studies. Many improvements of the technique regarding quantification and achievable lateral resolution have been achieved in the last years. Nevertheless, sample preparation is also of major importance and the optimal sample preparation strategy still has not been defined. While conventional histology knows a number of sample pre-treatment strategies, little is known about the effect of these approaches on the lateral distributions of elements and/or their quantities in tissues. The technique of formalin fixation and paraffin embedding (FFPE) has emerged as the gold standard in tissue preparation. However, the potential use for elemental distribution studies is questionable due to a large number of sample preparation steps. In this work, LA-ICP-MS was used to examine the applicability of the FFPE sample preparation approach for elemental distribution studies. Qualitative elemental distributions as well as quantitative concentrations in cryo-cut tissues as well as FFPE samples were compared. Results showed that some metals (especially Na and K) are severely affected by the FFPE process, whereas others (e.g., Mn, Ni) are less influenced. Based on these results, a general recommendation can be given: FFPE samples are completely unsuitable for the analysis of alkaline metals. When analyzing transition metals, FFPE samples can give comparable results to snap-frozen tissues. Graphical abstract Sample preparation strategies for biological tissues are compared with regard to the elemental distributions and average trace element concentrations.

Quantitative Analysis of Tissue Samples by Combining iTRAQ Isobaric Labeling with Selected/Multiple Reaction Monitoring (SRM/MRM).

PubMed

Narumi, Ryohei; Tomonaga, Takeshi

2016-01-01

Mass spectrometry-based phosphoproteomics is an indispensible technique used in the discovery and quantification of phosphorylation events on proteins in biological samples. The application of this technique to tissue samples is especially useful for the discovery of biomarkers as well as biological studies. We herein describe the application of a large-scale phosphoproteome analysis and SRM/MRM-based quantitation to develop a strategy for the systematic discovery and validation of biomarkers using tissue samples.

Contrast enhanced spectroscopic optical coherence tomography

NASA Technical Reports Server (NTRS)

Xu, Chenyang (Inventor); Boppart, Stephen A. (Inventor)

2010-01-01

A method of forming an image of a sample includes performing SOCT on a sample. The sample may include a contrast agent, which may include an absorbing agent and/or a scattering agent. A method of forming an image of tissue may include selecting a contrast agent, delivering the contrast agent to the tissue, acquiring SOCT data from the tissue, and converting the SOCT data into an image. The contributions to the SOCT data of an absorbing agent and a scattering agent in a sample may be quantified separately.

Improved results of LINE-1 methylation analysis in formalin-fixed, paraffin-embedded tissues with the application of a heating step during the DNA extraction process.

PubMed

Wen, Xianyu; Jeong, Seorin; Kim, Younghoon; Bae, Jeong Mo; Cho, Nam Yun; Kim, Jung Ho; Kang, Gyeong Hoon

2017-01-01

Formalin-fixed, paraffin-embedded (FFPE) tissues are important resources for profiling DNA methylation changes and for studying a variety of diseases. However, formalin fixation introduces inter-strand crosslinking, which might cause incomplete bisulfite conversion of unmethylated cytosines, which might lead to falsely elevated measurements of methylation levels in pyrosequencing assays. Long interspersed nucleotide element-1 (LINE-1) is a major constituent of repetitive transposable DNA elements, and its methylation is referred to correlates with global DNA methylation. To identify whether formalin fixation might impact the measured values of methylation in LINE-1 repetitive elements and whether prolonged heat-induced denaturation of DNA might reduce the artificial increases in measured values caused by formalin fixation, we analyzed paired fresh-frozen (FF) and FFPE xenograft tissue samples for their methylation levels in LINE-1 using a pyrosequencing assay. To further confirm the effect of a heating step in the measurement of LINE-1 or single gene methylation levels, we analyzed FFPE tissue samples of gastric cancer and colorectal cancer for their methylation status in LINE-1 and eight single genes, respectively. Formalin fixation led to an increase in the measured values of LINE-1 methylation regardless of the duration of fixation. Prolonged heating of the DNA at 95 °C for 30 min before bisulfite conversion was found (1) to decrease the discrepancy in the measured values between the paired FF and FFPE tissue samples, (2) to decrease the standard deviation of the measured value of LINE-1 methylation levels in FFPE tissue samples of gastric cancer, and (3) to improve the performance in the measurement of single gene methylation levels in FFPE tissue samples of colorectal cancer. Formalin fixation leads to artificial increases in the measured values of LINE-1 methylation, and the application of prolonged heating of DNA samples decreases the discrepancy in the measured values of LINE-1 methylation between paired FF and FFPE tissue samples. The application of prolonged heating of DNA samples improves bisulfite conversion-based measurement of LINE-1 or single gene methylation levels in FFPE tissue samples.

