Mass spectral analysis and imaging of tissue by ToF-SIMS--The role of buckminsterfullerene, C60+, primary ions

NASA Astrophysics Data System (ADS)

Jones, Emrys A.; Lockyer, Nicholas P.; Vickerman, John C.

2007-02-01

Recent developments in desorption/ionisation mass spectrometry techniques have made their application to biological analysis a realistic and successful proposition. Developments in primary ion source technology, mainly through the advent of polyatomic ion beams, have meant that the technique of secondary ion mass spectrometry (SIMS) can now access the depths of information required to allow biological imaging to be a viable option. Here the role of the primary ion C60+ is assessed with regard to molecular imaging of lipids and pharmaceuticals within tissue sections. High secondary ion yields and low surface damage accumulation are demonstrated on both model and real biological samples, indicating the high secondary ion efficiency afforded to the analyst by this primary ion when compared to other cluster ion beams used in imaging. The newly developed 40 keV C60+ ion source allows the beam to be focused such that high resolution imaging is demonstrated on a tissue sample, and the greater yields allow the molecular signal from the drug raclopride to be imaged within tissue section following in vivo dosing. The localisation shown for this drug alludes to issues regarding the chemical environment affecting the ionisation probability of the molecule; the importance of this effect is demonstrated with model systems and the possibility of using laser post-ionisation as a method for reducing this consequence of bio-sample complexity is demonstrated and discussed.

Presence of papillomavirus sequences in condylomatous lesions of the mamillae and in invasive carcinoma of the breast

PubMed Central

de Villiers, Ethel-Michele; Sandstrom, Robert E; zur Hausen, Harald; Buck, Charles E

2005-01-01

Background Viruses including Epstein–Barr virus (EBV), a human equivalent of murine mammary tumour virus (MMTV) and human papillomavirus (HPV) have been implicated in the aetiology of human breast cancer. We report the presence of HPV DNA sequences in areolar tissue and tumour tissue samples from female patients with breast carcinoma. The presence of virus in the areolar–nipple complex suggests to us a potential pathogenic mechanism. Methods Polymerase chain reaction (PCR) was undertaken to amplify HPV types in areolar and tumour tissue from breast cancer cases. In situ hybridisation supported the PCR findings and localised the virus in nipple, areolar and tumour tissue. Results Papillomavirus DNA was present in 25 of 29 samples of breast carcinoma and in 20 of 29 samples from the corresponding mamilla. The most prevalent type in both carcinomas and nipples was HPV 11, followed by HPV 6. Other types detected were HPV 16, 23, 27 and 57 (nipples and carcinomas), HPV 20, 21, 32, 37, 38, 66 and GA3-1 (nipples only) and HPV 3, 15, 24, 87 and DL473 (carcinomas only). Multiple types were demonstrated in seven carcinomas and ten nipple samples. Conclusions The data demonstrate the occurrence of HPV in nipple and areolar tissues in patients with breast carcinoma. The authors postulate a retrograde ductular pattern of viral spread that may have pathogenic significance. PMID:15642157

Application of FTA technology for sampling, recovery and molecular characterization of viral pathogens and virus-derived transgenes from plant tissues.

PubMed

Ndunguru, Joseph; Taylor, Nigel J; Yadav, Jitender; Aly, Haytham; Legg, James P; Aveling, Terry; Thompson, Graham; Fauquet, Claude M

2005-05-18

Plant viral diseases present major constraints to crop production. Effective sampling of the viruses infecting plants is required to facilitate their molecular study and is essential for the development of crop protection and improvement programs. Retaining integrity of viral pathogens within sampled plant tissues is often a limiting factor in this process, most especially when sample sizes are large and when operating in developing counties and regions remote from laboratory facilities. FTA is a paper-based system designed to fix and store nucleic acids directly from fresh tissues pressed into the treated paper. We report here the use of FTA as an effective technology for sampling and retrieval of DNA and RNA viruses from plant tissues and their subsequent molecular analysis. DNA and RNA viruses were successfully recovered from leaf tissues of maize, cassava, tomato and tobacco pressed into FTA Classic Cards. Viral nucleic acids eluted from FTA cards were found to be suitable for diagnostic molecular analysis by PCR-based techniques and restriction analysis, and for cloning and nucleotide sequencing in a manner equivalent to that offered by tradition isolation methods. Efficacy of the technology was demonstrated both from sampled greenhouse-grown plants and from leaf presses taken from crop plants growing in farmer's fields in East Africa. In addition, FTA technology was shown to be suitable for recovery of viral-derived transgene sequences integrated into the plant genome. Results demonstrate that FTA is a practical, economical and sensitive method for sampling, storage and retrieval of viral pathogens and plant genomic sequences, when working under controlled conditions and in the field. Application of this technology has the potential to significantly increase ability to bring modern analytical techniques to bear on the viral pathogens infecting crop plants.

Impact of Tumour Epithelial Subtype on Circulating microRNAs in Breast Cancer Patients

PubMed Central

Brougham, Cathy; Glynn, Claire L.; Wall, Deirdre; Hyland, Peter; Duignan, Maria; McLoughlin, Mark; Newell, John; Kerin, Michael J.

2014-01-01

While a range of miRNAs have been shown to be dysregulated in the circulation of patients with breast cancer, little is known about the relationship between circulating levels and tumour characteristics. The aim of this study was to analyse alterations in circulating miRNA expression during tumour progression in a murine model of breast cancer, and to detemine the clinical relevance of identified miRNAs at both tissue and circulating level in patient samples. Athymic nude mice received a subcutaneous or mammary fat pad injection of MDA-MB-231 cells. Blood sampling was performed at weeks 1, 3 and 6 following tumour induction, and microRNA extracted. MicroRNA microArray analysis was performed comparing samples harvested at week 1 to those collected at week 6 from the same animals. Significantly altered miRNAs were validated across all murine samples by RQ-PCR (n = 45). Three miRNAs of interest were then quantified in the circulation(n = 166) and tissue (n = 100) of breast cancer patients and healthy control individuals. MicroArray-based analysis of murine blood samples revealed levels of 77 circulating microRNAs to be changed during disease progression, with 44 demonstrating changes >2-fold. Validation across all samples revealed miR-138 to be significantly elevated in the circulation of animals during disease development, with miR-191 and miR-106a levels significantly decreased. Analysis of patient tissue and blood samples revealed miR-138 to be significantly up-regulated in the circulation of patients with breast cancer, with no change observed in the tissue setting. While not significantly changed overall in breast cancer patients compared to controls, circulating miR-106a and miR-191 were significantly decreased in patients with basal breast cancer. In tissue, both miRNAs were significantly elevated in breast cancer compared to normal breast tissue. The data demonstrates an impact of tumour epithelial subtype on circulating levels of miRNAs, and highlights divergent miRNA profiles between tissue and blood samples from breast cancer patients. PMID:24626163

Polarimetry based partial least square classification of ex vivo healthy and basal cell carcinoma human skin tissues.

PubMed

Ahmad, Iftikhar; Ahmad, Manzoor; Khan, Karim; Ikram, Masroor

2016-06-01

Optical polarimetry was employed for assessment of ex vivo healthy and basal cell carcinoma (BCC) tissue samples from human skin. Polarimetric analyses revealed that depolarization and retardance for healthy tissue group were significantly higher (p<0.001) compared to BCC tissue group. Histopathology indicated that these differences partially arise from BCC-related characteristic changes in tissue morphology. Wilks lambda statistics demonstrated the potential of all investigated polarimetric properties for computer assisted classification of the two tissue groups. Based on differences in polarimetric properties, partial least square (PLS) regression classified the samples with 100% accuracy, sensitivity and specificity. These findings indicate that optical polarimetry together with PLS statistics hold promise for automated pathology classification. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterisation of a fibre optic Raman probe within a hypodermic needle.

PubMed

Iping Petterson, Ingeborg E; Day, John C C; Fullwood, Leanne M; Gardner, Benjamin; Stone, Nick

2015-11-01

We demonstrate the first use of a multifibre Raman probe that fits inside the bore of a hypodermic needle. A Raman probe containing multiple collection fibres provides improved signal collection efficiency in biological samples compared with a previous two-fibre design. Furthermore, probe performance (signal-to-noise ratios) compared favourably with the performance achieved in previous Raman microscope experiments able to distinguish between benign lymph nodes, primary malignancies in lymph nodes and secondary malignancies in lymph nodes. The experimental measurements presented here give an indication of the sampling volume of the Raman needle probe in lymphoid tissues. Liquid tissue phantoms were used that contained scattering medium encompassing a range of scattering properties similar to those of a variety of tissue types, including lymph node tissues. To validate the appropriateness of the phantoms, the sampling depth of the probe was also measured in excised lymph node tissue. More than 50 % of Raman photons collected were found to originate from between the tip of the needle and a depth of 500 μm into the tissue. The needle probe presented here achieves spectral quality comparable to that in numerous studies previously demonstrating Raman disease discrimination. It is expected that this approach could achieve targeted subcutaneous tissue measurements and be viable for use for the in vivo Raman diagnostics of solid organs located within a few centimetres below the skin's surface. Graphical Abstract Schematic of multi-fibre Raman needle probe with disposible tips and proximal optical filtration.

Equilibrium Passive Sampling of POP in Lipid-Rich and Lean Fish Tissue: Quality Control Using Performance Reference Compounds.

PubMed

Rusina, Tatsiana P; Carlsson, Pernilla; Vrana, Branislav; Smedes, Foppe

2017-10-03

Passive sampling is widely used to measure levels of contaminants in various environmental matrices, including fish tissue. Equilibrium passive sampling (EPS) of persistent organic pollutants (POP) in fish tissue has been hitherto limited to application in lipid-rich tissue. We tested several exposure methods to extend EPS applicability to lean tissue. Thin-film polydimethylsiloxane (PDMS) passive samplers were exposed statically to intact fillet and fish homogenate and dynamically by rolling with cut fillet cubes. The release of performance reference compounds (PRC) dosed to passive samplers prior to exposure was used to monitor the exchange process. The sampler-tissue exchange was isotropic, and PRC were shown to be good indicators of sampler-tissue equilibration status. The dynamic exposures demonstrated equilibrium attainment in less than 2 days for all three tested fish species, including lean fish containing 1% lipid. Lipid-based concentrations derived from EPS were in good agreement with lipid-normalized concentrations obtained using conventional solvent extraction. The developed in-tissue EPS method is robust and has potential for application in chemical monitoring of biota and bioaccumulation studies.

Direct microCT imaging of non-mineralized connective tissues at high resolution.

PubMed

Naveh, Gili R S; Brumfeld, Vlad; Dean, Mason; Shahar, Ron; Weiner, Steve

2014-01-01

The 3D imaging of soft tissues in their native state is challenging, especially when high resolution is required. An X-ray-based microCT is, to date, the best choice for high resolution 3D imaging of soft tissues. However, since X-ray attenuation of soft tissues is very low, contrasting enhancement using different staining materials is needed. The staining procedure, which also usually involves tissue fixation, causes unwanted and to some extent unknown tissue alterations. Here, we demonstrate that a method that enables 3D imaging of soft tissues without fixing and staining using an X-ray-based bench-top microCT can be applied to a variety of different tissues. With the sample mounted in a custom-made loading device inside a humidity chamber, we obtained soft tissue contrast and generated 3D images of fresh, soft tissues with a resolution of 1 micron voxel size. We identified three critical conditions which make it possible to image soft tissues: humidified environment, mechanical stabilization of the sample and phase enhancement. We demonstrate the capability of the technique using different specimens: an intervertebral disc, the non-mineralized growth plate, stingray tessellated radials (calcified cartilage) and the collagenous network of the periodontal ligament. Since the scanned specimen is fresh an interesting advantage of this technique is the ability to scan a specimen under load and track the changes of the different structures. This method offers a unique opportunity for obtaining valuable insights into 3D structure-function relationships of soft tissues.

Surface-enhanced Raman scattering of rat tissues.

PubMed

Aydin, Omer; Kahraman, Mehmet; Kiliç, Ertuğul; Culha, Mustafa

2009-06-01

Surface-enhanced Raman scattering (SERS) is proven to be a powerful tool for investigation of biological structures. In this study, tissues obtained from different rat organs are examined using SERS. The tissue samples are crushed with a pestle after sudden freezing in liquid nitrogen and mixed with a concentrated colloidal silver nanoparticle suspension. The reproducibility of SERS spectra acquired from several tissue samples from different organs is demonstrated. The collected spectra are comparatively evaluated based on the physiological function of the organ from which the tissue is obtained. The spectra from the tissues show significant differences and indicate that they can be used for tissue characterization and differentiation. The identification of the origins of the bands on the spectra is also attempted. This study suggests that SERS can be used to monitor the changes at the molecular level during metabolic changes in an organ or tissue as a result of a disease or another cause.

Terahertz spectroscopy of liver cirrhosis: investigating the origin of contrast

NASA Astrophysics Data System (ADS)

Sy, Stanley; Huang, Shengyang; Wang, Yi-Xiang J.; Yu, Jun; Ahuja, Anil T.; Zhang, Yuan-ting; Pickwell-MacPherson, Emma

2010-12-01

We have previously demonstrated that terahertz pulsed imaging is able to distinguish between rat tissues from different healthy organs. In this paper we report our measurements of healthy and cirrhotic liver tissues using terahertz reflection spectroscopy. The water content of the fresh tissue samples was also measured in order to investigate the correlations between the terahertz properties, water content, structural changes and cirrhosis. Finally, the samples were fixed in formalin to determine whether water was the sole source of image contrast in this study. We found that the cirrhotic tissue had a higher water content and absorption coefficient than the normal tissue and that even after formalin fixing there were significant differences between the normal and cirrhotic tissues' terahertz properties. Our results show that terahertz pulsed imaging can distinguish between healthy and diseased tissue due to differences in absorption originating from both water content and tissue structure.

Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

PubMed

Eaton, Samantha L; Roche, Sarah L; Llavero Hurtado, Maica; Oldknow, Karla J; Farquharson, Colin; Gillingwater, Thomas H; Wishart, Thomas M

2013-01-01

Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB) to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls) are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate that normalisation using total protein analysis on samples run in parallel with stains such as Coomassie blue provides a more robust approach.

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.

Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue

PubMed Central

Robertson, David; Savage, Kay; Reis-Filho, Jorge S; Isacke, Clare M

2008-01-01

Background Investigating the expression of candidate genes in tissue samples usually involves either immunohistochemical labelling of formalin-fixed paraffin-embedded (FFPE) sections or immunofluorescence labelling of cryosections. Although both of these methods provide essential data, both have important limitations as research tools. Consequently, there is a demand in the research community to be able to perform routine, high quality immunofluorescence labelling of FFPE tissues. Results We present here a robust optimised method for high resolution immunofluorescence labelling of FFPE tissues, which involves the combination of antigen retrieval, indirect immunofluorescence and confocal laser scanning microscopy. We demonstrate the utility of this method with examples of immunofluorescence labelling of human kidney, human breast and a tissue microarray of invasive human breast cancers. Finally, we demonstrate that stained slides can be stored in the short term at 4°C or in the longer term at -20°C prior to images being collected. This approach has the potential to unlock a large in vivo database for immunofluorescence investigations and has the major advantages over immunohistochemistry in that it provides higher resolution imaging of antigen localization and the ability to label multiple antigens simultaneously. Conclusion This method provides a link between the cell biology and pathology communities. For the cell biologist, it will enable them to utilise the vast archive of pathology specimens to advance their in vitro data into in vivo samples, in particular archival material and tissue microarrays. For the pathologist, it will enable them to utilise multiple antibodies on a single section to characterise particular cell populations or to test multiple biomarkers in limited samples and define with greater accuracy cellular heterogeneity in tissue samples. PMID:18366689

NASA Bioreactor tissue culture

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 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. 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). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

High-resolution analysis of the mechanical behavior of tissue

NASA Astrophysics Data System (ADS)

Hudnut, Alexa W.; Armani, Andrea M.

2017-06-01

The mechanical behavior and properties of biomaterials, such as tissue, have been directly and indirectly connected to numerous malignant physiological states. For example, an increase in the Young's Modulus of tissue can be indicative of cancer. Due to the heterogeneity of biomaterials, it is extremely important to perform these measurements using whole or unprocessed tissue because the tissue matrix contains important information about the intercellular interactions and the structure. Thus, developing high-resolution approaches that can accurately measure the elasticity of unprocessed tissue samples is of great interest. Unfortunately, conventional elastography methods such as atomic force microscopy, compression testing, and ultrasound elastography either require sample processing or have poor resolution. In the present work, we demonstrate the characterization of unprocessed salmon muscle using an optical polarimetric elastography system. We compare the results of compression testing within different samples of salmon skeletal muscle with different numbers of collagen membranes to characterize differences in heterogeneity. Using the intrinsic collagen membranes as markers, we determine the resolution of the system when testing biomaterials. The device reproducibly measures the stiffness of the tissues at variable strains. By analyzing the amount of energy lost by the sample during compression, collagen membranes that are 500 μm in size are detected.

Analysis of human tissues by total reflection X-ray fluorescence. Application of chemometrics for diagnostic cancer recognition

NASA Astrophysics Data System (ADS)

Benninghoff, L.; von Czarnowski, D.; Denkhaus, E.; Lemke, K.

1997-07-01

For the determination of trace element distributions of more than 20 elements in malignant and normal tissues of the human colon, tissue samples (approx. 400 mg wet weight) were digested with 3 ml of nitric acid (sub-boiled quality) by use of an autoclave system. The accuracy of measurements has been investigated by using certified materials. The analytical results were evaluated by using a spreadsheet program to give an overview of the element distribution in cancerous samples and in normal colon tissues. A further application, cluster analysis of the analytical results, was introduced to demonstrate the possibility of classification for cancer diagnosis. To confirm the results of cluster analysis, multivariate three-way principal component analysis was performed. Additionally, microtome frozen sections (10 μm) were prepared from the same tissue samples to compare the analytical results, i.e. the mass fractions of elements, according to the preparation method and to exclude systematic errors depending on the inhomogeneity of the tissues.

Brain tumor imaging of rat fresh tissue using terahertz spectroscopy

NASA Astrophysics Data System (ADS)

Yamaguchi, Sayuri; Fukushi, Yasuko; Kubota, Oichi; Itsuji, Takeaki; Ouchi, Toshihiko; Yamamoto, Seiji

2016-07-01

Tumor imaging by terahertz spectroscopy of fresh tissue without dye is demonstrated using samples from a rat glioma model. The complex refractive index spectrum obtained by a reflection terahertz time-domain spectroscopy system can discriminate between normal and tumor tissues. Both the refractive index and absorption coefficient of tumor tissues are higher than those of normal tissues and can be attributed to the higher cell density and water content of the tumor region. The results of this study indicate that terahertz technology is useful for detecting brain tumor tissue.

Magnetoacoustic Imaging of Electrical Conductivity of Biological Tissues at a Spatial Resolution Better than 2 mm

PubMed Central

Hu, Gang; He, Bin

2011-01-01

Magnetoacoustic tomography with magnetic induction (MAT-MI) is an emerging approach for noninvasively imaging electrical impedance properties of biological tissues. The MAT-MI imaging system measures ultrasound waves generated by the Lorentz force, having been induced by magnetic stimulation, which is related to the electrical conductivity distribution in tissue samples. MAT-MI promises to provide fine spatial resolution for biological tissue imaging as compared to ultrasound resolution. In the present study, we first estimated the imaging spatial resolution by calculating the full width at half maximum (FWHM) of the system point spread function (PSF). The actual spatial resolution of our MAT-MI system was experimentally determined to be 1.51 mm by a parallel-line-source phantom with Rayleigh criterion. Reconstructed images made from tissue-mimicking gel phantoms, as well as animal tissue samples, were consistent with the morphological structures of the samples. The electrical conductivity value of the samples was determined directly by a calibrated four-electrode system. It has been demonstrated that MAT-MI is able to image the electrical impedance properties of biological tissues with better than 2 mm spatial resolution. These results suggest the potential of MAT-MI for application to early detection of small-size diseased tissues (e.g. small breast cancer). PMID:21858111

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.

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.

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

Surface-enhanced Raman spectroscopy for differentiation between benign and malignant thyroid tissues

NASA Astrophysics Data System (ADS)

Li, Zuanfang; Li, Chao; Lin, Duo; Huang, Zufang; Pan, Jianji; Chen, Guannan; Lin, Juqiang; Liu, Nenrong; Yu, Yun; Feng, Shangyuan; Chen, Rong

2014-04-01

The aim of this study was to evaluate the potential of applying silver nano-particle based surface-enhanced Raman scattering (SERS) to discriminate different types of human thyroid tissues. SERS measurements were performed on three groups of tissue samples including thyroid cancers (n = 32), nodular goiters (n = 20) and normal thyroid tissues (n = 25). Tentative assignments of the measured tissue SERS spectra suggest interesting cancer specific biomolecular differences. The principal component analysis (PCA) and linear discriminate analysis (LDA) together with the leave-one-out, cross-validated technique yielded diagnostic sensitivities of 92%, 75% and 87.5%; and specificities of 82.6%, 89.4% and 84.4%, respectively, for differentiation among normal, nodular and malignant thyroid tissue samples. This work demonstrates that tissue SERS spectroscopy associated with multivariate analysis diagnostic algorithms has great potential for detection of thyroid cancer at the molecular level.

Evaluating the Viscoelastic Properties of Tissue from Laser Speckle Fluctuations

PubMed Central

Hajjarian, Zeinab; Nadkarni, Seemantini K.

2012-01-01

Most pathological conditions such as atherosclerosis, cancer, neurodegenerative, and orthopedic disorders are accompanied with alterations in tissue viscoelasticity. Laser Speckle Rheology (LSR) is a novel optical technology that provides the invaluable potential for mechanical assessment of tissue in situ. In LSR, the specimen is illuminated with coherent light and the time constant of speckle fluctuations, τ, is measured using a high speed camera. Prior work indicates that τ is closely correlated with tissue microstructure and composition. Here, we investigate the relationship between LSR measurements of τ and sample mechanical properties defined by the viscoelastic modulus, G*. Phantoms and tissue samples over a broad range of viscoelastic properties are evaluated using LSR and conventional mechanical testing. Results demonstrate a strong correlation between τ and |G*| for both phantom (r = 0.79, p <0.0001) and tissue (r = 0.88, p<0.0001) specimens, establishing the unique capability of LSR in characterizing tissue viscoelasticity. PMID:22428085

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models.

PubMed

Blutke, Andreas; Wanke, Rüdiger

2018-03-06

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the "Orientator" and "Isector" methods, and vertical uniform random (VUR) sections.

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.

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.

Consistency of signal intensity and T2* in frozen ex vivo heart muscle, kidney, and liver tissue.

PubMed

Kaye, Elena A; Josan, Sonal; Lu, Aiming; Rosenberg, Jarrett; Daniel, Bruce L; Pauly, Kim Butts

2010-03-01

To investigate tissue dependence of the MRI-based thermometry in frozen tissue by quantification and comparison of signal intensity and T2* of ex vivo frozen tissue of three different types: heart muscle, kidney, and liver. Tissue samples were frozen and imaged on a 0.5 Tesla MRI scanner with ultrashort echo time (UTE) sequence. Signal intensity and T2* were determined as the temperature of the tissue samples was decreased from room temperature to approximately -40 degrees C. Statistical analysis was performed for (-20 degrees C, -5 degrees C) temperature interval. The findings of this study demonstrate that signal intensity and T2* are consistent across three types of tissue for (-20 degrees C, -5 degrees C) temperature interval. Both parameters can be used to calculate a single temperature calibration curve for all three types of tissue and potentially in the future serve as a foundation for tissue-independent MRI-based thermometry.

Histomorphometrical analysis following augmentation of infected extraction sites exhibiting severe bone loss and primarily closed by intrasocket reactive soft tissue.

PubMed

Mardinger, Ofer; Vered, Marilena; Chaushu, Gavriel; Nissan, Joseph

2012-06-01

Intrasocket reactive soft tissue can be used for primary closure during augmentation of infected extraction sites exhibiting severe bone loss prior to implant placement. The present study evaluated the histological characteristics of the initially used intrasocket reactive soft tissue, the overlying soft tissue, and the histomorphometry of the newly formed bone during implant placement. Thirty-six consecutive patients (43 sites) were included in the study. Extraction sites demonstrating extensive bone loss on preoperative periapical and panoramic radiographs served as inclusion criteria. Forty-three implants were inserted after a healing period of 6 months. Porous bovine xenograft bone mineral was used as a single bone substitute. The intrasocket reactive soft tissue was sutured over the grafting material to seal the coronal portion of the socket. Biopsies of the intrasocket reactive soft tissue at augmentation, healed mucosa, and bone cores at implant placement were retrieved and evaluated. The intrasocket reactive soft tissue demonstrated features compatible with granulation tissue and long junctional epithelium. The mucosal samples at implant placement demonstrated histopathological characteristics of keratinized mucosa with no residual elements of granulation tissue. Histomorphometrically, the mean composition of the bone cores was - vital bone 40 ± 19% (13.7-74.8%); bone substitute 25.7 ± 13% (0.6-51%); connective tissue 34.3 ± 15% (13.8-71.9%). Intrasocket reactive soft tissue used for primary closure following ridge augmentation is composed of granulation tissue and long junctional epithelium. At implant placement, clinical and histological results demonstrate its replacement by keratinized gingiva. The histomorphometrical results reveal considerable bone formation. Fresh extraction sites of hopeless teeth demonstrating chronic infection and severe bone loss may be grafted simultaneously with their removal. © 2010 Wiley Periodicals, Inc.

Structural locus of transmucosal albumin efflux in canine ileum. A fluorescent study.

PubMed

Granger, D N; Cook, B H; Taylor, A E

1976-12-01

This study demonstrates the effects of elevated intestinal venous pressure on the intestinal tissue spaces and the histological locus of the transmucosal albumin flux under such conditions. The authors were able to localize albumin in the tissues using an Evans blue-albumin fluorescence technique. This technique makes use of the fluorescence properties and albumin affinity of Evans blue dye (T-1824). Evans blue dye has a high affinity for albumin and emits a red-orange fluorescence at a wavelength of 720 nm. Evans blue was mixed with a solution of bovine serum albumin at concentrations that yield negligible amounts of free dye. Control ileal samples were obtained in order to visualize the natural tissue morphology and fluorescence. The Evans blue-albumin solution was injected and tissue samples were obtained 15 and 60 min postinjection, then venous outflow was occluded and after 15 and 60 min the tissues were sampled. Each sample was immediately frozen, freeze dried, embedded in paraffin, and 7-mu sections were made. The Evans blue-albumin was demonstrated histologically with a fluorescence microscope. No leakage sites were apparent at normal venous pressures. However, after elevation of venous pressure, Evans blue-albumin was observed in the interepithelial and/or intraepithelial spaces of villus tips, but no Evans blue-albumin was observed either between or within the epithelial cells of the crypts, or within the tubular crypt lumina. These results indicate that at elevated venous pressures, the transmucosal albumin flux occurs exclusively at the villus tip region, suggesting a great vulnerability of the cells found in this region to elevations in tissue pressure as compared to the crypt epithelial cells.

Quantitative micro-elastography: imaging of tissue elasticity using compression optical coherence elastography

PubMed Central

Kennedy, Kelsey M.; Chin, Lixin; McLaughlin, Robert A.; Latham, Bruce; Saunders, Christobel M.; Sampson, David D.; Kennedy, Brendan F.

2015-01-01

Probing the mechanical properties of tissue on the microscale could aid in the identification of diseased tissues that are inadequately detected using palpation or current clinical imaging modalities, with potential to guide medical procedures such as the excision of breast tumours. Compression optical coherence elastography (OCE) maps tissue strain with microscale spatial resolution and can delineate microstructural features within breast tissues. However, without a measure of the locally applied stress, strain provides only a qualitative indication of mechanical properties. To overcome this limitation, we present quantitative micro-elastography, which combines compression OCE with a compliant stress sensor to image tissue elasticity. The sensor consists of a layer of translucent silicone with well-characterized stress-strain behaviour. The measured strain in the sensor is used to estimate the two-dimensional stress distribution applied to the sample surface. Elasticity is determined by dividing the stress by the strain in the sample. We show that quantification of elasticity can improve the ability of compression OCE to distinguish between tissues, thereby extending the potential for inter-sample comparison and longitudinal studies of tissue elasticity. We validate the technique using tissue-mimicking phantoms and demonstrate the ability to map elasticity of freshly excised malignant and benign human breast tissues. PMID:26503225

Application of FTA technology for sampling, recovery and molecular characterization of viral pathogens and virus-derived transgenes from plant tissues

PubMed Central

Ndunguru, Joseph; Taylor, Nigel J; Yadav, Jitender; Aly, Haytham; Legg, James P; Aveling, Terry; Thompson, Graham; Fauquet, Claude M

2005-01-01

Background Plant viral diseases present major constraints to crop production. Effective sampling of the viruses infecting plants is required to facilitate their molecular study and is essential for the development of crop protection and improvement programs. Retaining integrity of viral pathogens within sampled plant tissues is often a limiting factor in this process, most especially when sample sizes are large and when operating in developing counties and regions remote from laboratory facilities. FTA is a paper-based system designed to fix and store nucleic acids directly from fresh tissues pressed into the treated paper. We report here the use of FTA as an effective technology for sampling and retrieval of DNA and RNA viruses from plant tissues and their subsequent molecular analysis. Results DNA and RNA viruses were successfully recovered from leaf tissues of maize, cassava, tomato and tobacco pressed into FTA® Classic Cards. Viral nucleic acids eluted from FTA cards were found to be suitable for diagnostic molecular analysis by PCR-based techniques and restriction analysis, and for cloning and nucleotide sequencing in a manner equivalent to that offered by tradition isolation methods. Efficacy of the technology was demonstrated both from sampled greenhouse-grown plants and from leaf presses taken from crop plants growing in farmer's fields in East Africa. In addition, FTA technology was shown to be suitable for recovery of viral-derived transgene sequences integrated into the plant genome. Conclusion Results demonstrate that FTA is a practical, economical and sensitive method for sampling, storage and retrieval of viral pathogens and plant genomic sequences, when working under controlled conditions and in the field. Application of this technology has the potential to significantly increase ability to bring modern analytical techniques to bear on the viral pathogens infecting crop plants. PMID:15904535

Preliminary characterization of IL32 in basal-like/triple negative compared to other types of breast cell lines and tissues

PubMed Central

2014-01-01

Background Triple negative breast cancer (TNBC) and often basal-like cancers are defined as negative for estrogen receptor, progesterone receptor and Her2 gene expression. Over the past few years an incredible amount of data has been generated defining the molecular characteristics of both cancers. The aim of these studies is to better understand the cancers and identify genes and molecular pathways that might be useful as targeted therapies. In an attempt to contribute to the understanding of basal-like/TNBC, we examined the Gene Expression Omnibus (GEO) public datasets in search of genes that might define basal-like/TNBC. The Il32 gene was identified as a candidate. Findings Analysis of several GEO datasets showed differential expression of IL32 in patient samples previously designated as basal and/or TNBC compared to normal and luminal breast samples. As validation of the GEO results, RNA and protein expression levels were examined using MCF7 and MDA MB231 cell lines and tissue microarrays (TMAs). IL32 gene expression levels were higher in MDA MB231 compared to MCF7. Analysis of TMAs showed 42% of TNBC tissues and 25% of the non-TNBC were positive for IL32, while non-malignant patient samples and all but one hyperplastic tissue sample demonstrated lower levels of IL32 protein expression. Conclusion Data obtained from several publically available GEO datasets showed overexpression of IL32 gene in basal-like/TNBC samples compared to normal and luminal samples. In support of these data, analysis of TMA clinical samples demonstrated a particular pattern of IL32 differential expression. Considered together, these data suggest IL32 is a candidate suitable for further study. PMID:25100201

Biological tissue imaging with a position and time sensitive pixelated detector.

PubMed

Jungmann, Julia H; Smith, Donald F; MacAleese, Luke; Klinkert, Ivo; Visser, Jan; Heeren, Ron M A

2012-10-01

We demonstrate the capabilities of a highly parallel, active pixel detector for large-area, mass spectrometric imaging of biological tissue sections. A bare Timepix assembly (512 × 512 pixels) is combined with chevron microchannel plates on an ion microscope matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI TOF-MS). The detector assembly registers position- and time-resolved images of multiple m/z species in every measurement frame. We prove the applicability of the detection system to biomolecular mass spectrometry imaging on biologically relevant samples by mass-resolved images from Timepix measurements of a peptide-grid benchmark sample and mouse testis tissue slices. Mass-spectral and localization information of analytes at physiologic concentrations are measured in MALDI-TOF-MS imaging experiments. We show a high spatial resolution (pixel size down to 740 × 740 nm(2) on the sample surface) and a spatial resolving power of 6 μm with a microscope mode laser field of view of 100-335 μm. Automated, large-area imaging is demonstrated and the Timepix' potential for fast, large-area image acquisition is highlighted.

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

Real-Time Reverse-Transcription Quantitative Polymerase Chain Reaction Assay Is a Feasible Method for the Relative Quantification of Heregulin Expression in Non-Small Cell Lung Cancer Tissue.

PubMed

Kristof, Jessica; Sakrison, Kellen; Jin, Xiaoping; Nakamaru, Kenji; Schneider, Matthias; Beckman, Robert A; Freeman, Daniel; Spittle, Cindy; Feng, Wenqin

2017-01-01

In preclinical studies, heregulin ( HRG ) expression was shown to be the most relevant predictive biomarker for response to patritumab, a fully human anti-epidermal growth factor receptor 3 monoclonal antibody. In support of a phase 2 study of erlotinib ± patritumab in non-small cell lung cancer (NSCLC), a reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay for relative quantification of HRG expression from formalin-fixed paraffin-embedded (FFPE) NSCLC tissue samples was developed and validated and described herein. Test specimens included matched FFPE normal lung and NSCLC and frozen NSCLC tissue, and HRG -positive and HRG -negative cell lines. Formalin-fixed paraffin-embedded tissue was examined for functional performance. Heregulin distribution was also analyzed across 200 NSCLC commercial samples. Applied Biosystems TaqMan Gene Expression Assays were run on the Bio-Rad CFX96 real-time PCR platform. Heregulin RT-qPCR assay specificity, PCR efficiency, PCR linearity, and reproducibility were demonstrated. The final assay parameters included the Qiagen FFPE RNA Extraction Kit for RNA extraction from FFPE NSCLC tissue, 50 ng of RNA input, and 3 reference (housekeeping) genes ( HMBS, IPO8 , and EIF2B1 ), which had expression levels similar to HRG expression levels and were stable among FFPE NSCLC samples. Using the validated assay, unimodal HRG distribution was confirmed across 185 evaluable FFPE NSCLC commercial samples. Feasibility of an RT-qPCR assay for the quantification of HRG expression in FFPE NSCLC specimens was demonstrated.

Pattern of somatostatin receptors expression in normal and bladder cancer tissue samples.

PubMed

Karavitakis, Markos; Msaouel, Pavlos; Michalopoulos, Vassilis; Koutsilieris, Michael

2014-06-01

Known risks factors for bladder cancer progression and recurrence are limited regarding their prognostic ability. Therefore identification of molecular determinants of disease progression could provide with more specific prognostic information and could be translated into new approaches for biomarker development. In the present study we evaluated, the expression patterns of somatostatin receptors 1-5 (SSTRs) in normal and tumor bladder tissues. The expression of SSTR1-5 was characterized in 45 normal and bladder cancer tissue samples using reverse transcriptase-polymerase chain reaction (RT-PCR). SSTR1 was expressed in 24 samples, SSTR2 in 15, SSTR3 in 23, SSTR4 in 16 and SSTR5 in all but one sample. Bladder cancer tissue samples expressed lower levels of SSTR3. Co-expression of SSTRs was associated with superficial disease. Our results demonstrate, for the first time, that there is expression of SSTR in normal and bladder cancer urothelium. Further studies are required to evaluate the prognostic and therapeutic significance of these findings. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Multimodal fiber-probe spectroscopy as a clinical tool for diagnosing and classifying biological tissues

NASA Astrophysics Data System (ADS)

Cicchi, Riccardo; Anand, Suresh; Fantechi, Riccardo; Giordano, Flavio; Gacci, Mauro; Conti, Valerio; Nesi, Gabriella; Buccoliero, Anna Maria; Carini, Marco; Guerrini, Renzo; Pavone, Francesco Saverio

2017-07-01

An optical fiber probe for multimodal spectroscopy was designed, developed and used for tissue diagnostics. The probe, based on a fiber bundle with optical fibers of various size and properties, allows performing spectroscopic measurements with different techniques, including fluorescence, Raman, and diffuse reflectance, using the same probe. Two visible laser diodes were used for fluorescence spectroscopy, a laser diode emitting in the NIR was used for Raman spectroscopy, and a fiber-coupled halogen lamp for diffuse reflectance. The developed probe was successfully employed for diagnostic purposes on various tissues, including brain and bladder. In particular, the device allowed discriminating healthy tissue from both tumor and dysplastic tissue as well as to perform tumor grading. The diagnostic capabilities of the method, determined using a cross-validation method with a leave-one-out approach, demonstrated high sensitivity and specificity for all the examined samples, as well as a good agreement with histopathological examination performed on the same samples. The obtained results demonstrated that the multimodal approach is crucial for improving diagnostic capabilities with respect to what can be obtained from individual techniques. The experimental setup presented here can improve diagnostic capabilities on a broad range of tissues and has the potential of being used clinically for guiding surgical resection in the near future.

Conductive carbon tape used for support and mounting of both whole animal and fragile heat-treated tissue sections for MALDI MS imaging and quantitation.

PubMed

Goodwin, Richard J A; Nilsson, Anna; Borg, Daniel; Langridge-Smith, Pat R R; Harrison, David J; Mackay, C Logan; Iverson, Suzanne L; Andrén, Per E

2012-08-30

Analysis of whole animal tissue sections by MALDI MS imaging (MSI) requires effective sample collection and transfer methods to allow the highest quality of in situ analysis of small or hard to dissect tissues. We report on the use of double-sided adhesive conductive carbon tape during whole adult rat tissue sectioning of carboxymethyl cellulose (CMC) embedded animals, with samples mounted onto large format conductive glass and conductive plastic MALDI targets, enabling MSI analysis to be performed on both TOF and FT-ICR MALDI mass spectrometers. We show that mounting does not unduly affect small molecule MSI detection by analyzing tiotropium abundance and distribution in rat lung tissues, with direct on-tissue quantitation achieved. Significantly, we use the adhesive tape to provide support to embedded delicate heat-stabilized tissues, enabling sectioning and mounting to be performed that maintained tissue integrity on samples that had previously been impossible to adequately prepare section for MSI analysis. The mapping of larger peptidomic molecules was not hindered by tape mounting samples and we demonstrate this by mapping the distribution of PEP-19 in both native and heat-stabilized rat brains. Furthermore, we show that without heat stabilization PEP-19 degradation fragments can detected and identified directly by MALDI MSI analysis. Copyright © 2012 Elsevier B.V. All rights reserved.

Flexible needle with integrated optical coherence tomography probe for imaging during transbronchial tissue aspiration

NASA Astrophysics Data System (ADS)

Li, Jiawen; Quirk, Bryden C.; Noble, Peter B.; Kirk, Rodney W.; Sampson, David D.; McLaughlin, Robert A.

2017-10-01

Transbronchial needle aspiration (TBNA) of small lesions or lymph nodes in the lung may result in nondiagnostic tissue samples. We demonstrate the integration of an optical coherence tomography (OCT) probe into a 19-gauge flexible needle for lung tissue aspiration. This probe allows simultaneous visualization and aspiration of the tissue. By eliminating the need for insertion and withdrawal of a separate imaging probe, this integrated design minimizes the risk of dislodging the needle from the lesion prior to aspiration and may facilitate more accurate placement of the needle. Results from in situ imaging in a sheep lung show clear distinction between solid tissue and two typical constituents of nondiagnostic samples (adipose and lung parenchyma). Clinical translation of this OCT-guided aspiration needle holds promise for improving the diagnostic yield of TBNA.

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 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. 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). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

An Adhesive Patch-Based Skin Biopsy Device for Molecular Diagnostics and Skin Microbiome Studies.

PubMed

Yao, Zuxu; Moy, Ronald; Allen, Talisha; Jansen, Burkhard

2017-10-01

A number of diagnoses in clinical dermatology are currently histopathologically confirmed and this image recognition-based confirmation generally requires surgical biopsies. The increasing ability of molecular pathology to corroborate or correct a clinical diagnosis based on objective gene expression, mutation analysis, or molecular microbiome data is on the horizon and would be further supported by a tool or procedure to collect samples non-invasively. This study characterizes such a tool in form of a 'bladeless' adhesive patch-based skin biopsy device. The performance of this device was evaluated through a variety of complementary technologies including assessment of sample biomass, electron microscopy demonstrating the harvesting of layers of epidermal tissue, and isolation of RNA and DNA from epidermal skin samples. Samples were obtained by application of adhesive patches to the anatomical area of interest. Biomass assessment demonstrated collection of approximately 0.3mg of skin tissue per adhesive patch and electron microscopy confirmed the nature of the harvested epidermal skin tissue. The obtained tissue samples are stored in a stable fashion on adhesive patches over a wide range of temperatures (-80oC to +60oC) and for extended periods of time (7 days or more). Total human RNA, human genomic DNA and microbiome DNA yields were 23.35 + 15.75ng, 27.72 + 20.71ng and 576.2 + 376.8pg, respectively, in skin samples obtained from combining 4 full patches collected non-invasively from the forehead of healthy volunteers. The adhesive patch skin sampling procedure is well tolerated and provides robust means to obtain skin tissue, RNA, DNA, and microbiome samples without involving surgical biopsies. The non-invasively obtained skin samples can be shipped cost effectively at ambient temperature by mail or standard courier service, and are suitable for a variety of molecular analyses of the skin microbiome as well as of keratinocytes, T cells, dendritic cells, melanocytes, and other skin cells involved in the pathology of various skin conditions and conditions where the skin can serve as a surrogate target organ.

J Drugs Dermatol. 2017;16(10):979-986.

Preparation of A Spaceflight: Apoptosis Search in Sutured Wound Healing Models.

PubMed

Riwaldt, Stefan; Monici, Monica; Graver Petersen, Asbjørn; Birk Jensen, Uffe; Evert, Katja; Pantalone, Desiré; Utpatel, Kirsten; Evert, Matthias; Wehland, Markus; Krüger, Marcus; Kopp, Sascha; Frandsen, Sofie; Corydon, Thomas; Sahana, Jayashree; Bauer, Johann; Lützenberg, Ronald; Infanger, Manfred; Grimm, Daniela

2017-12-03

To prepare the ESA (European Space Agency) spaceflight project "Wound healing and Sutures in Unloading Conditions", we studied mechanisms of apoptosis in wound healing models based on ex vivo skin tissue cultures, kept for 10 days alive in serum-free DMEM/F12 medium supplemented with bovine serum albumin, hydrocortisone, insulin, ascorbic acid and antibiotics at 32 °C. The overall goal is to test: (i) the viability of tissue specimens; (ii) the gene expression of activators and inhibitors of apoptosis and extracellular matrix components in wound and suture models; and (iii) to design analytical protocols for future tissue specimens after post-spaceflight download. Hematoxylin-Eosin and Elastica-van-Gieson staining showed a normal skin histology with no signs of necrosis in controls and showed a normal wound suture. TdT-mediated dUTP-biotin nick end labeling for detecting DNA fragmentation revealed no significant apoptosis. No activation of caspase-3 protein was detectable. FASL , FADD , CASP3 , CASP8 , CASP10 , BAX , BCL2 , CYC1 , APAF1 , LAMA3 and SPP1 mRNAs were not altered in epidermis and dermis samples with and without a wound compared to 0 day samples (specimens investigated directly post-surgery). BIRC5 , CASP9 , and FN1 mRNAs were downregulated in epidermis/dermis samples with and/or without a wound compared to 0 day samples. BIRC2 , BIRC3 were upregulated in 10 day wound samples compared to 0 day samples in epidermis/dermis. RELA/FAS mRNAs were elevated in 10 day wound and no wound samples compared to 0 day samples in dermis. In conclusion, we demonstrate that it is possible to maintain live skin tissue cultures for 10 days. The viability analysis showed no significant signs of cell death in wound and suture models. The gene expression analysis demonstrated the interplay of activators and inhibitors of apoptosis and extracellular matrix components, thereby describing important features in ex vivo sutured wound healing models. Collectively, the performed methods defining analytical protocols proved to be applicable for post-flight analyzes of tissue specimens after sample return.

Synergistically increased ILC2 and Th9 cells in lung tissue jointly promote the pathological process of asthma in mice.

PubMed

Ying, Xinyu; Su, Zhaoliang; Bie, Qingli; Zhang, Pan; Yang, Huijian; Wu, Yumin; Xu, Yunyun; Wu, Jing; Zhang, Mengying; Wang, Shengjun; Xu, Huaxi

2016-06-01

In recent years, T helper (Th) 9 cells have been demonstrated to be key mediators in immune responses in asthmatic lungs, and innate lymphoid cells 2 (ILC2s) have been described as a novel type of innate immunocyte with the ability to enhance immunoglobulin E (IgE) production. However, the interaction between ILC2s and Th9 cells in the pulmonary system of a mouse model of asthma remains to be elucidated. In the present study, the response state of lung tissue with regards to Th9 and ILC2s in a mouse model of asthma was investigated by detecting Th9‑ and ILC2‑associated cytokine receptors. The present study also investigated the association between the expression levels of the cytokine receptors in lung tissue samples and the IgE levels in sera samples from mouse models of asthma. Results from the present study demonstrated that the frequency of ILC2s and Th9 cells was significantly increased in the lung tissue samples, indicating that a Th2-type immune response had occurred. In addition, high mRNA expression levels of RAR‑related orphan receptor α, interleukin 1 receptor‑like 1, transcription factor PU.1 and interleukin (IL)‑9 were observed. Furthermore, IL‑5Rα, IL‑13Rα2 and high‑affinity IgE receptor were increased in mouse models of asthma, and a positive association was observed between the expression levels of ILC2‑ or Th9‑associated receptors in tissue samples and IgE levels in the sera. This indicated that ILC2s and Th9 were in a state of polarization and may promote each other in the lung tissue of mouse models of asthma, and that the lung tissue was responding to the two types of cells via increased expression of receptors.

Healing potential of transplanted allogeneic chondrocytes of three different sources in lesions of the avascular zone of the meniscus: a pilot study.

PubMed

Weinand, Christian; Peretti, Giuseppe M; Adams, Samuel B; Randolph, Mark A; Savvidis, Estafios; Gill, Thomas J

2006-11-01

Successful treatment of tears to the avascular region of the meniscus remains a challenge. Current repair techniques, such as sutures and anchors, are effective in stabilizing the peripheral, vascularized regions of the meniscus, but are not adequate for promoting healing in the avascular region. The purpose of this study was to demonstrate the healing ability of a tissue-engineered repair technique using allogenic chondrocytes from three different sources for the avascular zone of the meniscus. Articular, auricular, and costal chondrocytes were harvested from 3-month-old Yorkshire swine. A 1-cm bucket-handle lesion was created in the avascular zone of each three swine. A cell-scaffold construct, composed of a single chondrocyte cell type and Vicryl mesh, was implanted into the lesion and secured with two vertical mattress sutures. Controls consisted of each three sutured unseeded mesh implants, suture only, and untreated lesions. The swine were allowed immediate post-operative full weight bearing. Menisci and controls were harvested after 12 weeks. In all experimental samples, lesion closure was observed. Gross mechanical testing with two Adson forceps demonstrated bonding of the lesion. Histological analysis showed formation of new tissue in all three experimental samples. None of the control samples demonstrated closure and formation of new matrix. We present preliminary data that demonstrates the potential of a tissue-engineered, allogenic cellular repair to provide successful healing of lesions in the avascular zone in a large animal model.

The low-temperature method for study of coniferous tissues in the environmental scanning electron microscope.

PubMed

Neděla, Vilém; Tihlaříková, Eva; Hřib, Jiří

2015-01-01

The use of non-standard low-temperature conditions in environmental scanning electron microscopy might be promising for the observation of coniferous tissues in their native state. This study is aimed to analyse and evaluate the method based on the principle of low-temperature sample stabilization. We demonstrate that the upper mucous layer is sublimed and a microstructure of the sample surface can be observed with higher resolution at lower gas pressure conditions, thanks to a low-temperature method. An influence of the low-temperature method on sample stability was also studied. The results indicate that high-moisture conditions are not suitable for this method and often cause the collapse of samples. The potential improvement of stability to beam damage has been demonstrated by long-time observation at different operation parameters. We finally show high applicability of the low-temperature method on different types of conifers and Oxalis acetosella. © 2014 Wiley Periodicals, Inc.

Transcript levels of ten-eleven translocation type 1–3 in cervical cancer and non-cancerous cervical tissues

PubMed Central

Bronowicka-Kłys, Dorota Ewa; Roszak, Andrzej; Pawlik, Piotr; Sajdak, Stefan; Sowińska, Anna; Jagodziński, Paweł Piotr

2017-01-01

Decreased expression of ten-eleven translocation (TET1, TET2 and TET3) proteins has been reported in various types of cancer. However, the expression levels of TET proteins in cervical cancer (CC) remain to be elucidated. The present study determined the levels of TET1, TET2 and TET3 transcripts in cancerous (n=80) and non-cancerous cervical tissues (n=41). The results revealed a significant reduction in TET1 transcripts (P=0.0000001) in cervical tissue samples from patients with primary CC compared with samples from control patients. Significantly decreased TET1 transcript levels, as compared to non-cancerous cervical tissues, were also observed in tissue samples with the following characteristics: Stage I (P=0.016), II (P<0.0001), III (P=0.00007) and grade of differentiation G1 (P=0.026), G2 (P=0.00006), G3 (P=0.0007) and Gx (P=0.0004) and squamous histological type (P<0.00001). TET1 transcript levels were significantly lower in patients aged 45–60 years (P=0.0002) and patients age >60 years (P=0.003), as compared with non-cancerous cervical tissues. TET2 transcript levels were lower in cervical cancer tissues classified as stage II (P=0.043) and TET3 transcript levels were lower in stage III samples (P=0.010), tissue samples with a grade of differentiation of G3 (P=0.025) and tissue with squamous type histology (P=0.047), all compared with non-cancerous cervical tissues. The present study demonstrated a significantly reduced level of TET1 transcripts in cancerous cervical tissues, as compared with non-cancerous tissues. Furthermore, decreased TET1-3 transcript levels were identified when patients with CC were stratified by clinicopathological variables, as compared with non-cancerous cervical tissues. PMID:28521490

Comparison of microdialysis sampling perfusion fluid components on the foreign body reaction in rat subcutaneous tissue.

PubMed

Keeler, Geoffrey D; Durdik, Jeannine M; Stenken, Julie A

2014-06-16

Microdialysis sampling is a commonly used technique for collecting solutes from the extracellular space of tissues in laboratory animals and humans. Large molecular weight solutes can be collected using high molecular weight cutoff (MWCO) membranes (100kDa or greater). High MWCO membranes require addition of high molecular weight dextrans or albumin to the perfusion fluid to prevent fluid loss via ultrafiltration. While these perfusion fluid additives are commonly used during microdialysis sampling, the tissue response to the loss of these compounds across the membrane is poorly understood. Tissue reactions to implanted microdialysis sampling probes containing different microdialysis perfusion fluids were compared over a 7-day time period in rats. The base perfusion fluid was Ringer's solution supplemented with either bovine serum albumin (BSA), rat serum albumin (RSA), Dextran-70, or Dextran-500. A significant inflammatory response to Dextran-70 was observed. No differences in the tissue response between BSA and RSA were observed. Among these agents, the BSA, RSA, and Dextran-500 produced a significantly reduced inflammatory response compared to the Dextran-70. This work demonstrates that use of Dextran-70 in microdialysis sampling perfusion fluids should be eliminated and replaced with Dextran-500 or other alternatives. Copyright © 2013 Elsevier B.V. All rights reserved.

Crosslinking effect of dialdehyde starch (DAS) on decellularized porcine aortas for tissue engineering.

PubMed

Wang, Xu; Gu, Zhipeng; Qin, Huanhuan; Li, Li; Yang, Xu; Yu, Xixun

2015-08-01

Biological tissue-derived biomaterials must be chemically modified to avoid immediate degradation and immune response before being implanted in human body to replace malfunctioning organs. DAS with active aldehyde groups was employed to replace glutaraldehyde (GA), a most common synthetic crosslinking reagent in clinical practice, to fix bioprostheses for lower cytotoxicity. The aim of this research was to evaluate fixation effect of DAS. The tensile strength, crosslinking stability, cytotoxicity especially the anti-calcification capability of DAS-fixed tissues were investigated. The tensile strength and resistance to enzymatic degradation of samples were increased after DAS fixation, the values maintained stably in D-Hanks solution for several days. Meanwhile, ultrastructure of samples preserved well and the anti-calcification capability of samples were improved, the amount of positive staining points in the whole visual field of 15% DAS-fixed samples was only 0.576 times to GA-fixed ones. Moreover, both unreacted DAS and its hydrolytic products were nontoxic in cytotoxicity study. The results demonstrated DAS might be an effective crosslinking reagent to fix biological tissue-derived biomaterials in tissue engineering. Copyright © 2015 Elsevier B.V. All rights reserved.

Evaluating differential nuclear DNA yield rates and osteocyte numbers among human bone tissue types: A synchrotron radiation micro-CT approach.

PubMed

Andronowski, Janna M; Mundorff, Amy Z; Pratt, Isaac V; Davoren, Jon M; Cooper, David M L

2017-05-01

Molecular human identification has conventionally focused on DNA sampling from dense, weight-bearing cortical bone tissue, typically from femora or tibiae. A comparison of skeletal elements from three contemporary individuals demonstrated that elements with high quantities of cancellous bone yielded nuclear DNA at the highest rates, suggesting that preferentially sampling cortical bone may be suboptimal (Mundorff & Davoren, 2014). Despite these findings, the reason for the differential DNA yields between cortical and cancellous bone tissues remains unknown. The primary goal of this work is to ascertain whether differences in bone microstructure can be used to explain differential nuclear DNA yield among bone tissue types observed by Mundorff and Davoren (2014), with a focus on osteocytes and the three-dimensional (3D) quantification of their associated lacunae. Osteocytes and other bone cells are recognized to house DNA in bone tissue, thus examining the density of their lacunae may explain why nuclear DNA yield rates differ among bone tissue types. Lacunae were visualized and quantified using synchrotron radiation-based micro-Computed Tomographic imaging (SR micro-CT). Volumes of interest (VOIs) from cortical and cancellous bone tissues (n=129) were comparatively analyzed from the three skeletons sampled for Mundorff and Davoren's (2014) study. Analyses tested the primary hypothesis that the abundance and density of osteocytes (inferred from their lacunar spaces) vary between cortical and cancellous bone tissue types. Results demonstrated that osteocyte lacunar abundance and density vary between cortical and cancellous bone tissue types, with cortical bone VOIs containing a higher lacunar abundance and density. We found that the osteocyte lacunar density values are independent of nuclear DNA yield, suggesting an alternative explanation for the higher nuclear DNA yields from bones with greater quantities of cancellous bone tissue. The use of SR micro-CT allowed for a scale of analysis that revealed a high range of variation in lacunar abundance in both tissue types. Moreover, high-resolution SR micro-CT imaging revealed potential soft tissue remnants within marrow spaces not visible macroscopically. It is hypothesized that soft tissue remnants observed among the trabeculae of skeletal elements with high quantities of cancellous bone tissue are responsible for the high nuclear DNA yields. These findings have significant implications for bone-sample selection for nuclear DNA analysis in a forensic context when skeletal remains are recovered from the ground surface. Copyright © 2017 Elsevier B.V. All rights reserved.