Insulin Mediated 14C-Glucose Incorporation Into Adipose Tissue: An Undergraduate Biochemistry Experiment

ERIC Educational Resources Information Center

Landman, A. D.; Eskin, N. A. M.

1975-01-01

Describes an experiment in which rat adipose tissue samples are exposed to labeled glucose; insulin is added to one sample. Subsequent scintillation counting demonstrates the ability of insulin to facilitate the entry of glucose into the tissue. (MLH)

Microgravity

NASA Image and Video Library

1998-10-10

Breast tissue specimens in traditional sample dishes. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cells (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunourous tissues.

Glucose transporter-1 (GLUT-1) immunoreactivity in benign, premalignant and malignant lesions of the gallbladder.

PubMed

Legan, Mateja; Tevžič, Spela; Tolar, Ana; Luzar, Boštjan; Marolt, Vera Ferlan

2011-03-01

GLUT-1 is a transmembrane glucose transport protein that allows the facilitated transport of glucose into cells, normally expressed in tissues which depend mainly on glucose metabolism. Enhanced expression of GLUT-1 can also be found in a large spectrum of carcinomas. This study aimed to investigate GLUT-1 expression in gallbladder tissue: from normal tissue samples, hyperplasias, low-grade and high-grade dysplasias to gallbladder carcinomas. In all, 115 archived samples of gallbladder tissue from 68 patients, presented after cholecystectomy, were immunohistochemically stained for GLUT-1. According to the intensity of GLUT-1 immunoreactivity, samples were divided into negative (stained 0-10% of cells stained), positive with weak to moderate (10-50%) and positive with strong (>50%) GLUT-1 expression. The GLUT-1 immunoreactivity of the samples showed a characteristic increase from premalignant lesions to carcinomas. Normal gallbladder tissue samples did not express GLUT-1 (100%). Weak expression was shown only focally in hyperplasias, but to a greater extent with low-grade dysplasias (20%), high-grade dysplasias (40%) and carcinomas (51.8%). Normal gallbladder tissue is GLUT-1 negative. GLUT-1 expression in carcinoma tissue is significantly higher than in dysplastic lesions. Strong GLUT-1 expression indicates 100% specificity for detecting gallbladder carcinomas. Therefore, GLUT-1 is a candidate as a diagnostic as well as a tissue prognostic marker in gallbladder carcinoma patients.

King's Health Partners' Prostate Cancer Biobank (KHP PCaBB).

PubMed

Saifuddin, S R; Devlies, W; Santaolalla, A; Cahill, F; George, G; Enting, D; Rudman, S; Cathcart, P; Challacombe, B; Dasgupta, P; Galustian, C; Chandra, A; Chowdhury, S; Gillett, C; Van Hemelrijck, M

2017-11-22

The KHP PCaBB was established in 2013 and recruits donors from the Urology or Oncology Departments at Guy's Hospital in London (UK). Prostate cancer patients may be approached to give their consent for biobanking at any point in their treatment pathway, which allows residual material from their earlier diagnosis to be transferred and used by the Biobank. Currently, patients are specifically asked to donate samples of blood and surplus prostate tissue as well as permitting access to their clinical and pathological data that continues to be added throughout the course of their disease. Between 2013 and 2015, 549 prostate cancer patients gave their consent to the biobank and, the tissue repository collected 489 blood samples, 120 frozen prostate tissue samples and 1064 formalin fixed paraffin embedded diagnostic blocks.Prostate cancer has become a chronic disease in a large proportion of men, with many men receiving multiple subsequent treatments, and their treatment trajectory often spanning over decades. Therefore, this resource aims to provide an ideal research platform to explore potential variations in treatment response as well as disease markers in the different risk categories for prostate cancer.A recent audit of the KHP PCaBB revealed that between 2013 and 2015, 1796 patients were diagnosed with prostate cancer at King's Health Partners (KHP), out of which 549 (30.6%) gave their consent to KHP PCaBB. Comparisons between demographic and clinical characteristics of patients who had consented compared to the total patient population revealed that the KHP PCaBB is demographically representative of the total prostate cancer patient population seen in Guy's and St Thomas' NHS Foundation Trust (GSTT). We observed no differences in distribution of ethnicity (p = 0.507) and socioeconomic status (p = 0.097). Some differences were observed in clinical characteristics, specifically with treatment type - which differed significantly between the patients who had given consent and total patient population.The KHP PCaBB has thereby amassed a rich data and tissue repository that is largely reflective of both the demographic and clinical diversity within the total prostate cancer patient population seen at KHP, making it an ideal platform for prostate cancer research.

Correlations of low-field NMR and variable-field NMR parameters with osteoarthritis in human articular cartilage under load.