Few-mode fiber detection for tissue characterization in optical coherence tomography

NASA Astrophysics Data System (ADS)

Eugui, Pablo; Lichtenegger, Antonia; Augustin, Marco; Harper, Danielle J.; Fialová, Stanislava; Wartak, Andreas; Hitzenberger, Christoph K.; Baumann, Bernhard

2017-07-01

A few-mode fiber based detection for OCT systems is presented. The capability of few-mode fibers for delivering light through different fiber paths enables the application of these fibers for angular scattering tissue character- ization. Since the optical path lengths traveled in the fiber change between the fiber modes, the OCT image information will be reconstructed at different depth positions, separating the directly backscattered light from the light scattered at other angles. Using the proposed method, the relation between the angle of reflection from the sample and the respective modal intensity distribution was investigated. The system was demonstrated for imaging ex-vivo brain tissue samples of patients with Alzheimer's disease.

Connective tissue growth factor enhances the migration of gastric cancer through downregulation of E-cadherin via the NF-κB pathway.

PubMed

Mao, Zhengfa; Ma, Xiaoyan; Rong, Yefei; Cui, Lei; Wang, Xuqing; Wu, Wenchuan; Zhang, Jianxin; Jin, Dayong

2011-01-01

Local invasion and distant metastasis are difficult problems for surgical intervention and treatment in gastric cancer. Connective tissue growth factor (CTGF/CCN2) was considered to have an important role in this process. In this study, we demonstrated that expression of CTGF was significantly upregulated in clinical tissue samples of gastric carcinoma (GC) samples. Forced expression of CTGF in AGS GC cells promoted their migration in culture and significantly increased tumor metastasis in nude mice, whereas RNA interference-mediated knockdown of CTGF in GC cells significantly inhibited cell migration in vitro. We disclose that CTGF downregulated the expression of E-cadherin through activation of the nuclear factor-κappa B (NF-κB) pathway. The effects of CTGF in GC cells were abolished by dominant negative IκappaB. Collectively, these data reported here demonstrate CTGF could modulate the NF-κappaB pathway and perhaps be a promising therapeutic target for gastric cancer invasion and metastasis. © 2010 Japanese Cancer Association.

Ex vivo validation of photo-magnetic imaging.

PubMed

Luk, Alex; Nouizi, Farouk; Erkol, Hakan; Unlu, Mehmet B; Gulsen, Gultekin

2017-10-15

We recently introduced a new high-resolution diffuse optical imaging technique termed photo-magnetic imaging (PMI), which utilizes magnetic resonance thermometry (MRT) to monitor the 3D temperature distribution induced in a medium illuminated with a near-infrared light. The spatiotemporal temperature distribution due to light absorption can be accurately estimated using a combined photon propagation and heat diffusion model. High-resolution optical absorption images are then obtained by iteratively minimizing the error between the measured and modeled temperature distributions. We have previously demonstrated the feasibility of PMI with experimental studies using tissue simulating agarose phantoms. In this Letter, we present the preliminary ex vivo PMI results obtained with a chicken breast sample. Similarly to the results obtained on phantoms, the reconstructed images reveal that PMI can quantitatively resolve an inclusion with a 3 mm diameter embedded deep in a biological tissue sample with only 10% error. These encouraging results demonstrate the high performance of PMI in ex vivo biological tissue and its potential for in vivo imaging.

High resolution computational on-chip imaging of biological samples using sparsity constraint (Conference Presentation)

NASA Astrophysics Data System (ADS)

Rivenson, Yair; Wu, Chris; Wang, Hongda; Zhang, Yibo; Ozcan, Aydogan

2017-03-01

Microscopic imaging of biological samples such as pathology slides is one of the standard diagnostic methods for screening various diseases, including cancer. These biological samples are usually imaged using traditional optical microscopy tools; however, the high cost, bulkiness and limited imaging throughput of traditional microscopes partially restrict their deployment in resource-limited settings. In order to mitigate this, we previously demonstrated a cost-effective and compact lens-less on-chip microscopy platform with a wide field-of-view of >20-30 mm^2. The lens-less microscopy platform has shown its effectiveness for imaging of highly connected biological samples, such as pathology slides of various tissue samples and smears, among others. This computational holographic microscope requires a set of super-resolved holograms acquired at multiple sample-to-sensor distances, which are used as input to an iterative phase recovery algorithm and holographic reconstruction process, yielding high-resolution images of the samples in phase and amplitude channels. Here we demonstrate that in order to reconstruct clinically relevant images with high resolution and image contrast, we require less than 50% of the previously reported nominal number of holograms acquired at different sample-to-sensor distances. This is achieved by incorporating a loose sparsity constraint as part of the iterative holographic object reconstruction. We demonstrate the success of this sparsity-based computational lens-less microscopy platform by imaging pathology slides of breast cancer tissue and Papanicolaou (Pap) smears.

Detecting quinoxaline-2-carboxylic acid in animal tissues by using sensitive rapid enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay.

PubMed

Le, Tao; Yu, Huan; Niu, Xiaodong

2015-05-15

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and time-resolved fluoroimmunoassay (TR-FIA) based on an anti-N-butylquinoxaline-2-carboxamide (BQCA) monoclonal antibody were standardized and validated for quinoxaline-2-carboxylic acid (QCA) screening in animal tissues and its performance were compared to HPLC. The sensitivities obtained for edible tissue extracts were 1.62 and 1.12 ng ml(-1) for ic-ELISA and TR-FIA detection, respectively. Two samples were spiked with QCA and analyzed by both methods. The recovery values ranged from 92.6% to 112.2% and the coefficients of variation were less than 15% for QCA spiking into swine tissue samples at concentrations of 2.5-50.0 μg kg(-1). Excellent correlations (r(2)=0.987-0.996) of the ic-ELISA/HPLC and TR-FIA/HPLC data were observed for processed samples. The results demonstrated that the ic-ELISA and TR-FIA methods were rapid and accurate for the residue detection of QCA in animal tissues. Copyright © 2014 Elsevier Ltd. All rights reserved.

Various clinical application of phase contrast X-ray

NASA Astrophysics Data System (ADS)

Oh, Chilhwan; Park, Sangyong; Ha, Seunghan; Park, Gyuman; Lee, Gunwoo; Lee, Onseok; Je, Jungho

2008-02-01

In biomedical application study using phase contrast X-ray, both sample thickness or density and absorption difference are very important factors in aspects of contrast enhancement. We present experimental evidence that synchrotron hard X-ray are suitable for radiological imaging of biological samples down to the cellular level. We investigated the potential of refractive index radiology using un-monochromatized synchrotron hard X-rays for the imaging of cell and tissue in various diseases. Material had been adopted various medical field, such as apoE knockout mouse in cardiologic field, specimen from renal and prostatic carcinoma patient in urology, basal cell epithelioma in dermatology, brain tissue from autosy sample of pakinson's disease, artificially induced artilrtis tissue from rabbits and extracted tooth from patients of crack tooth syndrome. Formalin and paraffin fixed tissue blocks were cut in 3 mm thickness for the X-ray radiographic imaging. From adjacent areas, 4 μm thickness sections were also prepared for hematoxylin-eosin staining. Radiographic images of dissected tissues were obtained using the hard X-rays from the 7B2 beamline of the Pohang Light Source (PLS). The technique used for the study was the phase contrast images were compared with the optical microscopic images of corresponding histological slides. Radiographic images of various diseased tissues showed clear histological details of organelles in normal tissues. Most of cancerous lesions were well differentiated from adjacent normal tissues and detailed histological features of each tumor were clearly identified. Also normal microstructures were identifiable by the phase contrast imaging. Tissue in cancer or other disease showed clearly different findings from those of surrounding normal tissue. For the first time we successfully demonstrated that synchrotron hard X-rays can be used for radiological imaging of relatively thick tissue samples with great histological details.

3D on-chip microscopy of optically cleared tissue

NASA Astrophysics Data System (ADS)

Zhang, Yibo; Shin, Yoonjung; Sung, Kevin; Yang, Sam; Chen, Harrison; Wang, Hongda; Teng, Da; Rivenson, Yair; Kulkarni, Rajan P.; Ozcan, Aydogan

2018-02-01

Traditional pathology relies on tissue biopsy, micro-sectioning, immunohistochemistry and microscopic imaging, which are relatively expensive and labor-intensive, and therefore are less accessible in resource-limited areas. Low-cost tissue clearing techniques, such as the simplified CLARITY method (SCM), are promising to potentially reduce the cost of disease diagnosis by providing 3D imaging and phenotyping of thicker tissue samples with simpler preparation steps. However, the mainstream imaging approach for cleared tissue, fluorescence microscopy, suffers from high-cost, photobleaching and signal fading. As an alternative approach to fluorescence, here we demonstrate 3D imaging of SCMcleared tissue using on-chip holography, which is based on pixel-super-resolution and multi-height phase recovery algorithms to digitally compute the sample's amplitude and phase images at various z-slices/depths through the sample. The tissue clearing procedures and the lens-free imaging system were jointly optimized to find the best illumination wavelength, tissue thickness, staining solution pH, and the number of hologram heights to maximize the imaged tissue volume, minimize the amount of acquired data, while maintaining a high contrast-to-noise ratio for the imaged cells. After this optimization, we achieved 3D imaging of a 200-μm thick cleared mouse brain tissue over a field-of-view of <20mm2 , and the resulting 3D z-stack agrees well with the images acquired with a scanning lens-based microscope (20× 0.75NA). Moreover, the lens-free microscope achieves an order-of-magnitude better data efficiency compared to its lens-based counterparts for volumetric imaging of samples. The presented low-cost and high-throughput lens-free tissue imaging technique enabled by CLARITY can be used in various biomedical applications in low-resource-settings.

Preservation of pathological tissue specimens by freeze-drying for immunohistochemical staining and various molecular biological analyses.

PubMed

Matsuo, S; Sugiyama, T; Okuyama, T; Yoshikawa, K; Honda, K; Takahashi, R; Maeda, S

1999-05-01

Conditions of preserving DNA, RNA and protein in pathological specimens are of great importance as degradation of such macromolecules would critically affect results of molecular biological analysis. The feasibility of freeze-drying as a means of preserving pathological tissue samples for molecular analysis has previously been shown. In the present study, further tests on long-term storage conditions and analyses of freeze-dried samples by polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, western blotting and immunohistochemistry are reported. Rat chromosomal DNA of freeze-dried samples stored for 4 years showed slight degradation while RNA degradation was more prominently seen at an earlier stage of storage. However, these 4 year DNA and RNA samples were still able to serve as a template for some PCR and RT-PCR analyses, respectively. Overexpression of c-erbB-2 and p53 protein was demonstrated by western blotting and immunohistochemical staining using freeze-dried human breast cancer tissues. Although macromolecules in freeze-dried samples degrade to some extent during the preservation period, they should still be of value for certain molecular biological analyses and morphological examination; hence, providing more convenient and inexpensive ways of pathological tissue storage.

Nonlinear interferometric vibrational imaging of biological tissue

NASA Astrophysics Data System (ADS)

Jiang, Zhi; Marks, Daniel L.; Geddes, Joseph B., III; Boppart, Stephen A.

2008-02-01

We demonstrate imaging with the technique of nonlinear interferometric vibrational imaging (NIVI). Experimental images using this instrumentation and method have been acquired from both phantom and biological tissues. In our system, coherent anti-Stokes Raman scattering (CARS) signals are detected by spectral interferometry, which is able to fully restore high resolution Raman spectrum on each focal spot of a sample covering multiple Raman bands using broadband pump and Stokes laser beams. Spectral-domain detection has been demonstrated and allows for a significant increase in image acquiring speed, in signal-to-noise, and in interferometric signal stability.

Extraction of organic contaminants from marine sediments and tissues using microwave energy.

PubMed

Jayaraman, S; Pruell, R J; McKinney, R

2001-07-01

In this study, we compared microwave solvent extraction (MSE) to conventional methods for extracting organic contaminants from marine sediments and tissues with high and varying moisture content. The organic contaminants measured were polychlorinated biphenyl (PCB) congeners, chlorinated pesticides, and polycyclic aromatic hydrocarbons (PAHs). Initial experiments were conducted on dry standard reference materials (SRMs) and field collected marine sediments. Moisture content in samples greatly influenced the recovery of the analytes of interest. When wet sediments were included in a sample batch, low recoveries were often encountered in other samples in the batch, including the dry SRM. Experiments were conducted to test the effect of standardizing the moisture content in all samples in a batch prior to extraction. SRM1941a (marine sediment). SRM1974a (mussel tissue), as well as QA96SED6 (marine sediment), and QA96TIS7 (marine tissue), both from 1996 NIST Intercalibration Exercise were extracted using microwave and conventional methods. Moisture levels were adjusted in SRMs to match those of marine sediment and tissue samples before microwave extraction. The results demonstrated that it is crucial to standardize the moisture content in all samples, including dry reference material to ensure good recovery of organic contaminants. MSE yielded equivalent or superior recoveries compared to conventional methods for the majority of the compounds evaluated. The advantages of MSE over conventional methods are reduced solvent usage, higher sample throughput and the elimination of halogenated solvent usage.

Detection of beryllium in digested autopsy tissues by inductively coupled plasma mass spectrometry using a high matrix interface configuration.

PubMed

Larivière, Dominic; Tremblay, Mélodie; Durand-Jézéquel, Myriam; Tolmachev, Sergei

2012-04-01

This article describes a robust methodology using the combination of instrumental design (high matrix interface-HMI), sample dilution and internal standardization for the quantification of beryllium (Be) in various digested autopsy tissues using inductively coupled plasma mass spectrometry. The applicability of rhodium as a proper internal standard for Be was demonstrated in three types of biological matrices (i.e., femur, hair, lung tissues). Using HMI, it was possible to achieve instrumental detection limits and sensitivity of 0.6 ng L(-1) and 157 cps L ng(-1), respectively. Resilience to high salt matrices of the HMI setup was also highlighted using bone mimicking solution ([Ca(2+)] = 26 to 1,400 mg L(-1)), providing a 14-fold increase in tolerance and a 2.7-fold decrease in method detection limit compared to optimized experimental conditions obtained without the HMI configuration. Precision of the methodology to detect low levels of Be in autopsy samples was demonstrated using hair and blood certified reference materials. Be concentration ranging from 0.015 to 255 μg kg(-1) in autopsy samples obtained from the U.S. Transuranium and Uranium Registries were measured using the methodology presented.

Focusing light through biological tissue and tissue-mimicking phantoms up to 9.6 cm in thickness with digital optical phase conjugation

NASA Astrophysics Data System (ADS)

Shen, Yuecheng; Liu, Yan; Ma, Cheng; Wang, Lihong V.

2016-08-01

Optical phase conjugation (OPC)-based wavefront shaping techniques focus light through or within scattering media, which is critically important for deep-tissue optical imaging, manipulation, and therapy. However, to date, the sample thickness in OPC experiments has been limited to only a few millimeters. Here, by using a laser with a long coherence length and an optimized digital OPC system that can safely deliver more light power, we focused 532-nm light through tissue-mimicking phantoms up to 9.6 cm thick, as well as through ex vivo chicken breast tissue up to 2.5 cm thick. Our results demonstrate that OPC can be achieved even when photons have experienced on average 1000 scattering events. The demonstrated penetration of nearly 10 cm (˜100 transport mean free paths) has never been achieved before by any optical focusing technique, and it shows the promise of OPC for deep-tissue noninvasive optical imaging, manipulation, and therapy.

Differentiating fatty and non-fatty tissue using photoacoustic imaging

NASA Astrophysics Data System (ADS)

Pan, Leo; Rohling, Robert; Abolmaesumi, Purang; Salcudean, Septimiu; Tang, Shuo

2014-03-01

In this paper, we demonstrate a temporal-domain intensity-based photoacoustic imaging method that can differentiate between fatty and non-fatty tissues. PA pressure intensity is partly dependent on the tissue's speed of sound, which increases as temperature increases in non-fatty tissue and decreases in fatty tissue. Therefore, by introducing a temperature change in the tissue and subsequently monitoring the change of the PA intensity, it is possible to distinguish between the two types of tissue. A commercial ultrasound system with a 128-element 5-14 MHz linear array transducer and a tunable ND:YAG laser were used to produce PA images. Ex-vivo bovine fat and porcine liver tissues were precooled to below 10°C and then warmed to room-temperature over ~1 hour period. A thermocouple monitored the temperature rise while PA images were acquired at 0.5°C intervals. The averaged intensity of the illuminated tissue region at each temperature interval was plotted and linearly fitted. Liver samples showed a mean increase of 2.82 %/°C, whereas bovine fat had a mean decrease of 6.24 %/°C. These results demonstrate that this method has the potential to perform tissue differentiation in the temporal-domain.

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

The biocompatibility of carbon hydroxyapatite/β-glucan composite for bone tissue engineering studied with Raman and FTIR spectroscopic imaging.

PubMed

Sroka-Bartnicka, Anna; Kimber, James A; Borkowski, Leszek; Pawlowska, Marta; Polkowska, Izabela; Kalisz, Grzegorz; Belcarz, Anna; Jozwiak, Krzysztof; Ginalska, Grazyna; Kazarian, Sergei G

2015-10-01

The spectroscopic approaches of FTIR imaging and Raman mapping were applied to the characterisation of a new carbon hydroxyapatite/β-glucan composite developed for bone tissue engineering. The composite is an artificial bone material with an apatite-forming ability for the bone repair process. Rabbit bone samples were tested with an implanted bioactive material for a period of several months. Using spectroscopic and chemometric methods, we were able to determine the presence of amides and phosphates and the distribution of lipid-rich domains in the bone tissue, providing an assessment of the composite's bioactivity. Samples were also imaged in transmission using an infrared microscope combined with a focal plane array detector. CaF2 lenses were also used on the infrared microscope to improve spectral quality by reducing scattering artefacts, improving chemometric analysis. The presence of collagen and lipids at the bone/composite interface confirmed biocompatibility and demonstrate the suitability of FTIR microscopic imaging with lenses in studying these samples. It confirmed that the composite is a very good background for collagen growth and increases collagen maturity with the time of the bone growth process. The results indicate the bioactive and biocompatible properties of this composite and demonstrate how Raman and FTIR spectroscopic imaging have been used as an effective tool for tissue characterisation.

In vivo microsampling to capture the elusive exposome

NASA Astrophysics Data System (ADS)

Bessonneau, Vincent; Ings, Jennifer; McMaster, Mark; Smith, Richard; Bragg, Leslie; Servos, Mark; Pawliszyn, Janusz

2017-03-01

Loss and/or degradation of small molecules during sampling, sample transportation and storage can adversely impact biological interpretation of metabolomics data. In this study, we performed in vivo sampling using solid-phase microextraction (SPME) in combination with non-targeted liquid chromatography and high-resolution tandem mass spectrometry (LC-MS/MS) to capture the fish tissue exposome using molecular networking analysis, and the results were contrasted with molecular differences obtained with ex vivo SPME sampling. Based on 494 MS/MS spectra comparisons, we demonstrated that in vivo SPME sampling provided better extraction and stabilization of highly reactive molecules, such as 1-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoleoyl-glycero-3-phosphocholine, from fish tissue samples. This sampling approach, that minimizes sample handling and preparation, offers the opportunity to perform longitudinal monitoring of the exposome in biological systems and improve the reliability of exposure-measurement in exposome-wide association studies.

In vivo microsampling to capture the elusive exposome

PubMed Central

Bessonneau, Vincent; Ings, Jennifer; McMaster, Mark; Smith, Richard; Bragg, Leslie; Servos, Mark; Pawliszyn, Janusz

2017-01-01

Loss and/or degradation of small molecules during sampling, sample transportation and storage can adversely impact biological interpretation of metabolomics data. In this study, we performed in vivo sampling using solid-phase microextraction (SPME) in combination with non-targeted liquid chromatography and high-resolution tandem mass spectrometry (LC-MS/MS) to capture the fish tissue exposome using molecular networking analysis, and the results were contrasted with molecular differences obtained with ex vivo SPME sampling. Based on 494 MS/MS spectra comparisons, we demonstrated that in vivo SPME sampling provided better extraction and stabilization of highly reactive molecules, such as 1-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoleoyl-glycero-3-phosphocholine, from fish tissue samples. This sampling approach, that minimizes sample handling and preparation, offers the opportunity to perform longitudinal monitoring of the exposome in biological systems and improve the reliability of exposure-measurement in exposome-wide association studies. PMID:28266605

Histopathological findings in immunohistological staining of the granulomatous tissue reaction associated with tuberculosis.

PubMed

Karimi, Shirin; Shamaei, Masoud; Pourabdollah, Mihan; Sadr, Makan; Karbasi, Mehrdad; Kiani, Arda; Bahadori, Moslem

2014-01-01

Purpose. The histological diagnosis of Mycobacterium tuberculosis (MTB) remains a diagnostic challenge despite different methods. Immunohistochemistry (IHC) not only could confirm granulomatous tissue involvement but also can demonstrate MTB antigen immunolocalization. This study tries to clarify the details of immunohistochemical staining for MTB with pAbBCG. Materials/Methods. Twenty-three confirmed TB granulomatous tissue samples were studied by Ziehl-Neelsen and immunohistochemistry (IHC) staining with pAbBCG. Samples were selected from the archive of the Department of Pathology, National Research Institute of Tuberculosis and Lung Disease, Tehran, Iran. Results. IHC staining was positive in all samples, whereas Ziehl-Neelsen was positive in 9 cases out of 23 (39.1%). Tissue types used were pleural tissue, lymph nodes, and lung tissue. IHC showed positive coarse granular cytoplasmic and round, fragmented bacillary staining. In this study, epithelioid cells clearly showed more positive staining at the periphery of the granuloma rather than the center of granuloma. There is also positive staining in endothelial cells, fibroblasts, plasma cells, lymphocytes, and macrophages outside the granuloma. Conclusion. Considering the criteria of positive immunohistochemical staining of TB granulomatous reactions, this stain not only highlights the presence of mycobacterial antigens for tissue diagnosis, but also could morphologically localize its distribution in different cells.

Histopathological Findings in Immunohistological Staining of the Granulomatous Tissue Reaction Associated with Tuberculosis

PubMed Central

Karimi, Shirin; Pourabdollah, Mihan; Sadr, Makan; Karbasi, Mehrdad; Bahadori, Moslem

2014-01-01

Purpose. The histological diagnosis of Mycobacterium tuberculosis (MTB) remains a diagnostic challenge despite different methods. Immunohistochemistry (IHC) not only could confirm granulomatous tissue involvement but also can demonstrate MTB antigen immunolocalization. This study tries to clarify the details of immunohistochemical staining for MTB with pAbBCG. Materials/Methods. Twenty-three confirmed TB granulomatous tissue samples were studied by Ziehl-Neelsen and immunohistochemistry (IHC) staining with pAbBCG. Samples were selected from the archive of the Department of Pathology, National Research Institute of Tuberculosis and Lung Disease, Tehran, Iran. Results. IHC staining was positive in all samples, whereas Ziehl-Neelsen was positive in 9 cases out of 23 (39.1%). Tissue types used were pleural tissue, lymph nodes, and lung tissue. IHC showed positive coarse granular cytoplasmic and round, fragmented bacillary staining. In this study, epithelioid cells clearly showed more positive staining at the periphery of the granuloma rather than the center of granuloma. There is also positive staining in endothelial cells, fibroblasts, plasma cells, lymphocytes, and macrophages outside the granuloma. Conclusion. Considering the criteria of positive immunohistochemical staining of TB granulomatous reactions, this stain not only highlights the presence of mycobacterial antigens for tissue diagnosis, but also could morphologically localize its distribution in different cells. PMID:24511393

Dietary carotenoids in normal and pathological tissues of corpus uteri.

PubMed

Czeczuga-Semeniuk, Ewa; Wołczyński, Sławomir

2008-01-01

Carotenoids and retinyl esters are the source of vitamin A in the human body and its natural derivatives takes part in the regulation of cell replication and differentiation in the human endometrium, may induce the leiomyoma growth and has a role in differentiation of endometrial adenocarcinoma. The aim of the study was to demonstrate the presence of carotenoids in tissues from the normal uterus and from various tumors of the uterine corpus, as well as to compare the total content, major carotenoids and % of carotenoids belonging to the provitamin A group between the tissues examined. Using three independent methods of chromatography (CC, TLC, HPLC) we analysed 140 human samples. We identified 13 carotenoids belonging to the eg. provitamin A group and epoxy carotenoids. In all the samples beta-carotene, beta-cryptoxanthin, lutein, neoxanthin, violaxanthin and mutatoxanthin were isolated. In normal tissues, the mean carotenoid content was the highest in the follicular phase endometrium (9.9 microg/g), while the highest percentage of carotenoids belonging to provitamin A group was found in the luteal phase (18.2%). In the pathological group, the highest mean values were demonstrated for epithelial lesions (8.0 microg/g), and within this group - in endometrioid adenocarcinoma (10.8 microg/g). In both groups, violaxanthin, beta-cryptoxanthin, lutein epoxide and mutatoxanthin were the predominant carotenoids. We have demonstrated that all uterine tissues show a concentration of beta-carotene and beta-cryptoxanthin, being the source of vitamin A. The highest total values of carotenoids obtained in the group of endometrioid adenocarcinoma seem to confirm certain enzymatic defects in carotenoid metabolism in the course of the neoplastic process or some metabolic modifications. The finding of astaxanthin - the major antioxidant among carotenoids - in 63% of tissues examined is also significant.

Successful Application of Microarray Technology to Microdissected Formalin-Fixed, Paraffin-Embedded Tissue

PubMed Central

Coudry, Renata A.; Meireles, Sibele I.; Stoyanova, Radka; Cooper, Harry S.; Carpino, Alan; Wang, Xianqun; Engstrom, Paul F.; Clapper, Margie L.

2007-01-01

The establishment of a reliable method for using RNA from formalin-fixed, paraffin-embedded (FFPE) tissue would provide an opportunity to obtain novel gene expression data from the vast amounts of archived tissue. A custom-designed 22,000 oligonucleotide array was used in the present study to compare the gene expression profile of colonic epithelial cells isolated by laser capture microdissection from FFPE-archived samples with that of the same cell population from matched frozen samples, the preferred source of RNA. Total RNA was extracted from FFPE tissues, amplified, and labeled using the Paradise Reagent System. The quality of the input RNA was assessed by the Bioanalyzer profile, reverse transcriptase-polymerase chain reaction, and agarose gel electrophoresis. The results demonstrate that it is possible to obtain reliable microarray data from FFPE samples using RNA acquired by laser capture microdissection. The concordance between matched FFPE and frozen samples was evaluated and expressed as a Pearson’s correlation coefficient, with values ranging from 0.80 to 0.97. The presence of ribosomal RNA peaks in FFPE-derived RNA was reflected by a high correlation with paired frozen samples. A set of practical recommendations for evaluating the RNA integrity and quality in FFPE samples is reported. PMID:17251338

The Novel Application of Non-Lethal Citizen Science Tissue Sampling in Recreational Fisheries.