PubMed

Rössler, Erik; Mattea, Carlos; Saarakkala, Simo; Lehenkari, Petri; Finnilä, Mikko; Rieppo, Lassi; Karhula, Sakari; Nieminen, Miika T; Stapf, Siegfried

2017-08-01

NMR experiments carried out at magnetic fields below 1 T provide new relaxation parameters unavailable with conventional clinical scanners. Contrast of T 1 generally becomes larger towards low fields, as slow molecular reorientation processes dominate relaxation at the corresponding Larmor frequencies. This advantage has to be considered in the context of lower sensitivity and frequently reduced spatial resolution. The layered structure of cartilage is one example where a particularly strong variation of T 1 across the tissue occurs, being affected by degenerative diseases such as osteoarthritis (OA). Furthermore, the presence of 1 H- 14  N cross-relaxation, leading to so-called quadrupolar dips in the 1 H relaxation time dispersion, provide insight into the concentration and mobility of proteoglycans and collagen in cartilage, both being affected by OA. In this study, low-field imaging and variable-field NMR relaxometry were combined for the first time for tissue samples, employing unidirectional load to probe the mechanical properties. 20 human knee cartilage samples were placed in a compression cell, and studied by determining relaxation profiles without and with applied pressure (0.6 MPa) at 50 μm in-plane resolution, and comparing with volume-averaged T 1 dispersion. Samples were subsequently stored in formalin, prepared for histology and graded according to the Mankin score system. Quadrupolar dips and thickness change under load showed the strongest correlation with Mankin grade. Average T 1 and change of maximum T 1 under load, as well as its position, correlate with thickness and thickness change. Furthermore, T 1 (ω) above 25 mT was found to correlate with thickness change. While volume-averaged T 1 is not a suitable indicator for OA, its change due to mechanical load and its extreme values are suggested as biomarkers available in low-field MRI systems. The shape of the dispersion T 1 (ω) represents a promising access to understanding and quantifying molecular dynamics in tissue, pointing toward future in vivo tissue studies. Copyright © 2017 John Wiley & Sons, Ltd.

Distribution of volatile branched-chain fatty acids in various lamb tissues.

PubMed

Brennand, C P; Lindsay, R C

1992-01-01

Volatile fatty acids (C4-C11) including even-, odd-, and branched-chain members in lamb tissues were quantitatively analyzed. Volatile branched-chain fatty acids (BCFA) were more concentrated in subcutaneous adipose tissue samples (rump, shoulder, breast) than in perinepheric adipose or muscle tissues. Perinepheric adipose tissue contained relatively high quantities of n-chain, even-numbered fatty acids and very low levels of BCFA. Greater variation existed in fatty acid profiles among similar subcutaneous adipose tissues from different lambs than between samples of adipose tissue from different carcass sites from a given lamb sample. 4-Methyl- and 4-ethyloctanoic acids were present at concentrations greatly above threshold levels in all lamb fats tested, and thus upon hydrolysis would contribute species-related flavors to lamb. 4-Methylnonanoic concentrations in lamb fats ranged from nondetectable to greater than the threshold level, and therefore this compound would not always contribute to the species-related flavors of lamb. Lean meat samples contained very low concentrations of 4-methyl- and 4-ethyloctanoic acids. Copyright © 1992. Published by Elsevier Ltd.

Contaminant levels in fish tissue from San Francisco Bay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fairey, R.; Taberski, K.

1995-12-31

Edible fish species were collected from thirteen locations throughout San Francisco Bay, during the spring of 1994, for determination of contaminants levels in muscle tissue. Species collected included white croaker, surfperch, leopard and brown smoothhound sharks, striped bass, white sturgeon and halibut Sixty six composite tissue samples were analyzed for the presence of PAHs, PCBs, pesticides, trace elements and dioxin/furans. The US EPA approach to assessing chemical contaminant data for fish tissue consumption was used for identifying the primary chemicals of concern. Six chemicals or chemical groups were found to exceed screening levels established using the US EPA approach. PCBsmore » (as total Aroclors) exceeded the screening level of 3 ppb in all sixty six tissue samples, with the highest concentrations (638 ppb) found near San Francisco`s industrial areas. Mercury was elevated (> 0.14 ppm) in forty of the sixty-six samples with the highest levels (1.26 ppm) occurring in shark muscle tissues. Concentrations of the organochlorine pesticides dieldrin, total chlordanes and total DDTs exceeded screening levels in a number of samples. Dioxin/furans (as TEQs) were elevated (above 0.15 ppt) in 16 of the 19 samples analyzed. Fish with high lipid content (croaker and surfperch) in their muscle tissue generally exhibited higher contaminant levels while fish with low lipid levels (halibut and shark) exhibited lower organic contaminant levels. Tissue samples taken from North Bay stations most often exhibited high levels of chemical contamination. The California Office of Health Hazard Assessment is currently evaluating the results of this study and has issued an interim Health Advisory concerning the human consumption of fish tissue from San Francisco Bay.« less

Effects of soft X-ray radiation damage on paraffin-embedded rat tissues supported on ultralene: a chemical perspective.

PubMed

Bedolla, Diana E; Mantuano, Andrea; Pickler, Arissa; Mota, Carla Lemos; Braz, Delson; Salata, Camila; Almeida, Carlos Eduardo; Birarda, Giovanni; Vaccari, Lisa; Barroso, Regina Cély; Gianoncelli, Alessandra

2018-05-01

Radiation damage is an important aspect to be considered when analysing biological samples with X-ray techniques as it can induce chemical and structural changes in the specimens. This work aims to provide new insights into the soft X-ray induced radiation damage of the complete sample, including not only the biological tissue itself but also the substrate and embedding medium, and the tissue fixation procedure. Sample preparation and handling involves an unavoidable interaction with the sample matrix and could play an important role in the radiation-damage mechanism. To understand the influence of sample preparation and handling on radiation damage, the effects of soft X-ray exposure at different doses on ultralene, paraffin and on paraffin-embedded rat tissues were studied using Fourier-transform infrared (FTIR) microspectroscopy and X-ray microscopy. Tissues were preserved with three different commonly used fixatives: formalin, glutaraldehyde and Karnovsky. FTIR results showed that ultralene and paraffin undergo a dose-dependent degradation of their vibrational profiles, consistent with radiation-induced oxidative damage. In addition, formalin fixative has been shown to improve the preservation of the secondary structure of proteins in tissues compared with both glutaraldehyde and Karnovsky fixation. However, conclusive considerations cannot be drawn on the optimal fixation protocol because of the interference introduced by both substrate and embedding medium in the spectral regions specific to tissue lipids, nucleic acids and carbohydrates. Notably, despite the detected alterations affecting the chemical architecture of the sample as a whole, composed of tissue, substrate and embedding medium, the structural morphology of the tissues at the micrometre scale is essentially preserved even at the highest exposure dose.

Comparison between thaw-mounting and use of conductive tape for sample preparation in ToF-SIMS imaging of lipids in Drosophila microRNA-14 model.

PubMed

Le, Minh Uyen Thi; Son, Jin Gyeong; Shon, Hyun Kyoung; Park, Jeong Hyang; Lee, Sung Bae; Lee, Tae Geol

2018-03-30

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging elucidates molecular distributions in tissue sections, providing useful information about the metabolic pathways linked to diseases. However, delocalization of the analytes and inadequate tissue adherence during sample preparation are among some of the unfortunate phenomena associated with this technique due to their role in the reduction of the quality, reliability, and spatial resolution of the ToF-SIMS images. For these reasons, ToF-SIMS imaging requires a more rigorous sample preparation method in order to preserve the natural state of the tissues. The traditional thaw-mounting method is particularly vulnerable to altered distributions of the analytes due to thermal effects, as well as to tissue shrinkage. In the present study, the authors made comparisons of different tissue mounting methods, including the thaw-mounting method. The authors used conductive tape as the tissue-mounting material on the substrate because it does not require heat from the finger for the tissue section to adhere to the substrate and can reduce charge accumulation during data acquisition. With the conductive-tape sampling method, they were able to acquire reproducible tissue sections and high-quality images without redistribution of the molecules. Also, the authors were successful in preserving the natural states and chemical distributions of the different components of fat metabolites such as diacylglycerol and fatty acids by using the tape-supported sampling in microRNA-14 (miR-14) deleted Drosophila models. The method highlighted here shows an improvement in the accuracy of mass spectrometric imaging of tissue samples.

3D imaging of optically cleared tissue using a simplified CLARITY method and on-chip microscopy

PubMed Central

Zhang, Yibo; Shin, Yoonjung; Sung, Kevin; Yang, Sam; Chen, Harrison; Wang, Hongda; Teng, Da; Rivenson, Yair; Kulkarni, Rajan P.; Ozcan, Aydogan

2017-01-01

High-throughput sectioning and optical imaging of tissue samples using traditional immunohistochemical techniques can be costly and inaccessible in resource-limited areas. We demonstrate three-dimensional (3D) imaging and phenotyping in optically transparent tissue using lens-free holographic on-chip microscopy as a low-cost, simple, and high-throughput alternative to conventional approaches. The tissue sample is passively cleared using a simplified CLARITY method and stained using 3,3′-diaminobenzidine to target cells of interest, enabling bright-field optical imaging and 3D sectioning of thick samples. The lens-free computational microscope uses pixel super-resolution and multi-height phase recovery algorithms to digitally refocus throughout the cleared tissue and obtain a 3D stack of complex-valued images of the sample, containing both phase and amplitude information. We optimized the tissue-clearing and imaging system by finding the optimal illumination wavelength, tissue thickness, sample preparation parameters, and the number of heights of the lens-free image acquisition and implemented a sparsity-based denoising algorithm to maximize the imaging volume and minimize the amount of the acquired data while also preserving the contrast-to-noise ratio of the reconstructed images. As a proof of concept, we achieved 3D imaging of neurons in a 200-μm-thick cleared mouse brain tissue over a wide field of view of 20.5 mm2. The lens-free microscope also achieved more than an order-of-magnitude reduction in raw data compared to a conventional scanning optical microscope imaging the same sample volume. Being low cost, simple, high-throughput, and data-efficient, we believe that this CLARITY-enabled computational tissue imaging technique could find numerous applications in biomedical diagnosis and research in low-resource settings. PMID:28819645

Validation of a Radiography-Based Quantification Designed to Longitudinally Monitor Soft Tissue Calcification in Skeletal Muscle.