PubMed

Williams, Samuel M; Holmes, Bonnie J; Pepperell, Julian G

2015-01-01

Increasing fishing pressure and uncertainty surrounding recreational fishing catch and effort data promoted the development of alternative methods for conducting fisheries research. A pilot investigation was undertaken to engage the Australian game fishing community and promote the non-lethal collection of tissue samples from the black marlin Istiompax indica, a valuable recreational-only species in Australian waters, for the purpose of future genetic research. Recruitment of recreational anglers was achieved by publicizing the project in magazines, local newspapers, social media, blogs, websites and direct communication workshops at game fishing tournaments. The Game Fishing Association of Australia and the Queensland Game Fishing Association were also engaged to advertise the project and recruit participants with a focus on those anglers already involved in the tag-and-release of marlin. Participants of the program took small tissue samples using non-lethal methods which were stored for future genetic analysis. The program resulted in 165 samples from 49 participants across the known distribution of I. indica within Australian waters which was a sufficient number to facilitate a downstream population genetic analysis. The project demonstrated the potential for the development of citizen science sampling programs to collect tissue samples using non-lethal methods in order to achieve targeted research objects in recreationally caught species.

TU-FG-BRB-02: The Impact of Using Dual-Energy CT for Determining Proton Stopping Powers: Comparison Between Theory and Experiments

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

Baer, E; Jee, K; Zhang, R

Purpose: To evaluate the clinical performance of dual-energy CT (DECT) in determining proton stopping power ratios (SPR) and demonstrate advantages over conventional single-energy CT (SECT). Methods: SECT and DECT scans of tissue-equivalent plastics as well as animal meat samples are performed with a Siemens SOMATOM Definition Flash. The methods of Schneider et al. (1996) and Bourque et al. (2014) are used to determine proton SPR on SECT and DECT images, respectively. Waterequivalent path length (WEPL) measurements of plastics and tissue samples are performed with a 195 MeV proton beam. WEPL values are determined experimentally using the depth-dose shift and dosemore » extinction methods. Results: Comparison between CT-based and experimental WEPL is performed for 12 tissue-equivalent plastic as well as 6 meat boxes containing animal liver, kidney, heart, stomach, muscle and bones. For plastic materials, results show a systematic improvement in determining SPR with DECT, with a mean absolute error of 0.4% compared to 1.7% for SECT. For the meat samples, preliminary results show the ability for DECT to determine WEPL with a mean absolute value of 1.1% over all meat boxes. Conclusion: This work demonstrates the potential in using DECT for determining proton SPR with plastic materials in a clinical context. Further work is required to show the benefits of DECT for tissue samples. While experimental uncertainties could be a limiting factor to show the benefits of DECT over SECT for the meat samples, further work is required to adapt the DECT formalism in the context of clinical use, where noise and artifacts play an important role.« less

Vibrational Profiling of Brain Tumors and Cells

PubMed Central

Nelson, Sultan L; Proctor, Dustin T; Ghasemloonia, Ahmad; Lama, Sanju; Zareinia, Kourosh; Ahn, Younghee; Al-Saiedy, Mustafa R; Green, Francis HY; Amrein, Matthias W; Sutherland, Garnette R

2017-01-01

This study reports vibration profiles of neuronal cells and tissues as well as brain tumor and neocortical specimens. A contact-free method and analysis protocol was designed to convert an atomic force microscope into an ultra-sensitive microphone with capacity to record and listen to live biological samples. A frequency of 3.4 Hz was observed for both cultured rat hippocampal neurons and tissues and vibration could be modulated pharmacologically. Malignant astrocytoma tissue samples obtained from operating room, transported in artificial cerebrospinal fluid, and tested within an hour, vibrated with a much different frequency profile and amplitude, compared to meningioma or lateral temporal cortex providing a quantifiable measurement to accurately distinguish the three tissues in real-time. Vibration signals were converted to audible sound waves by frequency modulation, thus demonstrating, acoustic patterns unique to meningioma, malignant astrocytoma and neocortex. PMID:28744324

3D resolved mapping of optical aberrations in thick tissues

PubMed Central

Zeng, Jun; Mahou, Pierre; Schanne-Klein, Marie-Claire; Beaurepaire, Emmanuel; Débarre, Delphine

2012-01-01

We demonstrate a simple method for mapping optical aberrations with 3D resolution within thick samples. The method relies on the local measurement of the variation in image quality with externally applied aberrations. We discuss the accuracy of the method as a function of the signal strength and of the aberration amplitude and we derive the achievable resolution for the resulting measurements. We then report on measured 3D aberration maps in human skin biopsies and mouse brain slices. From these data, we analyse the consequences of tissue structure and refractive index distribution on aberrations and imaging depth in normal and cleared tissue samples. The aberration maps allow the estimation of the typical aplanetism region size over which aberrations can be uniformly corrected. This method and data pave the way towards efficient correction strategies for tissue imaging applications. PMID:22876353

Quantitative multiplex quantum dot in-situ hybridisation based gene expression profiling in tissue microarrays identifies prognostic genes in acute myeloid leukaemia

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

Tholouli, Eleni; MacDermott, Sarah; Hoyland, Judith

2012-08-24

Highlights: Black-Right-Pointing-Pointer Development of a quantitative high throughput in situ expression profiling method. Black-Right-Pointing-Pointer Application to a tissue microarray of 242 AML bone marrow samples. Black-Right-Pointing-Pointer Identification of HOXA4, HOXA9, Meis1 and DNMT3A as prognostic markers in AML. -- Abstract: Measurement and validation of microarray gene signatures in routine clinical samples is problematic and a rate limiting step in translational research. In order to facilitate measurement of microarray identified gene signatures in routine clinical tissue a novel method combining quantum dot based oligonucleotide in situ hybridisation (QD-ISH) and post-hybridisation spectral image analysis was used for multiplex in-situ transcript detection inmore » archival bone marrow trephine samples from patients with acute myeloid leukaemia (AML). Tissue-microarrays were prepared into which white cell pellets were spiked as a standard. Tissue microarrays were made using routinely processed bone marrow trephines from 242 patients with AML. QD-ISH was performed for six candidate prognostic genes using triplex QD-ISH for DNMT1, DNMT3A, DNMT3B, and for HOXA4, HOXA9, Meis1. Scrambled oligonucleotides were used to correct for background staining followed by normalisation of expression against the expression values for the white cell pellet standard. Survival analysis demonstrated that low expression of HOXA4 was associated with poorer overall survival (p = 0.009), whilst high expression of HOXA9 (p < 0.0001), Meis1 (p = 0.005) and DNMT3A (p = 0.04) were associated with early treatment failure. These results demonstrate application of a standardised, quantitative multiplex QD-ISH method for identification of prognostic markers in formalin-fixed paraffin-embedded clinical samples, facilitating measurement of gene expression signatures in routine clinical samples.« less

Combined spectroscopic imaging and chemometric approach for automatically partitioning tissue types in human prostate tissue biopsies

NASA Astrophysics Data System (ADS)

Haka, Abigail S.; Kidder, Linda H.; Lewis, E. Neil

2001-07-01

We have applied Fourier transform infrared (FTIR) spectroscopic imaging, coupling a mercury cadmium telluride (MCT) focal plane array detector (FPA) and a Michelson step scan interferometer, to the investigation of various states of malignant human prostate tissue. The MCT FPA used consists of 64x64 pixels, each 61 micrometers 2, and has a spectral range of 2-10.5 microns. Each imaging data set was collected at 16-1 resolution, resulting in 512 image planes and a total of 4096 interferograms. In this article we describe a method for separating different tissue types contained within FTIR spectroscopic imaging data sets of human prostate tissue biopsies. We present images, generated by the Fuzzy C-Means clustering algorithm, which demonstrate the successful partitioning of distinct tissue type domains. Additionally, analysis of differences in the centroid spectra corresponding to different tissue types provides an insight into their biochemical composition. Lastly, we demonstrate the ability to partition tissue type regions in a different data set using centroid spectra calculated from the original data set. This has implications for the use of the Fuzzy C-Means algorithm as an automated technique for the separation and examination of tissue domains in biopsy samples.

Three Dimensional Imaging of Paraffin Embedded Human Lung Tissue Samples by Micro-Computed Tomography

PubMed Central

Scott, Anna E.; Vasilescu, Dragos M.; Seal, Katherine A. D.; Keyes, Samuel D.; Mavrogordato, Mark N.; Hogg, James C.; Sinclair, Ian; Warner, Jane A.; Hackett, Tillie-Louise; Lackie, Peter M.

2015-01-01

Background Understanding the three-dimensional (3-D) micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT) has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffin embedded (FFPE) tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data. Methods FFPE human lung tissue samples (n = 4) were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm) was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging. Results The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15mm x 7mm). Resolution (voxel size 6.7 µm) in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections. Conclusion We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis. PMID:26030902

Near-infrared confocal micro-Raman spectroscopy combined with PCA-LDA multivariate analysis for detection of esophageal cancer

NASA Astrophysics Data System (ADS)

Chen, Long; Wang, Yue; Liu, Nenrong; Lin, Duo; Weng, Cuncheng; Zhang, Jixue; Zhu, Lihuan; Chen, Weisheng; Chen, Rong; Feng, Shangyuan

2013-06-01

The diagnostic capability of using tissue intrinsic micro-Raman signals to obtain biochemical information from human esophageal tissue is presented in this paper. Near-infrared micro-Raman spectroscopy combined with multivariate analysis was applied for discrimination of esophageal cancer tissue from normal tissue samples. Micro-Raman spectroscopy measurements were performed on 54 esophageal cancer tissues and 55 normal tissues in the 400-1750 cm-1 range. The mean Raman spectra showed significant differences between the two groups. Tentative assignments of the Raman bands in the measured tissue spectra suggested some changes in protein structure, a decrease in the relative amount of lactose, and increases in the percentages of tryptophan, collagen and phenylalanine content in esophageal cancer tissue as compared to those of a normal subject. The diagnostic algorithms based on principal component analysis (PCA) and linear discriminate analysis (LDA) achieved a diagnostic sensitivity of 87.0% and specificity of 70.9% for separating cancer from normal esophageal tissue samples. The result demonstrated that near-infrared micro-Raman spectroscopy combined with PCA-LDA analysis could be an effective and sensitive tool for identification of esophageal cancer.

Validation of a Plasma-Based Comprehensive Cancer Genotyping Assay Utilizing Orthogonal Tissue- and Plasma-Based Methodologies.

PubMed

Odegaard, Justin I; Vincent, John J; Mortimer, Stefanie; Vowles, James V; Ulrich, Bryan C; Banks, Kimberly C; Fairclough, Stephen R; Zill, Oliver A; Sikora, Marcin; Mokhtari, Reza; Abdueva, Diana; Nagy, Rebecca J; Lee, Christine E; Kiedrowski, Lesli A; Paweletz, Cloud P; Eltoukhy, Helmy; Lanman, Richard B; Chudova, Darya I; Talasaz, AmirAli

2018-04-24

Purpose: To analytically and clinically validate a circulating cell-free tumor DNA sequencing test for comprehensive tumor genotyping and demonstrate its clinical feasibility. Experimental Design: Analytic validation was conducted according to established principles and guidelines. Blood-to-blood clinical validation comprised blinded external comparison with clinical droplet digital PCR across 222 consecutive biomarker-positive clinical samples. Blood-to-tissue clinical validation comprised comparison of digital sequencing calls to those documented in the medical record of 543 consecutive lung cancer patients. Clinical experience was reported from 10,593 consecutive clinical samples. Results: Digital sequencing technology enabled variant detection down to 0.02% to 0.04% allelic fraction/2.12 copies with ≤0.3%/2.24-2.76 copies 95% limits of detection while maintaining high specificity [prevalence-adjusted positive predictive values (PPV) >98%]. Clinical validation using orthogonal plasma- and tissue-based clinical genotyping across >750 patients demonstrated high accuracy and specificity [positive percent agreement (PPAs) and negative percent agreement (NPAs) >99% and PPVs 92%-100%]. Clinical use in 10,593 advanced adult solid tumor patients demonstrated high feasibility (>99.6% technical success rate) and clinical sensitivity (85.9%), with high potential actionability (16.7% with FDA-approved on-label treatment options; 72.0% with treatment or trial recommendations), particularly in non-small cell lung cancer, where 34.5% of patient samples comprised a directly targetable standard-of-care biomarker. Conclusions: High concordance with orthogonal clinical plasma- and tissue-based genotyping methods supports the clinical accuracy of digital sequencing across all four types of targetable genomic alterations. Digital sequencing's clinical applicability is further supported by high rates of technical success and biomarker target discovery. Clin Cancer Res; 1-11. ©2018 AACR. ©2018 American Association for Cancer Research.

Thyroid peroxidase (TPO) expressed in thyroid and breast tissues shows similar antigenic properties.

PubMed

Godlewska, Marlena; Arczewska, Katarzyna D; Rudzińska, Magdalena; Łyczkowska, Anna; Krasuska, Wanda; Hanusek, Karolina; Ruf, Jean; Kiedrowski, Mirosław; Czarnocka, Barbara

2017-01-01

Thyroid peroxidase (TPO) is essential for physiological function of the thyroid gland. The high prevalence of thyroid peroxidase antibodies (TPOAbs) in patients with breast cancer and their protective role had previously been demonstrated, indicating a link between breast cancer and thyroid autoimmunity. Recently, TPO was shown to be present in breast cancer tissue samples but its antigenicity has not been analyzed. In this study, we investigated TPO expression levels in a series of fifty-six breast cancer samples paired with normal (peri-tumoral) tissue and its antigenic activity using a panel of well-characterized murine anti-human TPOAbs. We have shown that TPO transcripts were present in both normal and cancer tissue samples, although the amounts in the latter were reduced. Additionally, we observed that TPO levels are lower in more advanced cancers. TPO protein expression was confirmed in all tissue samples, both normal and cancerous. We also found that the antigenicity of the immunodominant regions (IDRs) in breast TPO resembles that of thyroid TPO, which is crucial for effective interactions with human TPOAbs. Expression of TPO in breast cancer together with its antigenic activity may have beneficial effects in TPOAb-positive breast cancer patients. However, further studies are needed to confirm the beneficial role of TPOAbs and to better understand the underlying mechanism.

Detection of Nanophyetus salmincola in water, snails, and fish tissues by quantitative polymerase chain reaction

USGS Publications Warehouse

Purcell, Maureen K.; Powers, Rachel L.; Besijn, Bonnie; Hershberger, Paul K.

2017-01-01

We report the development and validation of two quantitative PCR (qPCR) assays to detect Nanophyetus salmincola DNA in water samples and in fish and snail tissues. Analytical and diagnostic validation demonstrated good sensitivity, specificity, and repeatability of both qPCR assays. The N. salmincola DNA copy number in kidney tissue was significantly correlated with metacercaria counts based on microscopy. Extraction methods were optimized for the sensitive qPCR detection of N. salmincola DNA in settled water samples. Artificially spiked samples suggested that the 1-cercaria/L threshold corresponded to an estimated log10 copies per liter ≥ 6.0. Significant correlation of DNA copy number per liter and microscopic counts indicated that the estimated qPCR copy number was a good predictor of the number of waterborne cercariae. However, the detection of real-world samples below the estimated 1-cercaria/L threshold suggests that the assays may also detect other N. salmincola life stages, nonintact cercariae, or free DNA that settles with the debris. In summary, the qPCR assays reported here are suitable for identifying and quantifying all life stages of N. salmincola that occur in fish tissues, snail tissues, and water.

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.

Mechanical Barriers Restrict Invasion of Herpes Simplex Virus 1 into Human Oral Mucosa

PubMed Central

Thier, Katharina; Petermann, Philipp; Rahn, Elena; Rothamel, Daniel; Bloch, Wilhelm

2017-01-01

ABSTRACT Oral mucosa is one of the main target tissues of the human pathogen herpes simplex virus 1 (HSV-1). How the virus overcomes the protective epithelial barriers and penetrates the tissue to reach its receptors and initiate infection is still unclear. Here, we established an ex vivo infection assay with human oral mucosa that allows viral entry studies in a natural target tissue. The focus was on the susceptibility of keratinocytes in the epithelium and the characterization of cellular receptors that mediate viral entry. Upon ex vivo infection of gingiva or vestibular mucosa, we observed that intact human mucosa samples were protected from viral invasion. In contrast, the basal layer of the oral epithelium was efficiently invaded once the connective tissue and the basement membrane were removed. Later during infection, HSV-1 spread from basal keratinocytes to upper layers, demonstrating the susceptibility of the stratified squamous epithelium to HSV-1. The analysis of potential receptors revealed nectin-1 on most mucosal keratinocytes, whereas herpesvirus entry mediator (HVEM) was found only on a subpopulation of cells, suggesting that nectin-1 acts as primary receptor for HSV-1 in human oral mucosa. To mimic the supposed entry route of HSV-1 via microlesions in vivo, we mechanically wounded the mucosa prior to infection. While we observed a limited number of infected keratinocytes in some wounded mucosa samples, other samples showed no infected cells. Thus, we conclude that mechanical wounding of mucosa is insufficient for the virus to efficiently overcome epithelial barriers and to make entry-mediating receptors accessible. IMPORTANCE To invade the target tissue of its human host during primary infection, herpes simplex virus (HSV) must overcome the epithelial barriers of mucosa, skin, or cornea. For most viruses, the mechanisms underlying the invasion into the target tissues of their host organism are still open. Here, we established an ex vivo infection model of human oral mucosa to explore how HSV can enter its target tissue. Our results demonstrate that intact mucosa samples and even compromised tissue allow only very limited access of HSV to keratinocytes. Detailed understanding of barrier functions is an essential precondition to unravel how HSV bypasses the barriers and approaches its receptors in tissue and why it is beneficial for the virus to use a cell-cell adhesion molecule, such as nectin-1, as a receptor. PMID:28878080

Mechanical Barriers Restrict Invasion of Herpes Simplex Virus 1 into Human Oral Mucosa.

PubMed

Thier, Katharina; Petermann, Philipp; Rahn, Elena; Rothamel, Daniel; Bloch, Wilhelm; Knebel-Mörsdorf, Dagmar

2017-11-15

Oral mucosa is one of the main target tissues of the human pathogen herpes simplex virus 1 (HSV-1). How the virus overcomes the protective epithelial barriers and penetrates the tissue to reach its receptors and initiate infection is still unclear. Here, we established an ex vivo infection assay with human oral mucosa that allows viral entry studies in a natural target tissue. The focus was on the susceptibility of keratinocytes in the epithelium and the characterization of cellular receptors that mediate viral entry. Upon ex vivo infection of gingiva or vestibular mucosa, we observed that intact human mucosa samples were protected from viral invasion. In contrast, the basal layer of the oral epithelium was efficiently invaded once the connective tissue and the basement membrane were removed. Later during infection, HSV-1 spread from basal keratinocytes to upper layers, demonstrating the susceptibility of the stratified squamous epithelium to HSV-1. The analysis of potential receptors revealed nectin-1 on most mucosal keratinocytes, whereas herpesvirus entry mediator (HVEM) was found only on a subpopulation of cells, suggesting that nectin-1 acts as primary receptor for HSV-1 in human oral mucosa. To mimic the supposed entry route of HSV-1 via microlesions in vivo , we mechanically wounded the mucosa prior to infection. While we observed a limited number of infected keratinocytes in some wounded mucosa samples, other samples showed no infected cells. Thus, we conclude that mechanical wounding of mucosa is insufficient for the virus to efficiently overcome epithelial barriers and to make entry-mediating receptors accessible. IMPORTANCE To invade the target tissue of its human host during primary infection, herpes simplex virus (HSV) must overcome the epithelial barriers of mucosa, skin, or cornea. For most viruses, the mechanisms underlying the invasion into the target tissues of their host organism are still open. Here, we established an ex vivo infection model of human oral mucosa to explore how HSV can enter its target tissue. Our results demonstrate that intact mucosa samples and even compromised tissue allow only very limited access of HSV to keratinocytes. Detailed understanding of barrier functions is an essential precondition to unravel how HSV bypasses the barriers and approaches its receptors in tissue and why it is beneficial for the virus to use a cell-cell adhesion molecule, such as nectin-1, as a receptor. Copyright © 2017 American Society for Microbiology.

The relationship between decorrelation time and sample thickness in acute rat brain tissue slices (Conference Presentation)

NASA Astrophysics Data System (ADS)

Brake, Joshua; Jang, Mooseok; Yang, Changhuei

2016-03-01

The optical opacity of biological tissue has long been a challenge in biomedical optics due to the strong scattering nature of tissue in the optical regime. While most conventional optical techniques attempt to gate out multiply scattered light and use only unscattered light, new approaches in the field of wavefront shaping exploit the time reversible symmetry of optical scattering in order to focus light inside or through scattering media. While these approaches have been demonstrated effectively on static samples, it has proven difficult to apply them to dynamic biological samples since even small changes in the relative positions of the scatterers within will cause the time symmetry that wavefront shaping relies upon to decorrelate. In this paper we investigate the decorrelation curves of acute rat brain slices for thicknesses in the range 1-3 mm (1/e decorrelation time on the order of seconds) using multi-speckle diffusing wave spectroscopy (MSDWS) and compare the results with theoretical predictions. The results of this study demonstrate that the 1/L^2 relationship between decorrelation time and thickness predicted by diffusing wave spectroscopy provides a good rule of thumb for estimating how the decorrelation of a sample will change with increasing thickness. Understanding this relationship will provide insight to guide the future development of biophotonic wavefront shaping tools by giving an estimate of how fast wavefront shaping systems need to operate to overcome the dynamic nature of biological samples.

Substrate-Mediated Laser Ablation under Ambient Conditions for Spatially-Resolved Tissue Proteomics

PubMed Central

Fatou, Benoit; Wisztorski, Maxence; Focsa, Cristian; Salzet, Michel; Ziskind, Michael; Fournier, Isabelle

2015-01-01

Numerous applications of ambient Mass Spectrometry (MS) have been demonstrated over the past decade. They promoted the emergence of various micro-sampling techniques such as Laser Ablation/Droplet Capture (LADC). LADC consists in the ablation of analytes from a surface and their subsequent capture in a solvent droplet which can then be analyzed by MS. LADC is thus generally performed in the UV or IR range, using a wavelength at which analytes or the matrix absorb. In this work, we explore the potential of visible range LADC (532 nm) as a micro-sampling technology for large-scale proteomics analyses. We demonstrate that biomolecule analyses using 532 nm LADC are possible, despite the low absorbance of biomolecules at this wavelength. This is due to the preponderance of an indirect substrate-mediated ablation mechanism at low laser energy which contrasts with the conventional direct ablation driven by sample absorption. Using our custom LADC system and taking advantage of this substrate-mediated ablation mechanism, we were able to perform large-scale proteomic analyses of micro-sampled tissue sections and demonstrated the possible identification of proteins with relevant biological functions. Consequently, the 532 nm LADC technique offers a new tool for biological and clinical applications. PMID:26674367

Diagnosis of duck plague in waterfowl by polymerase chain reaction

USGS Publications Warehouse

Hansen, W.R.; Nashold, S.W.; Docherty, D.E.; Brown, S.E.; Knudson, D.L.

2000-01-01

A recently developed polymerase chain reaction (PCR) assay was used for diagnosis of duck plague in waterfowl tissues from past and current cases of waterfowl mortality and to identify duck plague virus in combined cloacal/oral-pharyngeal swab samples from healthy mallards (Anas platyrhynchos) after a disease outbreak. The PCR was able to detect viral DNA from all the individual or pooled tissues assayed from 10 waterfowl, including liver and spleen samples from three Muscovy ducks (Cairina moschata domesticus) that did not yield virus isolates. The strong staining intensity of the PCR products from the waterfowl tissues indicated that large amounts of virus were present, even when virus was not isolated. Duck plague DNA was also detected in a cloacal swab sample from a wood duck (Aix sponsa) carcass submitted for diagnosis. The PCR assay identified duck plague DNA in 13 swab samples that produced virus isolates from carrier mallards sampled in 1981 after a duck plague die-off. The duck plague PCR clearly demonstrated the ability to quickly diagnose duck plague in suspect mortality cases and to detect virus shed by carrier waterfowl.

Primary cilia are increased in number and demonstrate structural abnormalities in human cancer.

PubMed

Yasar, Binnaz; Linton, Kim; Slater, Christian; Byers, Richard

2017-07-01

Primary cilia play an important role in the regulation of cell signalling pathways and are thought to have a role in cancer but have seldom been studied in human cancer samples. Primary cilia were visualised by dual immunofluorescence for anti-CROCC (ciliary rootlet coiled-coil) and anti-tubulin in a range of human cancers (including carcinomas of stomach, pancreas, prostate, lung and colon, lobular and ductal breast cancers and follicular lymphoma) and in matched normal tissue (stomach, pancreas, lung, large and small intestines, breast and reactive lymph nodes) samples using a tissue microarray; their frequency, association with proliferation, was measured by Ki-67 staining and their structure was analysed. Compared with normal tissues, primary cilia frequency was significantly elevated in adenocarcinoma of the lung (2.75% vs 1.85%, p=0.016), adenocarcinoma of the colon (3.80% vs 2.43%, respectively, p=0.017), follicular lymphoma (1.18% vs 0.83%, p=0.003) and pancreatic adenocarcinoma (7.00% vs 5.26%, p=0.002); there was no statistically significant difference compared with normal control tissue for gastric and prostatic adenocarcinomas or for lobular and ductal breast cancers. Additionally, structural abnormalities of primary cilia were identified in cancer tissues, including elongation of the axoneme, multiple basal bodies and branching of the axoneme. Ki-67 scores ranged from 0.7% to 78.4% and showed no statistically significant correlation with primary cilia frequency across all tissues (p=0.1501). The results show upregulation of primary cilia and the presence of structural defects in a wide range of human cancer tissue samples demonstrating association of dysregulation of primary cilia with human cancer. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

The Hit and Away technique: optimal usage of the ultrasonic scalpel in laparoscopic gastrectomy.

PubMed

Irino, Tomoyuki; Hiki, Naoki; Ohashi, Manabu; Nunobe, Souya; Sano, Takeshi; Yamaguchi, Toshiharu

2016-01-01

Thermal injury and unexpected bleeding caused by ultrasonic scalpels can lead to fatal complications in laparoscopic gastrectomy (LG), such as postoperative pancreatic fistulas (POPF). In this study, we developed the "Hit and Away" protocol for optimal usage of the ultrasonic scalpel, which in essence involves dividing tissues and vessels in batches using the tip of the scalpel to control tissue temperature. To assess the effectiveness of the technique, the surface temperature of the mesocolon of female swine after ultrasonic scalpel activations was measured, and tissue samples were collected to evaluate microscopic thermal injury to the pancreas. In parallel, we retrospectively surveyed 216 patients who had undergone LG before or after the introduction of this technique and assessed the ability of this technique to reduce POPF. The tissue temperature of the swine mesocolon reached 43 °C, a temperature at which adipose tissue melted but fibrous tissue, including vessels, remained intact. The temperature returned to baseline within 3 s of turning off the ultrasonic scalpel, demonstrating the advantage of using ultrasonic scalpel in a pulsatile manner. Tissue samples from the pancreas demonstrated that the extent of thermal injury post-procedure was limited to the capsule of the pancreas. Moreover, with respect to the clinical outcomes before and after the introduction of this technique, POPF incidence decreased significantly from 7.8 to 1.0% (p = 0.021). The "Hit and Away" technique can reduce blood loss and thermal injury to the pancreas and help to ensure the safety of lymph node dissection in LG.