PubMed

Moore, Stephanie N; Hawley, Gregory D; Smith, Emily N; Mignemi, Nicholas A; Ihejirika, Rivka C; Yuasa, Masato; Cates, Justin M M; Liu, Xulei; Schoenecker, Jonathan G

2016-01-01

Soft tissue calcification, including both dystrophic calcification and heterotopic ossification, may occur following injury. These lesions have variable fates as they are either resorbed or persist. Persistent soft tissue calcification may result in chronic inflammation and/or loss of function of that soft tissue. The molecular mechanisms that result in the development and maturation of calcifications are uncertain. As a result, directed therapies that prevent or resorb soft tissue calcifications remain largely unsuccessful. Animal models of post-traumatic soft tissue calcification that allow for cost-effective, serial analysis of an individual animal over time are necessary to derive and test novel therapies. We have determined that a cardiotoxin-induced injury of the muscles in the posterior compartment of the lower extremity represents a useful model in which soft tissue calcification develops remote from adjacent bones, thereby allowing for serial analysis by plain radiography. The purpose of the study was to design and validate a method for quantifying soft tissue calcifications in mice longitudinally using plain radiographic techniques and an ordinal scoring system. Muscle injury was induced by injecting cardiotoxin into the posterior compartment of the lower extremity in mice susceptible to developing soft tissue calcification. Seven days following injury, radiographs were obtained under anesthesia. Multiple researchers applied methods designed to standardize post-image processing of digital radiographs (N = 4) and quantify soft tissue calcification (N = 6) in these images using an ordinal scoring system. Inter- and intra-observer agreement for both post-image processing and the scoring system used was assessed using weighted kappa statistics. Soft tissue calcification quantifications by the ordinal scale were compared to mineral volume measurements (threshold 450.7mgHA/cm3) determined by μCT. Finally, sample-size calculations necessary to discriminate between a 25%, 50%, 75%, and 100% difference in STiCSS score 7 days following burn/CTX induced muscle injury were determined. Precision analysis demonstrated substantial to good agreement for both post-image processing (κ = 0.73 to 0.90) and scoring (κ = 0.88 to 0.93), with low inter- and intra-observer variability. Additionally, there was a strong correlation in quantification of soft tissue calcification between the ordinal system and by mineral volume quantification by μCT (Spearman r = 0.83 to 0.89). The ordinal scoring system reliably quantified soft tissue calcification in a burn/CTX-induced soft tissue calcification model compared to non-injured controls (Mann-Whitney rank test: P = 0.0002, ***). Sample size calculations revealed that 6 mice per group would be required to detect a 50% difference in STiCSS score with a power of 0.8. Finally, the STiCSS was demonstrated to reliably quantify soft tissue calcification [dystrophic calcification and heterotopic ossification] by radiographic analysis, independent of the histopathological state of the mineralization. Radiographic analysis can discriminate muscle injury-induced soft tissue calcification from adjacent bone and follow its clinical course over time without requiring the sacrifice of the animal. While the STiCSS cannot identify the specific type of soft tissue calcification present, it is still a useful and valid method by which to quantify the degree of soft tissue calcification. This methodology allows for longitudinal measurements of soft tissue calcification in a single animal, which is relatively less expensive, less time-consuming, and exposes the animal to less radiation than in vivo μCT. Therefore, this high-throughput, longitudinal analytic method for quantifying soft tissue calcification is a viable alternative for the study of soft tissue calcification.

Validation of a Radiography-Based Quantification Designed to Longitudinally Monitor Soft Tissue Calcification in Skeletal Muscle

PubMed Central

Moore, Stephanie N.; Hawley, Gregory D.; Smith, Emily N.; Mignemi, Nicholas A.; Ihejirika, Rivka C.; Yuasa, Masato; Cates, Justin M. M.; Liu, Xulei; Schoenecker, Jonathan G.