Untargeted UPLC-MS Profiling Pipeline to Expand Tissue Metabolome Coverage: Application to Cardiovascular Disease

PubMed Central

2015-01-01

Metabolic profiling studies aim to achieve broad metabolome coverage in specific biological samples. However, wide metabolome coverage has proven difficult to achieve, mostly because of the diverse physicochemical properties of small molecules, obligating analysts to seek multiplatform and multimethod approaches. Challenges are even greater when it comes to applications to tissue samples, where tissue lysis and metabolite extraction can induce significant systematic variation in composition. We have developed a pipeline for obtaining the aqueous and organic compounds from diseased arterial tissue using two consecutive extractions, followed by a different untargeted UPLC-MS analysis method for each extract. Methods were rationally chosen and optimized to address the different physicochemical properties of each extract: hydrophilic interaction liquid chromatography (HILIC) for the aqueous extract and reversed-phase chromatography for the organic. This pipeline can be generic for tissue analysis as demonstrated by applications to different tissue types. The experimental setup and fast turnaround time of the two methods contributed toward obtaining highly reproducible features with exceptional chromatographic performance (CV % < 0.5%), making this pipeline suitable for metabolic profiling applications. We structurally assigned 226 metabolites from a range of chemical classes (e.g., carnitines, α-amino acids, purines, pyrimidines, phospholipids, sphingolipids, free fatty acids, and glycerolipids) which were mapped to their corresponding pathways, biological functions and known disease mechanisms. The combination of the two untargeted UPLC-MS methods showed high metabolite complementarity. We demonstrate the application of this pipeline to cardiovascular disease, where we show that the analyzed diseased groups (n = 120) of arterial tissue could be distinguished based on their metabolic profiles. PMID:25664760

Inter-comparison of boron concentration measurements at INFN-University of Pavia (Italy) and CNEA (Argentina).

PubMed

Portu, Agustina; Postuma, Ian; Gadan, Mario Alberto; Saint Martin, Gisela; Olivera, María Silvina; Altieri, Saverio; Protti, Nicoletta; Bortolussi, Silva

2015-11-01

An inter-comparison of three boron determination techniques was carried out between laboratories from INFN-University of Pavia (Italy) and CNEA (Argentina): alpha spectrometry (alpha-spect), neutron capture radiography (NCR) and quantitative autoradiography (QTA). Samples of different nature were analysed: liquid standards, liver homogenates and tissue samples from different treatment protocols. The techniques showed a good agreement in a concentration range of interest in BNCT (1-100ppm), thus demonstrating their applicability as precise methods to quantify boron and determine its distribution in tissues. Copyright © 2015 Elsevier Ltd. All rights reserved.

Whole Transcriptome Sequencing Enables Discovery and Analysis of Viruses in Archived Primary Central Nervous System Lymphomas

PubMed Central

DeBoever, Christopher; Reid, Erin G.; Smith, Erin N.; Wang, Xiaoyun; Dumaop, Wilmar; Harismendy, Olivier; Carson, Dennis; Richman, Douglas; Masliah, Eliezer; Frazer, Kelly A.

2013-01-01

Primary central nervous system lymphomas (PCNSL) have a dramatically increased prevalence among persons living with AIDS and are known to be associated with human Epstein Barr virus (EBV) infection. Previous work suggests that in some cases, co-infection with other viruses may be important for PCNSL pathogenesis. Viral transcription in tumor samples can be measured using next generation transcriptome sequencing. We demonstrate the ability of transcriptome sequencing to identify viruses, characterize viral expression, and identify viral variants by sequencing four archived AIDS-related PCNSL tissue samples and analyzing raw sequencing reads. EBV was detected in all four PCNSL samples and cytomegalovirus (CMV), JC polyomavirus (JCV), and HIV were also discovered, consistent with clinical diagnoses. CMV was found to express three long non-coding RNAs recently reported as expressed during active infection. Single nucleotide variants were observed in each of the viruses observed and three indels were found in CMV. No viruses were found in several control tumor types including 32 diffuse large B-cell lymphoma samples. This study demonstrates the ability of next generation transcriptome sequencing to accurately identify viruses, including DNA viruses, in solid human cancer tissue samples. PMID:24023918

Mathematical modeling of the malignancy of cancer using graph evolution.

PubMed

Gunduz-Demir, Cigdem

2007-10-01

We report a novel computational method based on graph evolution process to model the malignancy of brain cancer called glioma. In this work, we analyze the phases that a graph passes through during its evolution and demonstrate strong relation between the malignancy of cancer and the phase of its graph. From the photomicrographs of tissues, which are diagnosed as normal, low-grade cancerous and high-grade cancerous, we construct cell-graphs based on the locations of cells; we probabilistically generate an edge between every pair of cells depending on the Euclidean distance between them. For a cell-graph, we extract connectivity information including the properties of its connected components in order to analyze the phase of the cell-graph. Working with brain tissue samples surgically removed from 12 patients, we demonstrate that cell-graphs generated for different tissue types evolve differently and that they exhibit different phase properties, which distinguish a tissue type from another.

Selenium and mining in the Powder River Basin, Wyoming: Phase III - a preliminary survey of selenium concentrations in deer mice (Peromyscus maniculatus) livers

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

Raisbeck, M.L.; Vance, G.F.; Steward, D.G.

1995-09-01

Samples of liver tissue from deer mice trapped on not-yet-mined areas and reclaimed areas at five surface coal mines in the Powder River Basin of northeastern Wyoming were analyzed for selenium. The overall mean concentration of selenium in wet weight liver tissue was 1.685 ppm. The mean value from not-yet-mined areas was 1.437 ppm; the mean value from reclaimed areas was 1.910 ppm (significant at p<0.1016). When one not-yet-mined outlier was removed, significance rose to p<0.0004. Mine-to-mine comparison of samples stratified by type (that is, by not-yet-mined or reclaimed), showed average tissue concentrations from the reclaimed area of Mine 1more » were also higher (p<0.0143) then not-yet-mined area samples at Mine 1. No statistically significant differences were found between mines for samples from not-yet-mined areas, and no statistically significant differences were found between Mines 2, 3, 4, and 5 for samples from reclaimed areas. Multiple analysis of variance using the factors: site (mine) and type (not-yet-mined or reclaimed) was not significantly significant (p<0.2115). Simple linear regression showed that selenium concentrations in dry tissue could easily be predicted from wet tissue selenium (r2=0.9775), demonstrating that percent water in the samples was relatively constant. Animal body weight in general was not a predictor for either wet or dry tissue selenium concentrations, but was related to body weight at the higher tissue concentrations of selenium encountered in samples from the reclaimed area at Mine 1. Mouse body weights at Mine 1 were higher on the reclaimed area than mouse body weights from the not-yet-mined area.« less

The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens.

PubMed

Kim, Dong-Young; Sato, Ayuko; Fukuyama, Satoshi; Sagara, Hiroshi; Nagatake, Takahiro; Kong, Il Gyu; Goda, Kaoru; Nochi, Tomonori; Kunisawa, Jun; Sato, Shintaro; Yokota, Yoshifumi; Lee, Chul Hee; Kiyono, Hiroshi

2011-04-01

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2(+/-) mice. The titers of Ag-specific Abs were as high in Id2(-/-) mice as in Id2(+/-) mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.

Deep tissue optical focusing and optogenetic modulation with time-reversed ultrasonically encoded light

PubMed Central

Ruan, Haowen; Brake, Joshua; Robinson, J. Elliott; Liu, Yan; Jang, Mooseok; Xiao, Cheng; Zhou, Chunyi; Gradinaru, Viviana; Yang, Changhuei

2017-01-01

Noninvasive light focusing deep inside living biological tissue has long been a goal in biomedical optics. However, the optical scattering of biological tissue prevents conventional optical systems from tightly focusing visible light beyond several hundred micrometers. The recently developed wavefront shaping technique time-reversed ultrasonically encoded (TRUE) focusing enables noninvasive light delivery to targeted locations beyond the optical diffusion limit. However, until now, TRUE focusing has only been demonstrated inside nonliving tissue samples. We present the first example of TRUE focusing in 2-mm-thick living brain tissue and demonstrate its application for optogenetic modulation of neural activity in 800-μm-thick acute mouse brain slices at a wavelength of 532 nm. We found that TRUE focusing enabled precise control of neuron firing and increased the spatial resolution of neuronal excitation fourfold when compared to conventional lens focusing. This work is an important step in the application of TRUE focusing for practical biomedical uses. PMID:29226248

Confocal multispot microscope for fast and deep imaging in semicleared tissues

NASA Astrophysics Data System (ADS)

Adam, Marie-Pierre; Müllenbroich, Marie Caroline; Di Giovanna, Antonino Paolo; Alfieri, Domenico; Silvestri, Ludovico; Sacconi, Leonardo; Pavone, Francesco Saverio

2018-02-01

Although perfectly transparent specimens are imaged faster with light-sheet microscopy, less transparent samples are often imaged with two-photon microscopy leveraging its robustness to scattering; however, at the price of increased acquisition times. Clearing methods that are capable of rendering strongly scattering samples such as brain tissue perfectly transparent specimens are often complex, costly, and time intensive, even though for many applications a slightly lower level of tissue transparency is sufficient and easily achieved with simpler and faster methods. Here, we present a microscope type that has been geared toward the imaging of semicleared tissue by combining multispot two-photon excitation with rolling shutter wide-field detection to image deep and fast inside semicleared mouse brain. We present a theoretical and experimental evaluation of the point spread function and contrast as a function of shutter size. Finally, we demonstrate microscope performance in fixed brain slices by imaging dendritic spines up to 400-μm deep.

Reduction of Brochothrix thermosphacta on beef surfaces following immobilization of nisin in calcium alginate gels.

PubMed

Cutter, C N; Siragusa, G R

1996-07-01

Lean and adipose beef carcass tissues inoculated with Brochothrix thermosphacta (BT) (approx. 4.50 log10 cfu cm-2) were left untreated (U) or treated with 100 micrograms ml-1 nisin (N), calcium alginate (A) or 100 micrograms ml-1 nisin immobilized in a calcium alginate gel (AN). Tissue samples were refrigerated after treatments and bacterial populations and nisin activity were determined at 0, 1, 2 and 7 d. U, A and N treatments of lean and adipose tissues did not suppress bacterial growth ( > 6 log10 cfu cm-2 by day 7) while treatments of lean and adipose tissues with AN suppressed bacteria ( > 2.42 log10 cfu cm-2 by day 7). Bacteriocin titres from both tissues were higher in AN vs N samples after the 7 d incubation. This study demonstrates that immobilization of nisin in a gel may be a more effective delivery system of a bacteriocin to the carcass surface than direct application.

Photoacoustic-based sO2 estimation through excised bovine prostate tissue with interstitial light delivery.

PubMed

Mitcham, Trevor; Taghavi, Houra; Long, James; Wood, Cayla; Fuentes, David; Stefan, Wolfgang; Ward, John; Bouchard, Richard

2017-09-01

Photoacoustic (PA) imaging is capable of probing blood oxygen saturation (sO 2 ), which has been shown to correlate with tissue hypoxia, a promising cancer biomarker. However, wavelength-dependent local fluence changes can compromise sO 2 estimation accuracy in tissue. This work investigates using PA imaging with interstitial irradiation and local fluence correction to assess precision and accuracy of sO 2 estimation of blood samples through ex vivo bovine prostate tissue ranging from 14% to 100% sO 2 . Study results for bovine blood samples at distances up to 20 mm from the irradiation source show that local fluence correction improved average sO 2 estimation error from 16.8% to 3.2% and maintained an average precision of 2.3% when compared to matched CO-oximeter sO 2 measurements. This work demonstrates the potential for future clinical translation of using fluence-corrected and interstitially driven PA imaging to accurately and precisely assess sO 2 at depth in tissue with high resolution.

Cancer testis antigen OY-TES-1: analysis of protein expression in ovarian cancer with tissue microarrays.

PubMed

Fan, R; Huang, W; Luo, B; Zhang, Q M; Xiao, S W; Xie, X X

2015-01-01

Revised manuscript accepted for publication March 5, Objectives: The purpose of this study was to determine the potential of cancer testis antigen OY-TES-1 as a vaccine for ovarian cancer (OC). A tissue microarray (TMA) containing 107 samples from OC tissues and 48 samples from OC adjacent tissues was analyzed by immunohistochemistry with the OY-TES-1 polyclonal antibody. The correlation between OY-TES-1 and clinic pathological traits of OC was statistically analyzed. The expression of OY-TES-1 protein was found in 81% (87/107) of OC tissues and 56% (27/48) of OC adjacent tissues. The immunostaining intensity of OY-TES-1 in OC tissues was significantly higher than that in OC adjacent tissues tested (p = 0.040). OC adjacent tissues only demonstrated lower immunostaining intensity, whereas some of OC tissues presented higher immunostaining intensity and majority showed the heterogeneity of protein distribution. There was no statistically significant correlation found between OY-TES-1 expression and any other clinicopathological traits such as age, FIGO stage, pathological grade, and histological type. OY-TES-1 was expressed in OC tissues with a high proportion, and some of OC tissues presented OY-TES-1 expression in high level vs OC adjacent tissues. OY-TES-1 could be an attractive target for immunotherapy for OC in the future.

Differential expression of glucose transporters in normal and pathologic thyroid tissue.

PubMed

Matsuzu, Kenichi; Segade, Fernando; Matsuzu, Utako; Carter, Aaron; Bowden, Donald W; Perrier, Nancy D

2004-10-01

Malignant cells demonstrate increased glucose uptake and utilization. Immunohistochemical studies have suggested that enhanced glucose uptake in cancer cells may be caused by the overexpression of glucose transporters (GLUTs), in most cases GLUT1 and/or GLUT3. The aim of this study was to examine in detail the expression pattern and levels of GLUT genes in normal and pathologic thyroid tissues and to evaluate the clinical significance of GLUT mRNA levels. One hundred fifty-two surgically resected thyroid tissue samples from 103 patients were evaluated. Samples included: normal thyroid tissue (n = 58), benign thyroid disease (n = 61), and thyroid carcinoma (n = 33). Expression of the GLUT1, GLUT2, GLUT3, GLUT4, and GLUT10 genes were examined by reverse transcription-polymerase chain reaction (RT-PCR) and mRNA levels were quantitated by real-time RT-PCR. All thyroid parenchymal cells expressed GLUT1, GLUT3, GLUT4, and GLUT10. GLUT1 showed increased expression in carcinoma cases (p < 0.0001) and also in comparison with paired normal tissue samples from the same patient (p < 0.0001). Other GLUTs were statistically unchanged in pathologic tissues. These results are consistent with the theory that GLUT1 is upregulated during carcinogenesis and may play a major role in enhanced glucose uptake in thyroid cancer cells.

Investigation of the effect of hydration on dermal collagen in ex vivo human skin tissue using second harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Samatham, Ravikant; Wang, Nicholas K.; Jacques, Steven L.

2016-02-01

Effect of hydration on the dermal collagen structure in human skin was investigated using second harmonic generation microscopy. Dog ears from the Mohs micrographic surgery department were procured for the study. Skin samples with subject aged between 58-90 years old were used in the study. Three dimensional Multiphoton (Two-photon and backward SHG) control data was acquired from the skin samples. After the control measurement, the skin tissue was either soaked in deionized water for 2 hours (Hydration) or kept at room temperature for 2 hours (Desiccation), and SHG data was acquired. The data was normalized for changes in laser power and detector gain. The collagen signal per unit volume from the dermis was calculated. The desiccated skin tissue gave higher backward SHG compared to respective control tissue, while hydration sample gave a lower backward SHG. The collagen signal decreased with increase in hydration of the dermal collagen. Hydration affected the packing of the collagen fibrils causing a change in the backward SHG signal. In this study, the use of multiphoton microscopy to study the effect of hydration on dermal structure was demonstrated in ex vivo tissue.

Epithelial ovarian carcinoma diagnosis by desorption electrospray ionization mass spectrometry imaging

PubMed Central

Dória, Maria Luisa; McKenzie, James S.; Mroz, Anna; Phelps, David L.; Speller, Abigail; Rosini, Francesca; Strittmatter, Nicole; Golf, Ottmar; Veselkov, Kirill; Brown, Robert; Ghaem-Maghami, Sadaf; Takats, Zoltan

2016-01-01

Ovarian cancer is highly prevalent among European women, and is the leading cause of gynaecological cancer death. Current histopathological diagnoses of tumour severity are based on interpretation of, for example, immunohistochemical staining. Desorption electrospray mass spectrometry imaging (DESI-MSI) generates spatially resolved metabolic profiles of tissues and supports an objective investigation of tumour biology. In this study, various ovarian tissue types were analysed by DESI-MSI and co-registered with their corresponding haematoxylin and eosin (H&E) stained images. The mass spectral data reveal tissue type-dependent lipid profiles which are consistent across the n = 110 samples (n = 107 patients) used in this study. Multivariate statistical methods were used to classify samples and identify molecular features discriminating between tissue types. Three main groups of samples (epithelial ovarian carcinoma, borderline ovarian tumours, normal ovarian stroma) were compared as were the carcinoma histotypes (serous, endometrioid, clear cell). Classification rates >84% were achieved for all analyses, and variables differing statistically between groups were determined and putatively identified. The changes noted in various lipid types help to provide a context in terms of tumour biochemistry. The classification of unseen samples demonstrates the capability of DESI-MSI to characterise ovarian samples and to overcome existing limitations in classical histopathology. PMID:27976698

Immunohistochemical evaluation of neuroreceptors in healthy and pathological temporo-mandibular joint.

PubMed

Favia, Gianfranco; Corsalini, Massimo; Di Venere, Daniela; Pettini, Francesco; Favia, Giorgio; Capodiferro, Saverio; Maiorano, Eugenio

2013-01-01

A study was performed on the articular disk and periarticular tissues of the temporo-mandibular joint (TMJ) with immunohistochemical techniques to give evidence to the presence of neuroreceptors (NRec) in these sites. The study was carried out on tissue samples obtained from 10 subjects without TMJ disease and from 7 patients with severe TMJ arthritis and arthrosis. We use antibodies directed against following antigens: Gliofibrillary Acidic Protein (GFAP), Leu-7, Myelin Basic Protein (MBP), Neurofilaments 68 kD (NF), Neuron Specific Enolase (NSE), S-100 protein (S-100) and Synaptophysin (SYN). This study revealed that Ruffini's-like, Pacini's-like and Golgi's-like receptors can be demonstrated in TMJ periarticular tissues and that free nervous endings are present in the subsynovial tissues but not within the articular disk. We observed elongated cytoplamic processes of chondrocytes that demonstrated strong S-100 immunoreactivity but they were unreactive with all other antibodies. These cytoplamic processes were more abundant and thicker in the samples obtained from patients with disease TMJ. The results of this study confirm that different Nrec are detectable in TMJ periarticular tissues but they are absent within the articular disk. In the latter site, only condrocytic processes are evident, especially in diseased TMJ, and they might have been confused with nervous endings in previous morphological studies. Nevertheless the absence of immunoreactivity for NF, NSE and SYN proves that they are not of neural origin.

Bioelectrical Impedance and The Frequency Dependent Current Conduction Through Biological Tissues: A Short Review

NASA Astrophysics Data System (ADS)

Kanti Bera, Tushar

2018-03-01

Biological tissues are developed with biological cells which exhibit complex electrical impedance called electrical bioimpedance. Under an alternating electrical excitation the bioimpedance varies with the tissue anatomy, composition and the signal frequency. The current penetration and conduction paths vary with frequency of the applied signal. Bioimpedance spectroscopy is used to study the frequency response of the electrical impedance of biological materials noninvasively. In bioimpedance spectroscopy, a low amplitude electrical signal is injected to the tissue sample or body parts to characterization the sample in terms of its bioimpedance. The electrical current conduction phenomena, which is highly influenced by the tissue impedance and the signal frequency, is an important phenomena which should be studied to understand the bioimpedance techniques like bioelectrical impedance analysis (BIA), EIS, or else. In this paper the origin of bioelectrical impedance and current conduction phenomena has been reviewed to present a brief summary of bioelectrical impedance and the frequency dependent current conduction through biological tissues. Simulation studies are conducted with alternation current injection through a two dimensional model of biological tissues containing finite number of biological cells suspended in extracellular fluid. The paper demonstrates the simulation of alternating current conduction through biological tissues conducted by COMSOL Multiphysics. Simulation studies also show the frequency response of the tissue impedance for different tissue compositions.

A device for high-throughput monitoring of degradation in soft tissue samples.

PubMed

Tzeranis, D S; Panagiotopoulos, I; Gkouma, S; Kanakaris, G; Georgiou, N; Vaindirlis, N; Vasileiou, G; Neidlin, M; Gkousioudi, A; Spitas, V; Macheras, G A; Alexopoulos, L G

2018-06-06

This work describes the design and validation of a novel device, the High-Throughput Degradation Monitoring Device (HDD), for monitoring the degradation of 24 soft tissue samples over incubation periods of several days inside a cell culture incubator. The device quantifies sample degradation by monitoring its deformation induced by a static gravity load. Initial instrument design and experimental protocol development focused on quantifying cartilage degeneration. Characterization of measurement errors, caused mainly by thermal transients and by translating the instrument sensor, demonstrated that HDD can quantify sample degradation with <6 μm precision and <10 μm temperature-induced errors. HDD capabilities were evaluated in a pilot study that monitored the degradation of fresh ex vivo human cartilage samples by collagenase solutions over three days. HDD could robustly resolve the effects of collagenase concentration as small as 0.5 mg/ml. Careful sample preparation resulted in measurements that did not suffer from donor-to-donor variation (coefficient of variance <70%). Due to its unique combination of sample throughput, measurement precision, temporal sampling and experimental versality, HDD provides a novel biomechanics-based experimental platform for quantifying the effects of proteins (cytokines, growth factors, enzymes, antibodies) or small molecules on the degradation of soft tissues or tissue engineering constructs. Thereby, HDD can complement established tools and in vitro models in important applications including drug screening and biomaterial development. Copyright © 2018 Elsevier Ltd. All rights reserved.

Biochemical Fractionation and Stable Isotope Dilution Liquid Chromatography-mass Spectrometry for Targeted and Microdomain-specific Protein Quantification in Human Postmortem Brain Tissue*

PubMed Central

MacDonald, Matthew L.; Ciccimaro, Eugene; Prakash, Amol; Banerjee, Anamika; Seeholzer, Steven H.; Blair, Ian A.; Hahn, Chang-Gyu

2012-01-01

Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex. A majority of neuropsychiatric illnesses are complex trait disorders, in which multiple etiologic factors converge at the synapse via many signaling pathways. Investigating the protein composition of synaptic microdomains from human patient brain tissues will yield valuable insights into the interactions of risk genes in many disorders. These types of studies in postmortem tissues have been limited by the lack of proper study paradigms. Thus, it is necessary not only to develop strategies to quantify protein and post-translational modifications at the synapse, but also to rigorously validate them for use in postmortem human brain tissues. In this study we describe the development of a liquid chromatography-selected reaction monitoring method, using a stable isotope-labeled neuronal proteome standard prepared from the brain tissue of a stable isotope-labeled mouse, for the multiplexed quantification of target synaptic proteins in mammalian samples. Additionally, we report the use of this method to validate a biochemical approach for the preparation of synaptic microdomain enrichments from human postmortem prefrontal cortex. Our data demonstrate that a targeted mass spectrometry approach with a true neuronal proteome standard facilitates accurate and precise quantification of over 100 synaptic proteins in mammalian samples, with the potential to quantify over 1000 proteins. Using this method, we found that protein enrichments in subcellular fractions prepared from human postmortem brain tissue were strikingly similar to those prepared from fresh mouse brain tissue. These findings demonstrate that biochemical fractionation methods paired with targeted proteomic strategies can be used in human brain tissues, with important implications for the study of neuropsychiatric disease. PMID:22942359

Investigation of viability of plant tissue in the environmental scanning electron microscopy.

PubMed

Zheng, Tao; Waldron, K W; Donald, Athene M

2009-11-01

The advantages of environmental scanning electron microscopy (ESEM) make it a suitable technique for studying plant tissue in its native state. There have been few studies on the effects of ESEM environment and beam damage on the viability of plant tissue. A simple plant tissue, Allium cepa (onion) upper epidermal tissue was taken as the model for study. The change of moisture content of samples was studied at different relative humidities. Working with the electron beam on, viability tests were conducted for samples after exposure in the ESEM under different operating conditions to investigate the effect of electron beam dose on the viability of samples. The results suggested that without the electron beam, the ESEM chamber itself can prevent the loss of initial moisture if its relative humidity is maintained above 90%. With the electron beam on, the viability of Allium cepa (onion) cells depends both on the beam accelerating voltage and the electron dose/unit area hitting the sample. The dose can be controlled by several of the ESEM instrumental parameters. The detailed process of beam damage on cuticle-down and cuticle-up samples was investigated and compared. The results indicate that cuticular adhesion to the cell wall is relatively weak, but highly resistant to electron beam damage. Systematic study on the effect of ESEM operation parameters has been done. Results qualitatively support the intuitive expectations, but demonstrate quantitatively that Allium cepa epidermal cells are able to be kept in a hydrated and viable state under relevant operation condition inside ESEM, providing a basis for further in situ experiments on plant tissues.

A conformal, bio-interfaced class of silicon electronics for mapping cardiac electrophysiology.

PubMed

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

2010-03-24

In all current implantable medical devices such as pacemakers, deep brain stimulators, and epilepsy treatment devices, each electrode is independently connected to separate control systems. The ability of these devices to sample and stimulate tissues is hindered by this configuration and by the rigid, planar nature of the electronics and the electrode-tissue interfaces. Here, we report the development of a class of mechanically flexible silicon electronics for multiplexed measurement of signals in an intimate, conformal integrated mode on the dynamic, three-dimensional surfaces of soft tissues in the human body. We demonstrate 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 porcine heart in vivo. The devices sample with simultaneous submillimeter and submillisecond resolution through 288 amplified and multiplexed channels. We use this system 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 demonstration is one example of many possible uses of this technology in minimally invasive medical devices.

Microwave energy fixation for electron microscopy.

PubMed Central

Login, G. R.; Dvorak, A. M.

1985-01-01

We have demonstrated that microwave energy (MW) can be used in conjunction with chemical cross-linking agents in order to rapidly fix cell suspensions and tissue blocks for electron microscopy in 7-9 seconds. The optimal MW fixation method involved immersing tissues up to 1 cu cm in dilute aldehyde fixation and immediately irradiating the specimens in a conventional microwave oven for 9 seconds to 50 C. Ultrastructural preservation of samples irradiated by MW energy was comparable to that of the control samples immersed in aldehyde fixative for 2 hours at 25 C. Stereologic analysis showed that tissue blocks fixed by the MW fixation method did not cause organelles such as liver mitochondria and salivary gland granules to shrink or to swell. Potential applications for this new fixation technology include the investigation of rapid intracellular processes (eg, vesicular transport) and preservation of proteins that are difficult to demonstrate with routine fixation methods (eg, antigens and enzymes). Images Figure 4 Figure 5 Figure 2 Figure 3 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 PMID:3927740

Terahertz in-line digital holography of human hepatocellular carcinoma tissue.

PubMed

Rong, Lu; Latychevskaia, Tatiana; Chen, Chunhai; Wang, Dayong; Yu, Zhengping; Zhou, Xun; Li, Zeyu; Huang, Haochong; Wang, Yunxin; Zhou, Zhou

2015-02-13

Terahertz waves provide a better contrast in imaging soft biomedical tissues than X-rays, and unlike X-rays, they cause no ionisation damage, making them a good option for biomedical imaging. Terahertz absorption imaging has conventionally been used for cancer diagnosis. However, the absorption properties of a cancerous sample are influenced by two opposing factors: an increase in absorption due to a higher degree of hydration and a decrease in absorption due to structural changes. It is therefore difficult to diagnose cancer from an absorption image. Phase imaging can thus be critical for diagnostics. We demonstrate imaging of the absorption and phase-shift distributions of 3.2 mm × 2.3 mm × 30-μm-thick human hepatocellular carcinoma tissue by continuous-wave terahertz digital in-line holography. The acquisition time of a few seconds for a single in-line hologram is much shorter than that of other terahertz diagnostic techniques, and future detectors will allow acquisition of meaningful holograms without sample dehydration. The resolution of the reconstructions was enhanced by sub-pixel shifting and extrapolation. Another advantage of this technique is its relaxed minimal sample size limitation. The fibrosis indicated in the phase distribution demonstrates the potential of terahertz holographic imaging to obtain a more objective, early diagnosis of cancer.

Terahertz in-line digital holography of human hepatocellular carcinoma tissue

PubMed Central

Rong, Lu; Latychevskaia, Tatiana; Chen, Chunhai; Wang, Dayong; Yu, Zhengping; Zhou, Xun; Li, Zeyu; Huang, Haochong; Wang, Yunxin; Zhou, Zhou

2015-01-01

Terahertz waves provide a better contrast in imaging soft biomedical tissues than X-rays, and unlike X-rays, they cause no ionisation damage, making them a good option for biomedical imaging. Terahertz absorption imaging has conventionally been used for cancer diagnosis. However, the absorption properties of a cancerous sample are influenced by two opposing factors: an increase in absorption due to a higher degree of hydration and a decrease in absorption due to structural changes. It is therefore difficult to diagnose cancer from an absorption image. Phase imaging can thus be critical for diagnostics. We demonstrate imaging of the absorption and phase-shift distributions of 3.2 mm × 2.3 mm × 30-μm-thick human hepatocellular carcinoma tissue by continuous-wave terahertz digital in-line holography. The acquisition time of a few seconds for a single in-line hologram is much shorter than that of other terahertz diagnostic techniques, and future detectors will allow acquisition of meaningful holograms without sample dehydration. The resolution of the reconstructions was enhanced by sub-pixel shifting and extrapolation. Another advantage of this technique is its relaxed minimal sample size limitation. The fibrosis indicated in the phase distribution demonstrates the potential of terahertz holographic imaging to obtain a more objective, early diagnosis of cancer. PMID:25676705

Terahertz in-line digital holography of human hepatocellular carcinoma tissue

NASA Astrophysics Data System (ADS)

Rong, Lu; Latychevskaia, Tatiana; Chen, Chunhai; Wang, Dayong; Yu, Zhengping; Zhou, Xun; Li, Zeyu; Huang, Haochong; Wang, Yunxin; Zhou, Zhou

2015-02-01

Terahertz waves provide a better contrast in imaging soft biomedical tissues than X-rays, and unlike X-rays, they cause no ionisation damage, making them a good option for biomedical imaging. Terahertz absorption imaging has conventionally been used for cancer diagnosis. However, the absorption properties of a cancerous sample are influenced by two opposing factors: an increase in absorption due to a higher degree of hydration and a decrease in absorption due to structural changes. It is therefore difficult to diagnose cancer from an absorption image. Phase imaging can thus be critical for diagnostics. We demonstrate imaging of the absorption and phase-shift distributions of 3.2 mm × 2.3 mm × 30-μm-thick human hepatocellular carcinoma tissue by continuous-wave terahertz digital in-line holography. The acquisition time of a few seconds for a single in-line hologram is much shorter than that of other terahertz diagnostic techniques, and future detectors will allow acquisition of meaningful holograms without sample dehydration. The resolution of the reconstructions was enhanced by sub-pixel shifting and extrapolation. Another advantage of this technique is its relaxed minimal sample size limitation. The fibrosis indicated in the phase distribution demonstrates the potential of terahertz holographic imaging to obtain a more objective, early diagnosis of cancer.

High-resolution optical polarimetric elastography for measuring the mechanical properties of tissue

NASA Astrophysics Data System (ADS)

Hudnut, Alexa W.; Armani, Andrea M.

2018-02-01

Traditionally, chemical and molecular markers have been the predominate method in diagnostics. Recently, alternate methods of determining tissue and disease characteristics have been proposed based on testing the mechanical behavior of biomaterials. Existing methods for performing elastography measurements, such as atomic force microscopy, compression testing, and ultrasound elastography, require either extensive sample processing or have poor resolution. In the present work, we demonstrate an optical polarimetric elastography device to characterize the mechanical properties of salmon skeletal muscle. A fiber-coupled 1550nm laser paired with an optical polarizer is used to create a fiber optic sensing region. By measuring the change in polarization from the initial state to the final state within the fiber sensing region with a polarimeter, the loading-unloading curves can be determined for the biomaterial. The device is used to characterize the difference between samples with a range of collagen membranes. The loading-unloading curves are used to determine the change in polarization phase and energy loss of the samples at 10%, 20% and 30% strain. As expected, the energy loss is a better metric for measuring the mechanical properties of the tissues because it incorporates the entire loading-unloading curve rather than a single point. Using this metric, it is demonstrated the device can repeatedly differentiate between the different membrane configurations.

A Method to Prevent Protein Delocalization in Imaging Mass Spectrometry of Non-Adherent Tissues: Application to Small Vertebrate Lens Imaging

PubMed Central

Anderson, David M. G.; Floyd, Kyle A.; Barnes, Stephen; Clark, Judy M.; Clark, John I.; Mchaourab, Hassane; Schey, Kevin L.

2015-01-01

MALDI imaging requires careful sample preparation to obtain reliable, high quality images of small molecules, peptides, lipids, and proteins across tissue sections. Poor crystal formation, delocalization of analytes, and inadequate tissue adherence can affect the quality, reliability, and spatial resolution of MALDI images. We report a comparison of tissue mounting and washing methods that resulted in an optimized method using conductive carbon substrates that avoids thaw mounting or washing steps, minimizes protein delocalization, and prevents tissue detachment from the target surface. Application of this method to image ocular lens proteins of small vertebrate eyes demonstrates the improved methodology for imaging abundant crystallin protein products. This method was demonstrated for tissue sections from rat, mouse, and zebrafish lenses resulting in good quality MALDI images with little to no delocalization. The images indicate, for the first time in mouse and zebrafish, discrete localization of crystallin protein degradation products resulting in concentric rings of distinct protein contents that may be responsible for the refractive index gradient of vertebrate lenses. PMID:25665708

Increased Expression of Herpes Virus-Encoded hsv1-miR-H18 and hsv2-miR-H9-5p in Cancer-Containing Prostate Tissue Compared to That in Benign Prostate Hyperplasia Tissue

PubMed Central

Shinn, Helen Ki; Yan, Chunri; Kim, Tae-Hwan; Kim, Sang Tae; Kim, Won Tae; Lee, Ok-Jun; Moon, Sung-Kwon; Kim, Nam-Hyung; Kim, Jayoung; Cha, Eun-Jong

2016-01-01

Purpose: Previously, we reported the presence of virus-encoded microRNAs (miRNAs) in the urine of prostate cancer (CaP) patients. In this study, we investigated the expression of two herpes virus-encoded miRNAs in prostate tissue. Methods: A total of 175 tissue samples from noncancerous benign prostatic hyperplasia (BPH), 248 tissue samples from patients with CaP and BPH, and 50 samples from noncancerous surrounding tissues from these same patients were analyzed for the expression of two herpes virus-encoded miRNAs by real-time polymerase chain reaction (PCR) and immunocytochemistry using nanoparticles as molecular beacons. Results: Real-time reverse transcription-PCR results revealed significantly higher expression of hsv1-miR-H18 and hsv2-miRH9- 5p in surrounding noncancerous and CaP tissues than that in BPH tissue (each comparison, P<0.001). Of note, these miRNA were expressed equivalently in the CaP tissues and surrounding noncancerous tissues. Moreover, immunocytochemistry clearly demonstrated a significant enrichment of both hsv1-miR-H18 and hsv2-miR-H9 beacon-labeled cells in CaP and surrounding noncancerous tissue compared to that in BPH tissue (each comparison, P<0.05 for hsv1-miR-H18 and hsv2- miR-H9). Conclusions: These results suggest that increased expression of hsv1-miR-H18 and hsv2-miR-H95p might be associated with tumorigenesis in the prostate. Further studies will be required to elucidate the role of these miRNAs with respect to CaP and herpes viral infections. PMID:27377944

Increased Expression of Herpes Virus-Encoded hsv1-miR-H18 and hsv2-miR-H9-5p in Cancer-Containing Prostate Tissue Compared to That in Benign Prostate Hyperplasia Tissue.

PubMed

Yun, Seok Joong; Jeong, Pildu; Kang, Ho Won; Shinn, Helen Ki; Kim, Ye-Hwan; Yan, Chunri; Choi, Young-Ki; Kim, Dongho; Ryu, Dong Hee; Ha, Yun-Sok; Kim, Tae-Hwan; Kwon, Tae Gyun; Kim, Jung Min; Suh, Sang Heon; Kim, Seon-Kyu; Kim, Seon-Young; Kim, Sang Tae; Kim, Won Tae; Lee, Ok-Jun; Moon, Sung-Kwon; Kim, Nam-Hyung; Kim, Isaac Yi; Kim, Jayoung; Cha, Hee-Jae; Choi, Yung-Hyun; Cha, Eun-Jong; Kim, Wun-Jae

2016-06-01

Previously, we reported the presence of virus-encoded microRNAs (miRNAs) in the urine of prostate cancer (CaP) patients. In this study, we investigated the expression of two herpes virus-encoded miRNAs in prostate tissue. A total of 175 tissue samples from noncancerous benign prostatic hyperplasia (BPH), 248 tissue samples from patients with CaP and BPH, and 50 samples from noncancerous surrounding tissues from these same patients were analyzed for the expression of two herpes virus-encoded miRNAs by real-time polymerase chain reaction (PCR) and immunocytochemistry using nanoparticles as molecular beacons. Real-time reverse transcription-PCR results revealed significantly higher expression of hsv1-miR-H18 and hsv2-miRH9- 5p in surrounding noncancerous and CaP tissues than that in BPH tissue (each comparison, P<0.001). Of note, these miRNA were expressed equivalently in the CaP tissues and surrounding noncancerous tissues. Moreover, immunocytochemistry clearly demonstrated a significant enrichment of both hsv1-miR-H18 and hsv2-miR-H9 beacon-labeled cells in CaP and surrounding noncancerous tissue compared to that in BPH tissue (each comparison, P<0.05 for hsv1-miR-H18 and hsv2- miR-H9). These results suggest that increased expression of hsv1-miR-H18 and hsv2-miR-H95p might be associated with tumorigenesis in the prostate. Further studies will be required to elucidate the role of these miRNAs with respect to CaP and herpes viral infections.

Developing High-Frequency Quantitative Ultrasound Techniques to Characterize Three-Dimensional Engineered Tissues

NASA Astrophysics Data System (ADS)

Mercado, Karla Patricia E.

Tissue engineering holds great promise for the repair or replacement of native tissues and organs. Further advancements in the fabrication of functional engineered tissues are partly dependent on developing new and improved technologies to monitor the properties of engineered tissues volumetrically, quantitatively, noninvasively, and nondestructively over time. Currently, engineered tissues are evaluated during fabrication using histology, biochemical assays, and direct mechanical tests. However, these techniques destroy tissue samples and, therefore, lack the capability for real-time, longitudinal monitoring. The research reported in this thesis developed nondestructive, noninvasive approaches to characterize the structural, biological, and mechanical properties of 3-D engineered tissues using high-frequency quantitative ultrasound and elastography technologies. A quantitative ultrasound technique, using a system-independent parameter known as the integrated backscatter coefficient (IBC), was employed to visualize and quantify structural properties of engineered tissues. Specifically, the IBC was demonstrated to estimate cell concentration and quantitatively detect differences in the microstructure of 3-D collagen hydrogels. Additionally, the feasibility of an ultrasound elastography technique called Single Tracking Location Acoustic Radiation Force Impulse (STL-ARFI) imaging was demonstrated for estimating the shear moduli of 3-D engineered tissues. High-frequency ultrasound techniques can be easily integrated into sterile environments necessary for tissue engineering. Furthermore, these high-frequency quantitative ultrasound techniques can enable noninvasive, volumetric characterization of the structural, biological, and mechanical properties of engineered tissues during fabrication and post-implantation.

Telomerase activity as a marker for malignancy in feline tissues.

PubMed

Cadile, C D; Kitchell, B E; Biller, B J; Hetler, E R; Balkin, R G

2001-10-01

To establish the diagnostic significance of the telomeric repeat amplification protocol (TRAP) assay in detecting feline malignancies. Solid tissue specimens collected from 33 client-owned cats undergoing diagnostic or therapeutic procedures at the University of Illinois Veterinary Medical Teaching Hospital between July 1997 and September 1999 and an additional 20 tissue samples were collected from 3 clinically normal control cats euthanatized at the conclusion of an unrelated study. The TRAP assay was used for detection of telomerase activity. Each result was compared to its respective histopathologic diagnosis. Twenty-nine of 31 malignant and 1 of 22 benign or normal tissue samples had telomerase activity, indicating 94% sensitivity and 95% specificity of the TRAP assay in our laboratory. The diagnostic significance of telomerase activity has been demonstrated in humans and recently in dogs by our laboratory. We tested feline samples to determine whether similar patterns of telomerase activity exist. On the basis of our results, the TRAP assay may be clinically useful in providing a rapid diagnosis of malignancy in cats. The telomerase enzyme may also serve as a therapeutic target in feline tumors.

Effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in the digestive system of tilapia (Oreochromis niloticus).

PubMed

Dai, Wei; Du, Huahua; Fu, Linglin; Jin, Chengguan; Xu, Zirong; Liu, Huitao

2009-02-01

With the increasing occurrence of dietary lead (Pb) contamination in aquatic environment, threat of the dietary Pb toxicity to aquatic organisms attracted more attention. In this study, after being exposed to dietary Pb at concentrations of 0, 100, 400, and 800-microg/g dry weight for 60 days, the groups of tilapia (Oreochromis niloticus) were sacrificed and sampled to analyze the effects of dietary Pb on accumulation, histopathology, and digestive enzyme activities in tissues of the digestive system. The results showed that the Pb accumulation in tissues increased with the dietary Pb concentrations. Moreover, Pb accumulated in sampled tissues in the following order: intestine > stomach > liver. By observation of liver histological sections in optical microscope, lesions could be detected in the Pb-contaminated groups. It was also demonstrated that the inhibitory effect of dietary Pb on digestive enzyme activities was dietary Pb concentration dependent. Different degrees of inhibition of enzyme activities were exhibited in sampled tissues. It was indicated that digestive enzyme activities in the digestive system might be considered as the potential biomarkers of dietary Pb contamination in tilapia.

Epigenome overlap measure (EPOM) for comparing tissue/cell types based on chromatin states.

PubMed

Li, Wei Vivian; Razaee, Zahra S; Li, Jingyi Jessica

2016-01-11

The dynamics of epigenomic marks in their relevant chromatin states regulate distinct gene expression patterns, biological functions and phenotypic variations in biological processes. The availability of high-throughput epigenomic data generated by next-generation sequencing technologies allows a data-driven approach to evaluate the similarities and differences of diverse tissue and cell types in terms of epigenomic features. While ChromImpute has allowed for the imputation of large-scale epigenomic information to yield more robust data to capture meaningful relationships between biological samples, widely used methods such as hierarchical clustering and correlation analysis cannot adequately utilize epigenomic data to accurately reveal the distinction and grouping of different tissue and cell types. We utilize a three-step testing procedure-ANOVA, t test and overlap test to identify tissue/cell-type- associated enhancers and promoters and to calculate a newly defined Epigenomic Overlap Measure (EPOM). EPOM results in a clear correspondence map of biological samples from different tissue and cell types through comparison of epigenomic marks evaluated in their relevant chromatin states. Correspondence maps by EPOM show strong capability in distinguishing and grouping different tissue and cell types and reveal biologically meaningful similarities between Heart and Muscle, Blood & T-cell and HSC & B-cell, Brain and Neurosphere, etc. The gene ontology enrichment analysis both supports and explains the discoveries made by EPOM and suggests that the associated enhancers and promoters demonstrate distinguishable functions across tissue and cell types. Moreover, the tissue/cell-type-associated enhancers and promoters show enrichment in the disease-related SNPs that are also associated with the corresponding tissue or cell types. This agreement suggests the potential of identifying causal genetic variants relevant to cell-type-specific diseases from our identified associated enhancers and promoters. The proposed EPOM measure demonstrates superior capability in grouping and finding a clear correspondence map of biological samples from different tissue and cell types. The identified associated enhancers and promoters provide a comprehensive catalog to study distinct biological processes and disease variants in different tissue and cell types. Our results also find that the associated promoters exhibit more cell-type-specific functions than the associated enhancers do, suggesting that the non-associated promoters have more housekeeping functions than the non-associated enhancers.

Speckle-type POZ (pox virus and zinc finger protein) protein gene deletion in ovarian cancer: Fluorescence in situ hybridization analysis of a tissue microarray.

PubMed

Hu, Xiaoyu; Yang, Zhu; Zeng, Manman; Liu, Y I; Yang, Xiaotao; Li, Yanan; Li, X U; Yu, Qiubo

2016-07-01

The aim of the present study was to investigate the status of speckle-type POZ (pox virus and zinc finger protein) protein (SPOP) gene located on chromosome 17q21 in ovarian cancer (OC). The present study evaluated a tissue microarray, which contained 90 samples of ovarian cancer and 10 samples of normal ovarian tissue, using fluorescence in situ hybridization (FISH). FISH is a method where a SPOP-specific DNA red fluorescence probe was used for the experimental group and a centromere-specific DNA green fluorescence probe for chromosome 17 was used for the control group. The present study demonstrated that a deletion of the SPOP gene was observed in 52.27% (46/88) of the ovarian cancer tissues, but was not identified in normal ovarian tissues. Simultaneously, monosomy 17 was frequently identified in the ovarian cancer tissues, but not in the normal ovarian tissues. Furthermore, the present data revealed that the ovarian cancer histological subtype and grade were significantly associated with a deletion of the SPOP gene, which was assessed by the appearance of monosomy 17 in the ovarian cancer samples; the deletion of the SPOP gene was observed in a large proportion of serous epithelial ovarian cancer (41/61; 67.21%), particularly in grade 3 (31/37; 83.78%). In conclusion, deletion of the SPOP gene on chromosome 17 in ovarian cancer samples, which results from monosomy 17, indicates that the SPOP gene may serve as a tumor suppressor gene in ovarian cancer.

Towards optical spectroscopic anatomical mapping (OSAM) for lesion validation in cardiac tissue (Conference Presentation)

NASA Astrophysics Data System (ADS)

Singh-Moon, Rajinder P.; Zaryab, Mohammad; Hendon, Christine P.

2017-02-01

Electroanatomical mapping (EAM) is an invaluable tool for guiding cardiac radiofrequency ablation (RFA) therapy. The principle roles of EAM is the identification of candidate ablation sites by detecting regions of abnormal electrogram activity and lesion validation subsequent to RF energy delivery. However, incomplete lesions may present interim electrical inactivity similar to effective treatment in the acute setting, despite efforts to reveal them with pacing or drugs, such as adenosine. Studies report that the misidentification and recovery of such lesions is a leading cause of arrhythmia recurrence and repeat procedures. In previous work, we demonstrated spectroscopic characterization of cardiac tissues using a fiber optic-integrated RF ablation catheter. In this work, we introduce OSAM (optical spectroscopic anatomical mapping), the application of this spectroscopic technique to obtain 2-dimensional biodistribution maps. We demonstrate its diagnostic potential as an auxiliary method for lesion validation in treated swine preparations. Endocardial lesion sets were created on fresh swine cardiac samples using a commercial RFA system. An optically-integrated catheter console fabricated in-house was used for measurement of tissue optical spectra between 600-1000nm. Three dimensional, Spatio-spectral datasets were generated by raster scanning of the optical catheter across the treated sample surface in the presence of whole blood. Tissue optical parameters were recovered at each spatial position using an inverse Monte Carlo method. OSAM biodistribution maps showed stark correspondence with gross examination of tetrazolium chloride stained tissue specimens. Specifically, we demonstrate the ability of OSAM to readily distinguish between shallow and deeper lesions, a limitation faced by current EAM techniques. These results showcase the OSAMs potential for lesion validation strategies for the treatment of cardiac arrhythmias.

Tissue grown in space in NASA Bioreactor

NASA Technical Reports Server (NTRS)

2001-01-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens 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. Final samples from Mir and Earth appeared histologically cartilaginous throughout their entire cross sections (5-8 mm thick), with the exception of fibrous outer capsules. Constructs grown on Earth (A) appeared to have a more organized extracellular matrix with more uniform collagen orientation as compared with constructs grown on Mir (B), but the average collagen fiber diameter was similar in the two groups (22 +- 2 nm) and comparable to that previously reported for developing articular cartilage. Randomly oriented collagen in Mir samples would be consistent with previous reports that microgravity disrupts fibrillogenesis. These are transmission electron micrographs of constructs from Mir (A) and Earth (B) groups at magnifications of x3,500 and x120,000 (Inset). 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). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Credit: Proceedings of the National Academy of Sciences.

The serological screening of deceased tissue donors within the English Blood Service for infectious agents--a review of current outcomes and a more effective strategy for the future.

PubMed

Kitchen, A D; Gillan, H L

2010-04-01

The overall effectiveness of the NHSBT screening programme for infectious agents in deceased tissue donors is examined and evaluated in terms of current outcomes and how to improve upon these outcomes. The screening results and any subsequent confirmatory results from a total of 1659 samples from NHSBT deceased donors referred to NTMRL for screening for infectious agents were included in the analysis. Overall 1566/1659 (94.4%) of the samples were screen negative. A total of 93 were repeat reactive on screening for one or more of the mandatory markers screened for, of which only 12 (13%) were subsequently confirmed to be positive on confirmatory testing. The majority of the repeat reactive samples were demonstrating non-specific reactivity with the screening assays in use. Overall, the NHSBT screening programme for infectious agents in deceased tissue donors is very effective with a relatively low overall loss of donors because of non-specific reactivity. However, unnecessary loss of tissue products is not acceptable, and although this programme compares favourably with the outcomes of other such programmes, the confirmatory results obtained demonstrate both the need and the potential for improving the outcomes. This is particularly important as one donor may donate more than one product, and can be achieved very easily with a change to the screening algorithm followed, using the confirmatory data obtained to support and validate this change. CONTENTS SUMMARY: Critical analysis of the NHSBT screening programme for infectious agents in deceased tissue donors and a strategy involving the design and use of a different screening algorithm to improve these outcomes.

Photoacoustic resonance spectroscopy for biological tissue characterization

NASA Astrophysics Data System (ADS)

Gao, Fei; Feng, Xiaohua; Zheng, Yuanjin; Ohl, Claus-Dieter

2014-06-01

By "listening to photons," photoacoustics allows the probing of chromosomes in depth beyond the optical diffusion limit. Here we report the photoacoustic resonance effect induced by multiburst modulated laser illumination, which is theoretically modeled as a damped mass-string oscillator and a resistor-inductor-capacitor (RLC) circuit. Through sweeping the frequency of multiburst modulated laser, the photoacoustic resonance effect is observed experimentally on phantoms and porcine tissues. Experimental results demonstrate different spectra for each phantom and tissue sample to show significant potential for spectroscopic analysis, fusing optical absorption and mechanical vibration properties. Unique RLC circuit parameters are extracted to quantitatively characterize phantom and biological tissues.

Multilaboratory trial for determination of ceftiofur residues in bovine and swine kidney and muscle, and bovine milk.

PubMed

Hornish, Rex E; Hamlow, Philip J; Brown, Scott A

2003-01-01

A multilaboratory trial for determining ceftiofur-related residues in bovine and swine kidney and muscle, and bovine milk was conducted following regulatory guidelines of the U.S. Food and Drug Administration, Center for Veterinary Medicine. The methods convert all desfuroylceftiofur-related residues containing the intact beta-lactam ring to desfuroylceftiofur acetamide to establish ceftiofur residues in tissues. Four laboratories analyzed 5 sets of samples for each tissue. Each sample set consisted of a control/blank sample and 3 control samples fortified with ceftiofur at 0.5 Rm, Rm, and 2 Rm, respectively, where Rm is the U.S. tolerance assigned for ceftiofur residue in each tissue/matrix: 0.100 microg/mL for milk, 8.0 microg/g for kidney (both species), 1.0 microg/g for bovine muscle, and 2.0 microg/g for swine muscle. Each sample set also contained 2 samples of incurred-residue tissues (one > Rm and one < Rm) from animals treated with ceftiofur hydrochloride. All laboratories completed the method trial after a familiarization phase and test of system suitability in which they demonstrated > 80% recovery in pretrial fortified test samples. Results showed that the methods met all acceptable performance criteria for recovery, accuracy, and precision. Although sample preparation was easy, solid-phase extraction cartridge performance must be carefully evaluated before samples are processed. The liquid chromatography detection system was easily set up; however, the elution profile may require slight modifications. The procedures could clearly differentiate between violative (> Rm) and nonviolative (< Rm) ceftiofur residues. Participating laboratories found the procedures suitable for ceftiofur residue determination.

An Automated Platform for High-Resolution Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry

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

Lanekoff, Ingela T.; Heath, Brandi S.; Liyu, Andrey V.

2012-10-02

An automated platform has been developed for acquisition and visualization of mass spectrometry imaging (MSI) data using nanospray desorption electrospray ionization (nano-DESI). The new system enables robust operation of the nano-DESI imaging source over many hours. This is achieved by controlling the distance between the sample and the probe by mounting the sample holder onto an automated XYZ stage and defining the tilt of the sample plane. This approach is useful for imaging of relatively flat samples such as thin tissue sections. Custom software called MSI QuickView was developed for visualization of large data sets generated in imaging experiments. MSImore » QuickView enables fast visualization of the imaging data during data acquisition and detailed processing after the entire image is acquired. The performance of the system is demonstrated by imaging rat brain tissue sections. High resolution mass analysis combined with MS/MS experiments enabled identification of lipids and metabolites in the tissue section. In addition, high dynamic range and sensitivity of the technique allowed us to generate ion images of low-abundance isobaric lipids. High-spatial resolution image acquired over a small region of the tissue section revealed the spatial distribution of an abundant brain metabolite, creatine, in the white and gray matter that is consistent with the literature data obtained using magnetic resonance spectroscopy.« less

Thyroid peroxidase (TPO) expressed in thyroid and breast tissues shows similar antigenic properties

PubMed Central

Godlewska, Marlena; Arczewska, Katarzyna D.; Rudzińska, Magdalena; Łyczkowska, Anna; Krasuska, Wanda; Hanusek, Karolina; Ruf, Jean; Kiedrowski, Mirosław

2017-01-01

Background Thyroid peroxidase (TPO) is essential for physiological function of the thyroid gland. The high prevalence of thyroid peroxidase antibodies (TPOAbs) in patients with breast cancer and their protective role had previously been demonstrated, indicating a link between breast cancer and thyroid autoimmunity. Recently, TPO was shown to be present in breast cancer tissue samples but its antigenicity has not been analyzed. Methods In this study, we investigated TPO expression levels in a series of fifty-six breast cancer samples paired with normal (peri-tumoral) tissue and its antigenic activity using a panel of well-characterized murine anti-human TPOAbs. Results We have shown that TPO transcripts were present in both normal and cancer tissue samples, although the amounts in the latter were reduced. Additionally, we observed that TPO levels are lower in more advanced cancers. TPO protein expression was confirmed in all tissue samples, both normal and cancerous. We also found that the antigenicity of the immunodominant regions (IDRs) in breast TPO resembles that of thyroid TPO, which is crucial for effective interactions with human TPOAbs. Conclusions Expression of TPO in breast cancer together with its antigenic activity may have beneficial effects in TPOAb-positive breast cancer patients. However, further studies are needed to confirm the beneficial role of TPOAbs and to better understand the underlying mechanism. PMID:28575127

Visualizing molecular polar order in tissues via electromechanical coupling

PubMed Central

Denning, Denise; Alilat, Sofiane; Habelitz, Stefan; Fertala, Andrzej; Rodriguez, Brian J.