2016-01-01

Introduction Soft tissue calcification, including both dystrophic calcification and heterotopic ossification, may occur following injury. These lesions have variable fates as they are either resorbed or persist. Persistent soft tissue calcification may result in chronic inflammation and/or loss of function of that soft tissue. The molecular mechanisms that result in the development and maturation of calcifications are uncertain. As a result, directed therapies that prevent or resorb soft tissue calcifications remain largely unsuccessful. Animal models of post-traumatic soft tissue calcification that allow for cost-effective, serial analysis of an individual animal over time are necessary to derive and test novel therapies. We have determined that a cardiotoxin-induced injury of the muscles in the posterior compartment of the lower extremity represents a useful model in which soft tissue calcification develops remote from adjacent bones, thereby allowing for serial analysis by plain radiography. The purpose of the study was to design and validate a method for quantifying soft tissue calcifications in mice longitudinally using plain radiographic techniques and an ordinal scoring system. Methods Muscle injury was induced by injecting cardiotoxin into the posterior compartment of the lower extremity in mice susceptible to developing soft tissue calcification. Seven days following injury, radiographs were obtained under anesthesia. Multiple researchers applied methods designed to standardize post-image processing of digital radiographs (N = 4) and quantify soft tissue calcification (N = 6) in these images using an ordinal scoring system. Inter- and intra-observer agreement for both post-image processing and the scoring system used was assessed using weighted kappa statistics. Soft tissue calcification quantifications by the ordinal scale were compared to mineral volume measurements (threshold 450.7mgHA/cm3) determined by μCT. Finally, sample-size calculations necessary to discriminate between a 25%, 50%, 75%, and 100% difference in STiCSS score 7 days following burn/CTX induced muscle injury were determined. Results Precision analysis demonstrated substantial to good agreement for both post-image processing (κ = 0.73 to 0.90) and scoring (κ = 0.88 to 0.93), with low inter- and intra-observer variability. Additionally, there was a strong correlation in quantification of soft tissue calcification between the ordinal system and by mineral volume quantification by μCT (Spearman r = 0.83 to 0.89). The ordinal scoring system reliably quantified soft tissue calcification in a burn/CTX-induced soft tissue calcification model compared to non-injured controls (Mann-Whitney rank test: P = 0.0002, ***). Sample size calculations revealed that 6 mice per group would be required to detect a 50% difference in STiCSS score with a power of 0.8. Finally, the STiCSS was demonstrated to reliably quantify soft tissue calcification [dystrophic calcification and heterotopic ossification] by radiographic analysis, independent of the histopathological state of the mineralization. Conclusions Radiographic analysis can discriminate muscle injury-induced soft tissue calcification from adjacent bone and follow its clinical course over time without requiring the sacrifice of the animal. While the STiCSS cannot identify the specific type of soft tissue calcification present, it is still a useful and valid method by which to quantify the degree of soft tissue calcification. This methodology allows for longitudinal measurements of soft tissue calcification in a single animal, which is relatively less expensive, less time-consuming, and exposes the animal to less radiation than in vivo μCT. Therefore, this high-throughput, longitudinal analytic method for quantifying soft tissue calcification is a viable alternative for the study of soft tissue calcification. PMID:27438007

HPV frequency in penile carcinoma of Mexican patients: important contribution of HPV16 European variant.

PubMed

López-Romero, Ricardo; Iglesias-Chiesa, Candela; Alatorre, Brenda; Vázquez, Karla; Piña-Sánchez, Patricia; Alvarado, Isabel; Lazos, Minerva; Peralta, Raúl; González-Yebra, Beatriz; Romero, Anae; Salcedo, Mauricio

2013-01-01

The role of human papillomavirus (HPV) infection in penile carcinoma (PeC) is currently reported and about half of the PeC is associated with HPV16 and 18. We used a PCR-based strategy by using HPV general primers to analyze 86 penile carcinomas paraffin-embedded tissues. Some clinical data, the histological subtype, growth pattern, and differentiation degree were also collected. The amplified fragments were then sequenced to confirm the HPV type and for HPV16/18 variants. DNA samples were also subjected to relative real time PCR for hTERC gene copy number. Some clinical data were also collected. Global HPV frequency was 77.9%. Relative contributions was for HPV16 (85%), 31 (4.4%), 11 (4.4%), 58, 33, 18, and 59 (1.4% each one). Sequence analysis of HPV16 identified European variants and Asian-American (AAb-c) variants in 92% and in 8% of the samples, respectively. Furthermore hTERC gene amplification was observed in only 17% of the cases. Our results suggest that some members of HPV A9 group (represented by HPV16, 58, and 31) are the most frequent among PeC patients studied with an important contribution from HPV16 European variant. The hTERC gene amplification could be poorly related to penile epithelial tissue.

HPV frequency in penile carcinoma of Mexican patients: important contribution of HPV16 European variant

PubMed Central

López-Romero, Ricardo; Iglesias-Chiesa, Candela; Alatorre, Brenda; Vázquez, Karla; Piña-Sánchez, Patricia; Alvarado, Isabel; Lazos, Minerva; Peralta, Raúl; González-Yebra, Beatriz; Romero, AnaE; Salcedo, Mauricio

2013-01-01

The role of human papillomavirus (HPV) infection in penile carcinoma (PeC) is currently reported and about half of the PeC is associated with HPV16 and 18. We used a PCR-based strategy by using HPV general primers to analyze 86 penile carcinomas paraffin-embedded tissues. Some clinical data, the histological subtype, growth pattern, and differentiation degree were also collected. The amplified fragments were then sequenced to confirm the HPV type and for HPV16/18 variants. DNA samples were also subjected to relative real time PCR for hTERC gene copy number. Some clinical data were also collected. Global HPV frequency was 77.9%. Relative contributions was for HPV16 (85%), 31 (4.4%), 11 (4.4%), 58, 33, 18, and 59 (1.4% each one). Sequence analysis of HPV16 identified European variants and Asian-American (AAb-c) variants in 92% and in 8% of the samples, respectively. Furthermore hTERC gene amplification was observed in only 17% of the cases. Our results suggest that some members of HPV A9 group (represented by HPV16, 58, and 31) are the most frequent among PeC patients studied with an important contribution from HPV16 European variant. The hTERC gene amplification could be poorly related to penile epithelial tissue. PMID:23826423

Calnexin, an ER stress-induced protein, is a prognostic marker and potential therapeutic target in colorectal cancer.

PubMed

Ryan, Deborah; Carberry, Steven; Murphy, Áine C; Lindner, Andreas U; Fay, Joanna; Hector, Suzanne; McCawley, Niamh; Bacon, Orna; Concannon, Caoimhin G; Kay, Elaine W; McNamara, Deborah A; Prehn, Jochen H M

2016-07-01

Colorectal cancer (CRC) is a leading cause of cancer mortality in the Western world and commonly treated with genotoxic chemotherapy. Stress in the endoplasmic reticulum (ER) was implicated to contribute to chemotherapeutic resistance. Hence, ER stress related protein may be of prognostic or therapeutic significance. The expression levels of ER stress proteins calnexin, calreticulin, GRP78 and GRP94 were determined in n = 23 Stage II and III colon cancer fresh frozen tumour and matched normal tissue samples. Data were validated in a cohort of n = 11 rectal cancer patients treated with radiochemotherapy in the neoadjuvant setting. The calnexin gene was silenced using siRNA in HCT116 cells. There were no increased levels of ER stress proteins in tumour compared to matched normal tissue samples in Stage II or III CRC. However, increased calnexin protein levels were predictive of poor clinical outcome in the patient cohort. Data were validated in the rectal cancer cohort treated in the neoadjuvant setting. Calnexin gene-silencing significantly reduced cell survival and increased cancer cell susceptibility to 5FU chemotherapy. Increased tumour protein levels of calnexin may be of prognostic significance in CRC, and calnexin may represent a potential target for future therapies.

Preventing collapsing of vascular scaffolds: The mechanical behavior of PLA/PCL composite structure prostheses during in vitro degradation.

PubMed

Li, Chaojing; Wang, Fujun; Chen, Peifeng; Zhang, Ze; Guidoin, Robert; Wang, Lu

2017-11-01

The success of blood conduit replacement with synthetic graft is highly dependent on the architecture, and mechanical properties of the graft, especially for biodegradable grafts serving as scaffolds for in-situ tissue engineering. Particularly, the property of the radial compression recovery represents a critical to keep the patency during biointegration. Bi-component composite vascular grafts (cVG) made of polylactic acid (PLA) fabric and polycaprolactone (PCL) were developed with superior mechanical properties. In this research, the compressive and tensile properties of the prototypes were characterized when they were subjected to accelerated degradation. In addition, the prepared cVG were analyzed by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and wide angle X-ray diffraction (WAXD) to illustrate the gradual loss of mechanical properties. The results demonstrated that the cVG retained the circular cross-section even through its tensile strength decreased during degradation. The cVG samples containing a high percentage of PLA fibers lost their tensile strength faster, while the samples with lower PLA percentage lost the compressive resistance strength more quickly. This unique fabric-based composite biodegradable vascular prosthesis with an outstanding radical compression recovery could be a good candidate for in-situ formation of tissue engineered vascular graft. Copyright © 2017 Elsevier Ltd. All rights reserved.

A pilot study on the use of cerebrospinal fluid cell-free DNA in intramedullary spinal ependymoma.