2015-01-01

Electron microscopy (EM) and atomic force microscopy (AFM) techniques have long been used to characterize collagen fibril ordering and alignment in connective tissues. These techniques, however, are unable to map collagen fibril polarity, i.e., the polar orientation that is directed from the amine to the carboxyl termini. Using a voltage modulated AFM-based technique called piezoresponse force microscopy (PFM), we show it is possible to visualize both the alignment of collagen fibrils within a tissue and the polar orientation of the fibrils with minimal sample preparation. We demonstrate the technique on rat tail tendon and porcine eye tissues in ambient conditions. In each sample, fibrils are arranged into domains whereby neighboring domains exhibit opposite polarizations, which in some cases extend to the individual fibrillar level. Uniform polarity has not been observed in any of the tissues studied. Evidence of anti-parallel ordering of the amine to carboxyl polarity in bundles of fibrils or in individual fibrils is found in all tissues, which has relevance for understanding mechanical and biofunctional properties and the formation of connective tissues. The technique can be applied to any biological material containing piezoelectric biopolymers or polysaccharides. PMID:22985991

Depth-resolved fluorescence of human ectocervical tissue

NASA Astrophysics Data System (ADS)

Wu, Yicong; Xi, Peng; Cheung, Tak-Hong; Yim, So Fan; Yu, Mei-Yung; Qu, Jianan Y.

2005-04-01

The depth-resolved autofluorescence of normal and dysplastic human ectocervical tissue within 120um depth were investigated utilizing a portable confocal fluorescence spectroscopy with the excitations at 355nm and 457nm. From the topmost keratinizing layer of all ectocervical tissue samples, strong keratin fluorescence with the spectral characteristics similar to collagen was observed, which created serious interference in seeking the correlation between tissue fluorescence and tissue pathology. While from the underlying non-keratinizing epithelial layer, the measured NADH fluorescence induced by 355nm excitation and FAD fluorescence induced by 457nm excitation were strongly correlated to the tissue pathology. The ratios between NADH over FAD fluorescence increased statistically in the CIN epithelial relative to the normal and HPV epithelia, which indicated increased metabolic activity in precancerous tissue. This study demonstrates that the depth-resolved fluorescence spectroscopy can reveal fine structural information on epithelial tissue and potentially provide more accurate diagnostic information for determining tissue pathology.

Lead, selenium and nickel concentrations in epithelial ovarian cancer, borderline ovarian tumor and healthy ovarian tissues.

PubMed

Canaz, Emel; Kilinc, Metin; Sayar, Hamide; Kiran, Gurkan; Ozyurek, Eser

2017-09-01

Wide variation exists in ovarian cancer incidence rates suggesting the importance of environmental factors. Due to increasing environmental pollution, trace elements and heavy metals have drawn attention in studies defining the etiology of cancer, but scant data is available for ovarian cancer. Our aim was to compare the tissue concentrations of lead, selenium and nickel in epithelial ovarian cancer, borderline tumor and healthy ovarian tissues. The levels of lead, selenium and nickel were estimated using atomic absorption spectrophotometry in formalin-fixed paraffin-embedded tissue samples. Tests were carried out in 20 malignant epithelial ovarian cancer, 15 epithelial borderline tumor and 20 non-neoplastic healthy ovaries. Two samples were collected for borderline tumors, one from papillary projection and one from the smooth surface of cyst wall. Pb and Ni concentrations were found to be higher both in malignant and borderline tissues than those in healthy ovaries. Concentrations of Pb and Ni in malignant tissues, borderline papillary projections and capsular tissue samples were not different. Comparison of Se concentrations of malignant, borderline and healthy ovarian tissues did not reveal statistical difference. Studied metal levels were not found to be different in either papillary projection or in cyst wall of the borderline tumors. This study revealed the accumulation of lead and nickel in ovarian tissue is associated with borderline and malignant proliferation of the surface epithelium. Accumulation of these metals in epithelial ovarian cancer and borderline ovarian tumor has not been demonstrated before. Copyright © 2017 Elsevier GmbH. All rights reserved.

Lack of tissue renewal in human adult Achilles tendon is revealed by nuclear bomb 14C

PubMed Central

Heinemeier, Katja Maria; Schjerling, Peter; Heinemeier, Jan; Magnusson, Stig Peter; Kjaer, Michael

2013-01-01

Tendons are often injured and heal poorly. Whether this is caused by a slow tissue turnover is unknown, since existing data provide diverging estimates of tendon protein half-life that range from 2 mo to 200 yr. With the purpose of determining life-long turnover of human tendon tissue, we used the 14C bomb-pulse method. This method takes advantage of the dramatic increase in atmospheric levels of 14C, produced by nuclear bomb tests in 1955–1963, which is reflected in all living organisms. Levels of 14C were measured in 28 forensic samples of Achilles tendon core and 4 skeletal muscle samples (donor birth years 1945–1983) with accelerator mass spectrometry (AMS) and compared to known atmospheric levels to estimate tissue turnover. We found that Achilles tendon tissue retained levels of 14C corresponding to atmospheric levels several decades before tissue sampling, demonstrating a very limited tissue turnover. The tendon concentrations of 14C approximately reflected the atmospheric levels present during the first 17 yr of life, indicating that the tendon core is formed during height growth and is essentially not renewed thereafter. In contrast, 14C levels in muscle indicated continuous turnover. Our observation provides a fundamental premise for understanding tendon function and pathology, and likely explains the poor regenerative capacity of tendon tissue.—Heinemeier, K. M., Schjerling, P., Heinemeier, J., Magnusson, S. P., Kjaer, M. Lack of tissue renewal in human adult Achilles tendon is revealed by nuclear bomb 14C. PMID:23401563

Association between P16INK4a Promoter Methylation and Non-Small Cell Lung Cancer: A Meta-Analysis

PubMed Central

Zhu, Siwei; Hua, Feng; Zhao, Hui; Xu, Hongrui; You, Jiacong; Sun, Linlin; Wang, Weiqiang; Chen, Jun; Zhou, Qinghua

2013-01-01

Background Aberrant methylation of CpG islands acquired in tumor cells in promoter regions plays an important role in carcinogenesis. Accumulated evidence demonstrates P16INK4a gene promoter hypermethylation is involved in non-small cell lung carcinoma (NSCLC), indicating it may be a potential biomarker for this disease. The aim of this study is to evaluate the frequency of P16INK4a gene promoter methylation between cancer tissue and autologous controls by summarizing published studies. Methods By searching Medline, EMBSE and CNKI databases, the open published studies about P16INK4a gene promoter methylation and NSCLC were identified using a systematic search strategy. The pooled odds of P16INK4A promoter methylation in lung cancer tissue versus autologous controls were calculated by meta-analysis method. Results Thirty-four studies, including 2 652 NSCLC patients with 5 175 samples were included in this meta-analysis. Generally, the frequency of P16INK4A promoter methylation ranged from 17% to 80% (median 44%) in the lung cancer tissue and 0 to 80% (median 15%) in the autologous controls, which indicated the methylation frequency in cancer tissue was much higher than that in autologous samples. We also find a strong and significant correlation between tumor tissue and autologous controls of P16INK4A promoter methylation frequency across studies (Correlation coefficient 0.71, 95% CI:0.51–0.83, P<0.0001). And the pooled odds ratio of P16INK4A promoter methylation in cancer tissue was 3.45 (95% CI: 2.63–4.54) compared to controls under random-effect model. Conclusion Frequency of P16INK4a promoter methylation in cancer tissue was much higher than that in autologous controls, indicating promoter methylation plays an important role in carcinogenesis of the NSCLC. Strong and significant correlation between tumor tissue and autologous samples of P16INK4A promoter methylation demonstrated a promising biomarker for NSCLC. PMID:23577085

Model development and experimental validation for analyzing initial transients of irradiation of tissues during thermal therapy using short pulse lasers.

PubMed

Ganguly, Mohit; Miller, Stephanie; Mitra, Kunal

2015-11-01

Short pulse lasers with pulse durations in the range of nanoseconds and shorter are effective in the targeted delivery of heat energy for precise tissue heating and ablation. This photothermal therapy is useful where the removal of cancerous tissue sections is required. The objective of this paper is to use finite element modeling to demonstrate the differences in the thermal response of skin tissue to short-pulse and continuous wave laser irradiation in the initial stages of the irradiation. Models have been developed to validate the temperature distribution and heat affected zone during laser irradiation of excised rat skin samples and live anesthetized mouse tissue. Excised rat skin samples and live anesthetized mice were subjected to Nd:YAG pulsed laser (1,064 nm, 500 ns) irradiation of varying powers. A thermal camera was used to measure the rise in surface temperature as a result of the laser irradiation. Histological analyses of the heat affected zone created in the tissue samples due to the temperature rise were performed. The thermal interaction of the laser with the tissue was quantified by measuring the thermal dose delivered by the laser. Finite element geometries of three-dimensional tissue sections for continuum and vascular models were developed using COMSOL Multiphysics. Blood flow was incorporated into the vascular model to mimic the presence of discrete blood vessels and contrasted with the continuum model without blood perfusion. The temperature rises predicted by the continuum and the vascular models agreed with the temperature rises observed at the surface of the excised rat tissue samples and live anesthetized mice due to laser irradiation respectively. The vascular model developed was able to predict the cooling produced by the blood vessels in the region where the vessels were present. The temperature rise in the continuum model due to pulsed laser irradiation was higher than that due to continuous wave (CW) laser irradiation in the initial stages of the irradiation. The temperature rise due to pulsed and CW laser irradiation converged as the time of irradiation increased. A similar trend was observed when comparing the thermal dose for pulsed and CW laser irradiation in the vascular model. Finite element models (continuum and vascular) were developed that can be used to predict temperature rise and quantify the thermal dose resulting from laser irradiation of excised rat skin samples and live anesthetized mouse tissue. The vascular model incorporating blood perfusion effects predicted temperature rise better in the live animal tissue. The models developed demonstrated that pulsed lasers caused greater temperature rise and delivered a greater thermal dose than CW lasers of equal average power, especially during the initial transients of irradiation. This analysis will be beneficial for thermal therapy applications where maximum delivery of thermal dose over a short period of time is important. © 2015 Wiley Periodicals, Inc.

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.

Optoacoustic detection of thermal lesions

NASA Astrophysics Data System (ADS)

Arsenault, Michel G.; Kolios, Michael C.; Whelan, William M.

2009-02-01

Minimally invasive thermal therapy is being investigated as an alternative cancer treatment. It involves heating tissues to greater than 55°C over a period of a few minutes, which results in tissue coagulation. Optoacoustic (OA) imaging is a new imaging technique that involves exposing tissues to pulsed light and detecting the acoustic waves that are generated. In this study, adult bovine liver tissue samples were heated using continuous wave laser energy for various times, then scanned using an optoacoustic imaging system. Large optoacoustic signal variability was observed in the native tissue prior to heating. OA signal amplitude increased with maximum tissue temperature achieved, characterized by a correlation coefficient of 0.63. In this study we show that there are detectable changes in optoacoustic signal strength that arise from tissue coagulation, which demonstrates the potential of optoacoustic technology for the monitoring of thermal therapy delivery.

Spectromicroscopy of boron in human glioblastomas following administration of Na2B12H11SH.

PubMed

Gilbert, B; Perfetti, L; Fauchoux, O; Redondo, J; Baudat, P A; Andres, R; Neumann, M; Steen, S; Gabel, D; Mercanti, D; Ciotti, M T; Perfetti, P; Margaritondo, G; De Stasio, G

2000-07-01

Boron neutron capture therapy (BNCT) is an experimental, binary treatment for brain cancer which requires as the first step that tumor tissue is targeted with a boron-10 containing compound. Subsequent exposure to a thermal neutron flux results in destructive, short range nuclear reaction within 10 microm of the boron compound. The success of the therapy requires than the BNCT agents be well localized in tumor, rather than healthy tissue. The MEPHISTO spectromicroscope, which performs microchemical analysis by x-ray absorption near edge structure (XANES) spectroscopy from microscopic areas, has been used to study the distribution of trace quantities of boron in human brain cancer tissues surgically removed from patients first administered with the compound Na2B12H11SH (BSH). The interpretation of XANES spectra is complicated by interference from physiologically present sulfur and phosphorus, which contribute structure in the same energy range as boron. We addressed this problem with the present extensive set of spectra from S, B, and P in relevant compounds. We demonstrate that a linear combination of sulfate, phosphate and BSH XANES can be used to reproduce the spectra acquired on boron-treated human brain tumor tissues. We analyzed human glioblastoma tissue from two patients administered and one not administered with BSH. As well as weak signals attributed to BSH, x-ray absorption spectra acquired from tissue samples detected boron in a reduced chemical state with respect to boron in BSH. This chemical state was characterized by a sharp absorption peak at 188.3 eV. Complementary studies on BSH reference samples were not able to reproduce this chemical state of boron, indicating that it is not an artifact produced during sample preparation or x-ray exposure. These data demonstrate that the chemical state of BSH may be altered by in vivo metabolism.

Upregulation of Poly (ADP-Ribose) Polymerase-1 (PARP1) in Triple-Negative Breast Cancer and Other Primary Human Tumor Types

PubMed Central

Ossovskaya, Valeria; Koo, Ingrid Chou; Kaldjian, Eric P.; Alvares, Christopher; Sherman, Barry M.

2010-01-01

Poly (ADP-ribose) polymerase-1 (PARP1) is a key facilitator of DNA repair and is implicated in pathways of tumorigenesis. PARP inhibitors have gained recent attention as rationally designed therapeutics for the treatment of several malignancies, particularly those associated with dysfunctional DNA repair pathways, including triple-negative breast cancer (TNBC). We investigated the PARP1 gene expression profile in surgical samples from more than 8,000 primary malignant and normal human tissues. PARP1 expression was found to be significantly increased in several malignant tissues, including those isolated from patients with breast, uterine, lung, ovarian, and skin cancers, and non-Hodgkin’s lymphoma. Within breast infiltrating ductal carcinoma (IDC) samples tested, mean PARP1 expression was significantly higher relative to normal breast tissue, with over 30% of IDC samples demonstrating upregulation of PARP1, compared with 2.9% of normal tissues. Because of known DNA repair defects, including BRCA1 dysfunction, associated with TNBC, exploration of PARP1 expression in breast cancers related to expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) led to the observation that negative expression of any of the 3 receptors was associated with upregulation of PARP1 expression, compared with receptor-positive tissues. To validate these observations, an independent set of breast adenocarcinomas was evaluated and demonstrated >2-fold upregulation of PARP1 in approximately 70% of primary breast adenocarcinomas, including TNBC, compared with syngeneic nonmalignant breast tissues. Immunohistochemistry (IHC) showed that upregulation of the PARP1 gene was consistent with increased protein expression in TNBC. These analyses suggest a potential biological role for PARP1 in several distinct malignancies, including TNBC. Further investigation of PARP1 as a biomarker for the therapeutic activity of PARP inhibitor-based therapy is warranted. PMID:21779467

An enhanced droplet-based liquid microjunction surface sampling system coupled with HPLC-ESI-MS/MS for spatially resolved analysis

DOE PAGES

Van Berkel, Gary J.; Weiskittel, Taylor M.; Kertesz, Vilmos

2014-11-07

Droplet-based liquid microjunction surface sampling coupled with high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) for spatially resolved analysis provides the possibility of effective analysis of complex matrix samples and can provide a greater degree of chemical information from a single spot sample than is typically possible with a direct analysis of an extract. Described here is the setup and enhanced capabilities of a discrete droplet liquid microjunction surface sampling system employing a commercially available CTC PAL autosampler. The system enhancements include incorporation of a laser distance sensor enabling unattended analysis of samples and sample locations of dramatically disparatemore » height as well as reliably dispensing just 0.5 μL of extraction solvent to make the liquid junction to the surface, wherein the extraction spot size was confined to an area about 0.7 mm in diameter; software modifications improving the spatial resolution of sampling spot selection from 1.0 to 0.1 mm; use of an open bed tray system to accommodate samples as large as whole-body rat thin tissue sections; and custom sample/solvent holders that shorten sampling time to approximately 1 min per sample. Lastly, the merit of these new features was demonstrated by spatially resolved sampling, HPLC separation, and mass spectral detection of pharmaceuticals and metabolites from whole-body rat thin tissue sections and razor blade (“crude”) cut mouse tissue.« less

An enhanced droplet-based liquid microjunction surface sampling system coupled with HPLC-ESI-MS/MS for spatially resolved analysis

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

Van Berkel, Gary J.; Weiskittel, Taylor M.; Kertesz, Vilmos

Droplet-based liquid microjunction surface sampling coupled with high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) for spatially resolved analysis provides the possibility of effective analysis of complex matrix samples and can provide a greater degree of chemical information from a single spot sample than is typically possible with a direct analysis of an extract. Described here is the setup and enhanced capabilities of a discrete droplet liquid microjunction surface sampling system employing a commercially available CTC PAL autosampler. The system enhancements include incorporation of a laser distance sensor enabling unattended analysis of samples and sample locations of dramatically disparatemore » height as well as reliably dispensing just 0.5 μL of extraction solvent to make the liquid junction to the surface, wherein the extraction spot size was confined to an area about 0.7 mm in diameter; software modifications improving the spatial resolution of sampling spot selection from 1.0 to 0.1 mm; use of an open bed tray system to accommodate samples as large as whole-body rat thin tissue sections; and custom sample/solvent holders that shorten sampling time to approximately 1 min per sample. Lastly, the merit of these new features was demonstrated by spatially resolved sampling, HPLC separation, and mass spectral detection of pharmaceuticals and metabolites from whole-body rat thin tissue sections and razor blade (“crude”) cut mouse tissue.« less

Determination of the complex refractive index segments of turbid sample with multispectral spatially modulated structured light and models approximation

NASA Astrophysics Data System (ADS)

Meitav, Omri; Shaul, Oren; Abookasis, David

2017-09-01

Spectral data enabling the derivation of a biological tissue sample's complex refractive index (CRI) can provide a range of valuable information in the clinical and research contexts. Specifically, changes in the CRI reflect alterations in tissue morphology and chemical composition, enabling its use as an optical marker during diagnosis and treatment. In the present work, we report a method for estimating the real and imaginary parts of the CRI of a biological sample using Kramers-Kronig (KK) relations in the spatial frequency domain. In this method, phase-shifted sinusoidal patterns at single high spatial frequency are serially projected onto the sample surface at different near-infrared wavelengths while a camera mounted normal to the sample surface acquires the reflected diffuse light. In the offline analysis pipeline, recorded images at each wavelength are converted to spatial phase maps using KK analysis and are then calibrated against phase-models derived from diffusion approximation. The amplitude of the reflected light, together with phase data, is then introduced into Fresnel equations to resolve both real and imaginary segments of the CRI at each wavelength. The technique was validated in tissue-mimicking phantoms with known optical parameters and in mouse models of ischemic injury and heat stress. Experimental data obtained indicate variations in the CRI among brain tissue suffering from injury. CRI fluctuations correlated with alterations in the scattering and absorption coefficients of the injured tissue are demonstrated. This technique for deriving dynamic changes in the CRI of tissue may be further developed as a clinical diagnostic tool and for biomedical research applications. To the best of our knowledge, this is the first report of the estimation of the spectral CRI of a mouse head following injury obtained in the spatial frequency domain.

Spatially-Resolved Proteomics: Rapid Quantitative Analysis of Laser Capture Microdissected Alveolar Tissue Samples

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

Clair, Geremy; Piehowski, Paul D.; Nicola, Teodora

Global proteomics approaches allow characterization of whole tissue lysates to an impressive depth. However, it is now increasingly recognized that to better understand the complexity of multicellular organisms, global protein profiling of specific spatially defined regions/substructures of tissues (i.e. spatially-resolved proteomics) is essential. Laser capture microdissection (LCM) enables microscopic isolation of defined regions of tissues preserving crucial spatial information. However, current proteomics workflows entail several manual sample preparation steps and are challenged by the microscopic mass-limited samples generated by LCM, and that impact measurement robustness, quantification, and throughput. Here, we coupled LCM with a fully automated sample preparation workflow thatmore » with a single manual step allows: protein extraction, tryptic digestion, peptide cleanup and LC-MS/MS analysis of proteomes from microdissected tissues. Benchmarking against the current state of the art in ultrasensitive global proteomic analysis, our approach demonstrated significant improvements in quantification and throughput. Using our LCM-SNaPP proteomics approach, we characterized to a depth of more than 3,400 proteins, the ontogeny of protein changes during normal lung development in laser capture microdissected alveolar tissue containing ~4,000 cells per sample. Importantly, the data revealed quantitative changes for 350 low abundance transcription factors and signaling molecules, confirming earlier transcript-level observations and defining seven modules of coordinated transcription factor/signaling molecule expression patterns, suggesting that a complex network of temporal regulatory control directs normal lung development with epigenetic regulation fine-tuning pre-natal developmental processes. Our LCM-proteomics approach facilitates efficient, spatially-resolved, ultrasensitive global proteomics analyses in high-throughput that will be enabling for several clinical and biological applications.« less

Full scattering profile for detecting physiological tissue properties

NASA Astrophysics Data System (ADS)

Duadi, Hamootal; Fixler, Dror

2017-02-01

Light reflectance and transmission from soft tissue has been utilized in noninvasive clinical measurement devices such as the photoplethysmograph (PPG) and reflectance pulse oximeter. Most methods of near infrared (NIR) spectroscopy focus on the volume reflectance from a semi-infinite sample, while very few measure transmission. We have previously shown that examining the full scattering profile (FSP), which is the angular distribution of exiting photons, provides more comprehensive information when measuring from a cylindrical tissue, such as earlobe, fingertip and pinched tissue. Our hypothesis is that the change in blood vessel diameter is more significant than the change in optical properties. The findings of this work demonstrate a realistic model for optical tissue measurements such as NIR spectroscopy, PPG and pulse oximetery.

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.

Comparative evaluation of human pulp tissue dissolution by different concentrations of chlorine dioxide, calcium hypochlorite and sodium hypochlorite: An in vitro study

PubMed Central

Taneja, Sonali; Mishra, Neha; Malik, Shubhra

2014-01-01

Introduction: Irrigation plays an indispensable role in removal of tissue remnants and debris from the complicated root canal system. This study compared the human pulp tissue dissolution by different concentrations of chlorine dioxide, calcium hypochlorite and sodium hypochlorite. Materials and Methods: Pulp tissue was standardized to a weight of 9 mg for each sample. In all,60 samples obtained were divided into 6 groups according to the irrigating solution used- 2.5% sodium hypochlorite (NaOCl), 5.25% NaOCl, 5% calcium hypochlorite (Ca(OCl)2), 10% Ca(OCl)2, 5%chlorine dioxide (ClO2) and 13% ClO2. Pulp tissue was placed in each test tube carrying irrigants of measured volume (5ml) according to their specified subgroup time interval: 30 minutes (Subgroup A) and 60 minutes (Subgroup B). The solution from each sample test tube was filtered and was left for drying overnight. The residual weight was calculated by filtration method. Results: Mean tissue dissolution increases with increase in time period. Results showed 5.25% NaOCl to be most effective at both time intervals followed by 2.5% NaOCl at 60 minutes, 10%Ca(OCl)2 and 13% ClO2 at 60 minutes. Least amount of tissue dissolving ability was demonstrated by 5% Ca(OCl)2 and 5% ClO2 at 30 minutes. Distilled water showed no pulp tissue dissolution. Conclusion: Withinthe limitations of the study, NaOCl most efficiently dissolved the pulp tissue at both concentrations and at both time intervals. Mean tissue dissolution by Ca(OCl)2 and ClO2 gradually increased with time and with their increase in concentration. PMID:25506141

Nondestructive cryomicro-CT imaging enables structural and molecular analysis of human lung tissue.

PubMed

Vasilescu, Dragoş M; Phillion, André B; Tanabe, Naoya; Kinose, Daisuke; Paige, David F; Kantrowitz, Jacob J; Liu, Gang; Liu, Hanqiao; Fishbane, Nick; Verleden, Stijn E; Vanaudenaerde, Bart M; Lenburg, Marc; Stevenson, Christopher S; Spira, Avrum; Cooper, Joel D; Hackett, Tillie-Louise; Hogg, James C

2017-01-01

Micro-computed tomography (CT) enables three-dimensional (3D) imaging of complex soft tissue structures, but current protocols used to achieve this goal preclude cellular and molecular phenotyping of the tissue. Here we describe a radiolucent cryostage that permits micro-CT imaging of unfixed frozen human lung samples at an isotropic voxel size of (11 µm) 3 under conditions where the sample is maintained frozen at -30°C during imaging. The cryostage was tested for thermal stability to maintain samples frozen up to 8 h. This report describes the methods used to choose the materials required for cryostage construction and demonstrates that whole genome mRNA integrity and expression are not compromised by exposure to micro-CT radiation and that the tissue can be used for immunohistochemistry. The new cryostage provides a novel method enabling integration of 3D tissue structure with cellular and molecular analysis to facilitate the identification of molecular determinants of disease. The described micro-CT cryostage provides a novel way to study the three-dimensional lung structure preserved without the effects of fixatives while enabling subsequent studies of the cellular matrix composition and gene expression. This approach will, for the first time, enable researchers to study structural changes of lung tissues that occur with disease and correlate them with changes in gene or protein signatures. Copyright © 2017 the American Physiological Society.