PubMed

Connolly, Ian David; Li, Yingmei; Pan, Wenying; Johnson, Eli; You, Linya; Vogel, Hannes; Ratliff, John; Hayden Gephart, Melanie

2017-10-01

Cerebrospinal fluid (CSF) represents a promising source of cell-free DNA (cfDNA) for tumors of the central nervous system. A CSF-based liquid biopsy may obviate the need for riskier tissue biopsies and serve as a means for monitoring tumor recurrence or response to therapy. Spinal ependymomas most commonly occur in adults, and aggressive resection must be delicately balanced with the risk of injury to adjacent normal tissue. In patients with subtotal resection, recurrence commonly occurs. A CSF-based liquid biopsy matched to the patient's spinal ependymoma mutation profile has potential to be more sensitive then surveillance MRI, but the utility has not been well characterized for tumors of the spinal cord. In this study, we collected matched blood, tumor, and CSF samples from three adult patients with WHO grade II intramedullary spinal ependymoma. We performed whole exome sequencing on matched tumor and normal DNA to design Droplet Digital™ PCR (ddPCR) probes for tumor and wild-type mutations. We then interrogated CSF samples for tumor-derived cfDNA by performing ddPCR on extracted cfDNA. Tumor cfDNA was not reliably detected in the CSF of our cohort. Anatomic sequestration and low grade of intramedullary spinal cord tumors likely limits the role of CSF liquid biopsy.

Heavy metals and hydrocarbon concentrations in water, sediments and tissue of Cyclope neritea from two sites in Suez Canal, Egypt and histopathological effects.

PubMed

Sharaf, Hesham M; Shehata, Abdalla M

2015-01-01

Heavy metals and hydrocarbons are of the most common marine pollutants around the world. The present study aimed to assess the concentration of petroleum hydrocarbons and heavy metals in tissues of the snail cyclope neritea, water and sediments from two sites of the study area (Temsah lake and Suez canal) represent polluted and unpolluted sites respectively. The results showed that, the levels of the heavy metals (Pb, Cd, Co, Mg and Zn) in the polluted area have reached harmful limits recorded globally. Lead in water, sediment and tissue of the snail reached to 0.95 ppm, 4.54 ppm and 7.93 ppm respectively. Cadmium reached 0.31 ppm, 1.15 ppm and 3.08 ppm in the corresponding samples. Cobalt was not detected in water, but it reached 1.42 ppm and 10.36 ppm in the sediment and snails tissue respectively. Magnesium in water, sediment and tissue of the snail reached 3.73 ppm, 9.44 ppm and12.6 ppm respectively. Zinc reached 0.11 ppm, 3.89 ppm and 12.60ppm in the corresponding samples. Meanwhile, hydrocarbons in the polluted area (site1) reached 110.10 μg/L, 980.15 μg/g and 228.00 μg/g in water sediment and digestive gland tissues of the snails respectively. Whereas, hydrocarbons in the unpolluted area (site2) were estimated as 14.20 μg/L, 55.60 μg/g and 22.66 μg/g in water, sediment and tissue of the snails respectively. The combination of histopathological image with monitoring of the metal level in the digestive gland of the present snail provides an important tool for early detection of impending environmental problems and potential public health issues. Petroleum hydrocarbons are toxic to the marine fauna when present above certain limit in the marine water. The major detoxification organ in molluscs is the digestive gland, which has been used as a bioindicator organ for toxicity assessment. The effect of high crude oil on the digestive gland tubules of exposed snails when examined microscopically reveals a series of histological changes which indicates that the cellular compensatory mechanism is activated by hydrocarbons. These changes include vacuolation and presence of pyknotic nuclei.

Sample Preparation of Corn Seed Tissue to Prevent Analyte Relocations for Mass Spectrometry Imaging

NASA Astrophysics Data System (ADS)

Kim, Shin Hye; Kim, Jeongkwon; Lee, Young Jin; Lee, Tae Geol; Yoon, Sohee

2017-08-01

Corn seed tissue sections were prepared by the tape support method using an adhesive tape, and mass spectrometry imaging (MSI) was performed. The effect of heat generated during sample preparation was investigated by time-of-flight secondary mass spectrometry (TOF-SIMS) imaging of corn seed tissue prepared by the tape support and the thaw-mounted methods. Unlike thaw-mounted sample preparation, the tape support method does not cause imaging distortion because of the absence of heat, which can cause migration of the analytes on the sample. By applying the tape-support method, the corn seed tissue was prepared without structural damage and MSI with accurate spatial information of analytes was successfully performed.

Impact of holmium fibre laser radiation (λ = 2.1 μm) on the spinal cord dura mater and adipose tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Filatova, S A; Kamynin, V A; Ryabova, A V

The impact of holmium fibre laser radiation on the samples of biologic tissues (dura mater of spinal cord and adipose tissue with interlayers of muscle) is studied. The experimental results are evaluated by the size of carbonisation and coagulation necrosis zones. The experiment shows that in the case of irradiation of the spinal cord dura mater samples the size of carbonisation and coagulation necrosis zones is insignificant. In the adipose tissue the carbonisation zone is also insignificant, but the region of cellular structure disturbance is large. In the muscle tissue the situation is opposite. The cw laser operation provides clinicallymore » acceptable degree of destruction in tissue samples with a minimal carbonisation zone. (laser applications in medicine)« less