Immunohistochemical Evaluation of Neuroreceptors in Healthy and Pathological Temporo-Mandibular Joint

PubMed Central

Favia, Gianfranco; Corsalini, Massimo; Di Venere, Daniela; Pettini, Francesco; Favia, Giorgio; Capodiferro, Saverio; Maiorano, Eugenio

2013-01-01

Aim: A study was performed on the articular disk and periarticular tissues of the temporo-mandibular joint (TMJ) with immunohistochemical techniques to give evidence to the presence of neuroreceptors (NRec) in these sites. Methods: The study was carried out on tissue samples obtained from 10 subjects without TMJ disease and from 7 patients with severe TMJ arthritis and arthrosis. We use antibodies directed against following antigens: Gliofibrillary Acidic Protein (GFAP), Leu-7, Myelin Basic Protein (MBP), Neurofilaments 68 kD (NF), Neuron Specific Enolase (NSE), S-100 protein (S-100) and Synaptophysin (SYN). Results: This study revealed that Ruffini's-like, Pacini's-like and Golgi's-like receptors can be demonstrated in TMJ periarticular tissues and that free nervous endings are present in the subsynovial tissues but not within the articular disk. We observed elongated cytoplamic processes of chondrocytes that demonstrated strong S-100 immunoreactivity but they were unreactive with all other antibodies. These cytoplamic processes were more abundant and thicker in the samples obtained from patients with disease TMJ. Conclusion: The results of this study confirm that different Nrec are detectable in TMJ periarticular tissues but they are absent within the articular disk. In the latter site, only condrocytic processes are evident, especially in diseased TMJ, and they might have been confused with nervous endings in previous morphological studies. Nevertheless the absence of immunoreactivity for NF, NSE and SYN proves that they are not of neural origin. PMID:24151441

Evaluation of specimen preparation techniques for micro-PIXE localisation of elements in hyperaccumulating plants

NASA Astrophysics Data System (ADS)

Kachenko, Anthony G.; Siegele, Rainer; Bhatia, Naveen P.; Singh, Balwant; Ionescu, Mihail

2008-04-01

Hybanthus floribundus subsp. floribundus, a rare Australian Ni-hyperaccumulating shrub and Pityrogramma calomelanos var. austroamericana, an Australian naturalized As-hyperaccumulating fern are promising species for use in phytoremediation of contaminated sites. Micro-proton-induced X-ray emission (μ-PIXE) spectroscopy was used to map the elemental distribution of the accumulated metal(loid)s, Ca and K in leaf or pinnule tissues of the two plant species. Samples were prepared by two contrasting specimen preparation techniques: freeze-substitution in tetrahydrofuran (THF) and freeze-drying. The specimens were analysed to compare the suitability of each technique in preserving (i) the spatial elemental distribution and (ii) the tissue structure of the specimens. Further, the μ-PIXE results were compared with concentration of elements in the bulk tissue obtained by ICP-AES analysis. In H. floribundus subsp. floribundus, μ-PIXE analysis revealed Ni, Ca and K concentrations in freeze-dried leaf tissues were at par with bulk tissue concentrations. Elemental distribution maps illustrated that Ni was preferentially localised in the adaxial epidermal tissues (1% DW) and least concentration was found in spongy mesophyll tissues (0.53% DW). Conversely, elemental distribution maps of THF freeze-substituted tissues indicated significantly lower Ni, Ca and K concentrations than freeze-dried specimens and bulk tissue concentrations. Moreover, Ni concentrations were uniform across the whole specimen and no localisation was observed. In P. calomelanos var. austroamericana freeze-dried pinnule tissues, μ-PIXE revealed statistically similar As, Ca and K concentrations as compared to bulk tissue concentrations. Elemental distribution maps showed that As localisation was relatively uniform across the whole specimen. Once again, THF freeze-substituted tissues revealed a significant loss of As compared to freeze-dried specimens and the concentrations obtained by bulk tissue analysis. The results demonstrate that freeze-drying is a suitable sample preparation technique to study elemental distribution of ions in H. floribundus and P. calomelanos plant tissues using μ-PIXE spectroscopy. Furthermore, cellular structure was preserved in samples prepared using this technique.

Shaping field for deep tissue microscopy

NASA Astrophysics Data System (ADS)

Colon, J.; Lim, H.

2015-05-01

Information capacity of a lossless image-forming system is a conserved property determined by two imaging parameters - the resolution and the field of view (FOV). Adaptive optics improves the former by manipulating the phase, or wavefront, in the pupil plane. Here we describe a homologous approach, namely adaptive field microscopy, which aims to enhance the FOV by controlling the phase, or defocus, in the focal plane. In deep tissue imaging, the useful FOV can be severely limited if the region of interest is buried in a thick sample and not perpendicular to the optic axis. One must acquire many z-scans and reconstruct by post-processing, which exposes tissue to excessive radiation and is also time consuming. We demonstrate the effective FOV can be substantially enhanced by dynamic control of the image plane. Specifically, the tilt of the image plane is continuously adjusted in situ to match the oblique orientation of the sample plane within tissue. The utility of adaptive field microscopy is tested for imaging tissue with non-planar morphology. Ocular tissue of small animals was imaged by two-photon excited fluorescence. Our results show that adaptive field microscopy can utilize the full FOV. The freedom to adjust the image plane to account for the geometrical variations of sample could be extremely useful for 3D biological imaging. Furthermore, it could facilitate rapid surveillance of cellular features within deep tissue while avoiding photo damages, making it suitable for in vivo imaging.

Biochemical surface modification of Co-Cr-Mo.

PubMed

Puleo, D A

1996-01-01

Because of the limited mechanical properties of tissue substitutes formed by culturing cells on polymeric scaffolds, other approaches to tissue engineering must be explored for applications that require complete and immediate ability to bear weight, e.g. total joint replacements. Biochemical surface modification offers a way to partially regulate events at the bone-implant interface to obtain preferred tissue responses. Tresyl chloride, gamma-aminopropyltriethoxysilane (APS) and p-nitrophenyl chloroformate (p-NPC) immobilization schemes were used to couple a model enzyme, trypsin, on bulk samples of Co-Cr-Mo. For comparison, samples were simply adsorbed with protein. The three derivatization schemes resulted in different patterns and levels of activity. Tresyl chloride was not effective in immobilizing active enzyme on Co-Cr-Mo. Aqueous silanization with 12.5% APS resulted in optimal immobilized activity. Activity on samples derivatized with 0.65 mg p-NPC cm-2 was four to five times greater than that on samples simple adsorbed with enzyme or optimally derivatized with APS and was about eight times that on tresylated samples. This work demonstrates that, although different methods have different effectiveness, chemical derivatization can be used to alter the amount and/or stability of biomolecules immobilized on the surface of Co-Cr-Mo.

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

Errors in Measuring Water Potentials of Small Samples Resulting from Water Adsorption by Thermocouple Psychrometer Chambers 1

PubMed Central

Bennett, Jerry M.; Cortes, Peter M.

1985-01-01

The adsorption of water by thermocouple psychrometer assemblies is known to cause errors in the determination of water potential. Experiments were conducted to evaluate the effect of sample size and psychrometer chamber volume on measured water potentials of leaf discs, leaf segments, and sodium chloride solutions. Reasonable agreement was found between soybean (Glycine max L. Merr.) leaf water potentials measured on 5-millimeter radius leaf discs and large leaf segments. Results indicated that while errors due to adsorption may be significant when using small volumes of tissue, if sufficient tissue is used the errors are negligible. Because of the relationship between water potential and volume in plant tissue, the errors due to adsorption were larger with turgid tissue. Large psychrometers which were sealed into the sample chamber with latex tubing appeared to adsorb more water than those sealed with flexible plastic tubing. Estimates are provided of the amounts of water adsorbed by two different psychrometer assemblies and the amount of tissue sufficient for accurate measurements of leaf water potential with these assemblies. It is also demonstrated that water adsorption problems may have generated low water potential values which in prior studies have been attributed to large cut surface area to volume ratios. PMID:16664367

Errors in measuring water potentials of small samples resulting from water adsorption by thermocouple psychrometer chambers.

PubMed

Bennett, J M; Cortes, P M

1985-09-01

The adsorption of water by thermocouple psychrometer assemblies is known to cause errors in the determination of water potential. Experiments were conducted to evaluate the effect of sample size and psychrometer chamber volume on measured water potentials of leaf discs, leaf segments, and sodium chloride solutions. Reasonable agreement was found between soybean (Glycine max L. Merr.) leaf water potentials measured on 5-millimeter radius leaf discs and large leaf segments. Results indicated that while errors due to adsorption may be significant when using small volumes of tissue, if sufficient tissue is used the errors are negligible. Because of the relationship between water potential and volume in plant tissue, the errors due to adsorption were larger with turgid tissue. Large psychrometers which were sealed into the sample chamber with latex tubing appeared to adsorb more water than those sealed with flexible plastic tubing. Estimates are provided of the amounts of water adsorbed by two different psychrometer assemblies and the amount of tissue sufficient for accurate measurements of leaf water potential with these assemblies. It is also demonstrated that water adsorption problems may have generated low water potential values which in prior studies have been attributed to large cut surface area to volume ratios.

Simultaneous sampling of tissue oxygenation and oxygen consumption in skeletal muscle.

PubMed

Nugent, William H; Song, Bjorn K; Pittman, Roland N; Golub, Aleksander S

2016-05-01

Under physiologic conditions, microvascular oxygen delivery appears to be well matched to oxygen consumption in respiring tissues. We present a technique to measure interstitial oxygen tension (PISFO2) and oxygen consumption (VO2) under steady-state conditions, as well as during the transitions from rest to activity and back. Phosphorescence Quenching Microscopy (PQM) was employed with pneumatic compression cycling to achieve 1 to 10 Hz sampling rates of interstitial PO2 and simultaneous recurrent sampling of VO2 (3/min) in the exteriorized rat spinotrapezius muscle. The compression pressure was optimized to 120-130 mmHg without adverse effect on the tissue preparation. A cycle of 5s compression followed by 15s recovery yielded a resting VO2 of 0.98 ± 0.03 ml O2/100 cm(3)min while preserving microvascular oxygen delivery. The measurement system was then used to assess VO2 dependence on PISFO2 at rest and further tested under conditions of isometric muscle contraction to demonstrate a robust ability to monitor the on-kinetics of tissue respiration and the compensatory changes in PISFO2 during contraction and recovery. The temporal and spatial resolution of this approach is well suited to studies seeking to characterize microvascular oxygen supply and demand in thin tissues. Copyright © 2015 Elsevier Inc. All rights reserved.

Colorectal cancer detection by hyperspectral imaging using fluorescence excitation scanning

NASA Astrophysics Data System (ADS)

Leavesley, Silas J.; Deal, Joshua; Hill, Shante; Martin, Will A.; Lall, Malvika; Lopez, Carmen; Rider, Paul F.; Rich, Thomas C.; Boudreaux, Carole W.

2018-02-01

Hyperspectral imaging technologies have shown great promise for biomedical applications. These techniques have been especially useful for detection of molecular events and characterization of cell, tissue, and biomaterial composition. Unfortunately, hyperspectral imaging technologies have been slow to translate to clinical devices - likely due to increased cost and complexity of the technology as well as long acquisition times often required to sample a spectral image. We have demonstrated that hyperspectral imaging approaches which scan the fluorescence excitation spectrum can provide increased signal strength and faster imaging, compared to traditional emission-scanning approaches. We have also demonstrated that excitation-scanning approaches may be able to detect spectral differences between colonic adenomas and adenocarcinomas and normal mucosa in flash-frozen tissues. Here, we report feasibility results from using excitation-scanning hyperspectral imaging to screen pairs of fresh tumoral and nontumoral colorectal tissues. Tissues were imaged using a novel hyperspectral imaging fluorescence excitation scanning microscope, sampling a wavelength range of 360-550 nm, at 5 nm increments. Image data were corrected to achieve a NIST-traceable flat spectral response. Image data were then analyzed using a range of supervised and unsupervised classification approaches within ENVI software (Harris Geospatial Solutions). Supervised classification resulted in >99% accuracy for single-patient image data, but only 64% accuracy for multi-patient classification (n=9 to date), with the drop in accuracy due to increased false-positive detection rates. Hence, initial data indicate that this approach may be a viable detection approach, but that larger patient sample sizes need to be evaluated and the effects of inter-patient variability studied.

An allogenic cell-based implant for meniscal lesions.

PubMed

Weinand, Christian; Peretti, Giuseppe M; Adams, Samuel B; Bonassar, Lawrence J; Randolph, Mark A; Gill, Thomas J

2006-11-01

Meniscal tears in the avascular zones do not heal. Although tissue-engineering approaches using cells seeded onto scaffolds could expand the indication for meniscal repair, harvesting autologous cells could cause additional trauma to the patient. Allogenic cells, however, could provide an unlimited amount of cells. Allogenic cells from 2 anatomical sources can repair lesions in the avascular region of the meniscus. Controlled laboratory study. Both autologous and allogenic chondrocytes were seeded onto a Vicryl mesh scaffold and sutured into a bucket-handle lesion created in the medial menisci of 17 swine. Controls consisted of 3 swine knees treated with unseeded implants and controls from a previous experiment in which 4 swine were treated with suture only and 4 with no treatment. Menisci were harvested after 12 weeks and evaluated histologically for new tissue and percentage of interface healing surface; they were also evaluated statistically. The lesions were closed in 15 of 17 menisci. None of the control samples demonstrated healing. Histologic analysis of sequential cuts through the lesion showed formation of new scar-like tissue in all experimental samples. One of 8 menisci was completely healed in the allogenic group and 2 of 9 in the autologous group; the remaining samples were partially healed in both groups. No statistically significant differences in the percentage of healing were observed between the autologous and allogenic cell-based implants. Use of autologous and allogenic chondrocytes delivered via a biodegradable mesh enhanced healing of avascular meniscal lesions. This study demonstrates the potential of a tissue-engineered cellular repair of the meniscus using autologous and allogenic chondrocytes.

Measuring optical properties of a blood vessel model using optical coherence tomography

NASA Astrophysics Data System (ADS)

Levitz, David; Hinds, Monica T.; Tran, Noi; Vartanian, Keri; Hanson, Stephen R.; Jacques, Steven L.

2006-02-01

In this paper we develop the concept of a tissue-engineered optical phantom that uses engineered tissue as a phantom for calibration and optimization of biomedical optics instrumentation. With this method, the effects of biological processes on measured signals can be studied in a well controlled manner. To demonstrate this concept, we attempted to investigate how the cellular remodeling of a collagen matrix affected the optical properties extracted from optical coherence tomography (OCT) images of the samples. Tissue-engineered optical phantoms of the vascular system were created by seeding smooth muscle cells in a collagen matrix. Four different optical properties were evaluated by fitting the OCT signal to 2 different models: the sample reflectivity ρ and attenuation parameter μ were extracted from the single scattering model, and the scattering coefficient μ s and root-mean-square scattering angle θ rms were extracted from the extended Huygens-Fresnel model. We found that while contraction of the smooth muscle cells was clearly evident macroscopically, on the microscopic scale very few cells were actually embedded in the collagen. Consequently, no significant difference between the cellular and acellular samples in either set of measured optical properties was observed. We believe that further optimization of our tissue-engineering methods is needed in order to make the histology and biochemistry of the cellular samples sufficiently different from the acellular samples on the microscopic level. Once these methods are optimized, we can better verify whether the optical properties of the cellular and acellular collagen samples differ.

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

Detection of Pseudomonas aeruginosa biomarkers from thermally injured mice in situ using imaging mass spectrometry.

PubMed

Hamerly, Timothy; Everett, Jake A; Paris, Nina; Fisher, Steve T; Karunamurthy, Arivarasan; James, Garth A; Rumbaugh, Kendra P; Rhoads, Daniel D; Bothner, Brian

2017-12-15

Monitoring patients with burn wounds for infection is standard practice because failure to rapidly and specifically identify a pathogen can result in poor clinical outcomes, including death. Therefore, a method that facilitates detection and identification of pathogens in situ within minutes of biopsy would be a significant benefit to clinicians. Mass spectrometry is rapidly becoming a standard tool in clinical settings, capable of identifying specific pathogens from complex samples. Imaging mass spectrometry (IMS) expands the information content by enabling spatial resolution of biomarkers in tissue samples as in histology, without the need for specific stains/antibodies. Herein, a murine model of thermal injury was used to study infection of burn tissue by Pseudomonas aeruginosa. This is the first use of IMS to detect P. aeruginosa infection in situ from thermally injured tissue. Multiple molecular features could be spatially resolved to infected or uninfected tissue. This demonstrates the potential use of IMS in a clinical setting to aid doctors in identifying both presence and species of pathogens in tissue. Copyright © 2017. Published by Elsevier Inc.

High-resolution, 2- and 3-dimensional imaging of uncut, unembedded tissue biopsy samples.

PubMed

Torres, Richard; Vesuna, Sam; Levene, Michael J

2014-03-01

Despite continuing advances in tissue processing automation, traditional embedding, cutting, and staining methods limit our ability for rapid, comprehensive visual examination. These limitations are particularly relevant to biopsies for which immediate therapeutic decisions are most necessary, faster feedback to the patient is desired, and preservation of tissue for ancillary studies is most important. The recent development of improved tissue clearing techniques has made it possible to consider use of multiphoton microscopy (MPM) tools in clinical settings, which could address difficulties of established methods. To demonstrate the potential of MPM of cleared tissue for the evaluation of unembedded and uncut pathology samples. Human prostate, liver, breast, and kidney specimens were fixed and dehydrated by using traditional histologic techniques, with or without incorporation of nucleic acid fluorescent stains into dehydration steps. A benzyl alcohol/benzyl benzoate clearing protocol was substituted for xylene. Multiphoton microscopy was performed on a home-built system. Excellent morphologic detail was achievable with MPM at depths greater than 500 μm. Pseudocoloring produced images analogous to hematoxylin-eosin-stained images. Concurrent second-harmonic generation detection allowed mapping of collagen. Subsequent traditional section staining with hematoxylin-eosin did not reveal any detrimental morphologic effects. Sample immunostains on renal tissue showed preservation of normal reactivity. Complete reconstructions of 1-mm cubic samples elucidated 3-dimensional architectural organization. Multiphoton microscopy on cleared, unembedded, uncut biopsy specimens shows potential as a practical clinical tool with significant advantages over traditional histology while maintaining compatibility with gold standard techniques. Further investigation to address remaining implementation barriers is warranted.

Quantifying the ultrastructure of carotid arteries using high-resolution micro-diffusion tensor imaging—comparison of intact versus open cut tissue

NASA Astrophysics Data System (ADS)

Salman Shahid, Syed; Gaul, Robert T.; Kerskens, Christian; Flamini, Vittoria; Lally, Caitríona

2017-12-01

Diffusion magnetic resonance imaging (dMRI) can provide insights into the microstructure of intact arterial tissue. The current study employed high magnetic field MRI to obtain ultra-high resolution dMRI at an isotropic voxel resolution of 117 µm3 in less than 2 h of scan time. A parameter selective single shell (128 directions) diffusion-encoding scheme based on Stejskel-Tanner sequence with echo-planar imaging (EPI) readout was used. EPI segmentation was used to reduce the echo time (TE) and to minimise the susceptibility-induced artefacts. The study utilised the dMRI analysis with diffusion tensor imaging (DTI) framework to investigate structural heterogeneity in intact arterial tissue and to quantify variations in tissue composition when the tissue is cut open and flattened. For intact arterial samples, the region of interest base comparison showed significant differences in fractional anisotropy and mean diffusivity across the media layer (p  <  0.05). For open cut flat samples, DTI based directionally invariant indices did not show significant differences across the media layer. For intact samples, fibre tractography based indices such as calculated helical angle and fibre dispersion showed near circumferential alignment and a high degree of fibre dispersion, respectively. This study demonstrates the feasibility of fast dMRI acquisition with ultra-high spatial and angular resolution at 7 T. Using the optimised sequence parameters, this study shows that DTI based markers are sensitive to local structural changes in intact arterial tissue samples and these markers may have clinical relevance in the diagnosis of atherosclerosis and aneurysm.

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.

Polarization sensitive optical coherence tomography – a review [Invited

PubMed Central

de Boer, Johannes F.; Hitzenberger, Christoph K.; Yasuno, Yoshiaki

2017-01-01

Optical coherence tomography (OCT) is now a well-established modality for high-resolution cross-sectional and three-dimensional imaging of transparent and translucent samples and tissues. Conventional, intensity based OCT, however, does not provide a tissue-specific contrast, causing an ambiguity with image interpretation in several cases. Polarization sensitive (PS) OCT draws advantage from the fact that several materials and tissues can change the light’s polarization state, adding an additional contrast channel and providing quantitative information. In this paper, we review basic and advanced methods of PS-OCT and demonstrate its use in selected biomedical applications. PMID:28663869

Transgenic zebrafish reveal tissue-specific differences in estrogen signaling in response to environmental water samples.

PubMed

Gorelick, Daniel A; Iwanowicz, Luke R; Hung, Alice L; Blazer, Vicki S; Halpern, Marnie E

2014-04-01

Environmental endocrine disruptors (EEDs) are exogenous chemicals that mimic endogenous hormones such as estrogens. Previous studies using a zebrafish transgenic reporter demonstrated that the EEDs bisphenol A and genistein preferentially activate estrogen receptors (ERs) in the larval heart compared with the liver. However, it was not known whether the transgenic zebrafish reporter was sensitive enough to detect estrogens from environmental samples, whether environmental estrogens would exhibit tissue-specific effects similar to those of BPA and genistein, or why some compounds preferentially target receptors in the heart. We tested surface water samples using a transgenic zebrafish reporter with tandem estrogen response elements driving green fluorescent protein expression (5xERE:GFP). Reporter activation was colocalized with tissue-specific expression of ER genes by RNA in situ hybridization. We observed selective patterns of ER activation in transgenic fish exposed to river water samples from the Mid-Atlantic United States, with several samples preferentially activating receptors in embryonic and larval heart valves. We discovered that tissue specificity in ER activation was due to differences in the expression of ER subtypes. ERα was expressed in developing heart valves but not in the liver, whereas ERβ2 had the opposite profile. Accordingly, subtype-specific ER agonists activated the reporter in either the heart valves or the liver. The use of 5xERE:GFP transgenic zebrafish revealed an unexpected tissue-specific difference in the response to environmentally relevant estrogenic compounds. Exposure to estrogenic EEDs in utero was associated with adverse health effects, with the potentially unanticipated consequence of targeting developing heart valves.

Transgenic zebrafish reveal tissue-specific differences in estrogen signaling in response to environmental water samples

USGS Publications Warehouse

Gorelick, Daniel A.; Iwanowicz, Luke R.; Hung, Alice L.; Blazer, Vicki; Halpern, Marnie E.

2014-01-01

Background: Environmental endocrine disruptors (EED) are exogenous chemicals that mimic endogenous hormones, such as estrogens. Previous studies using a zebrafish transgenic reporter demonstrated that the EEDs bisphenol A and genistein preferentially activate estrogen receptors (ER) in the larval heart compared to the liver. However, it was not known whether the transgenic zebrafish reporter was sensitive enough to detect estrogens from environmental samples, whether environmental estrogens would exhibit similar tissue-specific effects as BPA and genistein or why some compounds preferentially target receptors in the heart. Methods: We tested surface water samples using a transgenic zebrafish reporter with tandem estrogen response elements driving green fluorescent protein expression (5xERE:GFP). Reporter activation was colocalized with tissue-specific expression of estrogen receptor genes by RNA in situ hybridization. Results: Selective patterns of ER activation were observed in transgenic fish exposed to river water samples from the Mid-Atlantic United States, with several samples preferentially activating receptors in embryonic and larval heart valves. We discovered that tissue-specificity in ER activation is due to differences in the expression of estrogen receptor subtypes. ERα is expressed in developing heart valves but not in the liver, whereas ERβ2 has the opposite profile. Accordingly, subtype-specific ER agonists activate the reporter in either the heart valves or the liver. Conclusion: The use of 5xERE:GFP transgenic zebrafish has revealed an unexpected tissue-specific difference in the response to environmentally relevant estrogenic compounds. Exposure to estrogenic EEDs in utero is associated with adverse health effects, with the potentially unanticipated consequence of targeting developing heart valves.

In vivo classification of human skin burns using machine learning and quantitative features captured by optical coherence tomography

NASA Astrophysics Data System (ADS)

Singla, Neeru; Srivastava, Vishal; Singh Mehta, Dalip

2018-02-01

We report the first fully automated detection of human skin burn injuries in vivo, with the goal of automatic surgical margin assessment based on optical coherence tomography (OCT) images. Our proposed automated procedure entails building a machine-learning-based classifier by extracting quantitative features from normal and burn tissue images recorded by OCT. In this study, 56 samples (28 normal, 28 burned) were imaged by OCT and eight features were extracted. A linear model classifier was trained using 34 samples and 22 samples were used to test the model. Sensitivity of 91.6% and specificity of 90% were obtained. Our results demonstrate the capability of a computer-aided technique for accurately and automatically identifying burn tissue resection margins during surgical treatment.

Identification of immune cell infiltration in hematoxylin-eosin stained breast cancer samples: texture-based classification of tissue morphologies

NASA Astrophysics Data System (ADS)

Turkki, Riku; Linder, Nina; Kovanen, Panu E.; Pellinen, Teijo; Lundin, Johan

2016-03-01

The characteristics of immune cells in the tumor microenvironment of breast cancer capture clinically important information. Despite the heterogeneity of tumor-infiltrating immune cells, it has been shown that the degree of infiltration assessed by visual evaluation of hematoxylin-eosin (H and E) stained samples has prognostic and possibly predictive value. However, quantification of the infiltration in H and E-stained tissue samples is currently dependent on visual scoring by an expert. Computer vision enables automated characterization of the components of the tumor microenvironment, and texture-based methods have successfully been used to discriminate between different tissue morphologies and cell phenotypes. In this study, we evaluate whether local binary pattern texture features with superpixel segmentation and classification with support vector machine can be utilized to identify immune cell infiltration in H and E-stained breast cancer samples. Guided with the pan-leukocyte CD45 marker, we annotated training and test sets from 20 primary breast cancer samples. In the training set of arbitrary sized image regions (n=1,116) a 3-fold cross-validation resulted in 98% accuracy and an area under the receiver-operating characteristic curve (AUC) of 0.98 to discriminate between immune cell -rich and - poor areas. In the test set (n=204), we achieved an accuracy of 96% and AUC of 0.99 to label cropped tissue regions correctly into immune cell -rich and -poor categories. The obtained results demonstrate strong discrimination between immune cell -rich and -poor tissue morphologies. The proposed method can provide a quantitative measurement of the degree of immune cell infiltration and applied to digitally scanned H and E-stained breast cancer samples for diagnostic purposes.

Identification of methylation haplotype blocks aids in deconvolution of heterogeneous tissue samples and tumor tissue-of-origin mapping from plasma DNA.

PubMed

Guo, Shicheng; Diep, Dinh; Plongthongkum, Nongluk; Fung, Ho-Lim; Zhang, Kang; Zhang, Kun

2017-04-01

Adjacent CpG sites in mammalian genomes can be co-methylated owing to the processivity of methyltransferases or demethylases, yet discordant methylation patterns have also been observed, which are related to stochastic or uncoordinated molecular processes. We focused on a systematic search and investigation of regions in the full human genome that show highly coordinated methylation. We defined 147,888 blocks of tightly coupled CpG sites, called methylation haplotype blocks, after analysis of 61 whole-genome bisulfite sequencing data sets and validation with 101 reduced-representation bisulfite sequencing data sets and 637 methylation array data sets. Using a metric called methylation haplotype load, we performed tissue-specific methylation analysis at the block level. Subsets of informative blocks were further identified for deconvolution of heterogeneous samples. Finally, using methylation haplotypes we demonstrated quantitative estimation of tumor load and tissue-of-origin mapping in the circulating cell-free DNA of 59 patients with lung or colorectal cancer.

Evaluation of a multi-fibre needle Raman probe for tissue analysis

NASA Astrophysics Data System (ADS)

Fullwood, Leanne M.; Iping Petterson, Ingeborg E.; Dudgeon, Alexander P.; Lloyd, Gavin R.; Kendall, Catherine; Hall, Charlie; Day, John C. C.; Stone, Nick

2016-03-01

Raman spectroscopy is a rapid technique for the identification of cancers. Its coupling with a hypodermic needle provides a minimally invasive instrument with the potential to aid real time assessment of suspicious lesions in vivo and guide surgery. A fibre optic Raman needle probe was utilised in this study to evaluate the classification ability of the instrument as a diagnostic tool together with multivariate analysis, through measurements of tissues from different animal species as well as various different porcine tissue types. Cross validation was performed and preliminary classification accuracies were calculated as 100% for the identification of tissue type and 97.5% for the identification of animal species. A lymph node sample was also measured using the needle probe to assess the use of the technique for human tissue and hence its efficiency as a clinical instrument. This needle probe has been demonstrated to have the capabilities to classify tissue samples based on their biochemical components. The Raman needle probe also has the potential to act as a diagnostic and surgical tool to delineate cancerous from non-cancerous cells in real time, thus assisting complete removal of a tumour.

Automated tumor analysis for molecular profiling in lung cancer

PubMed Central

Boyd, Clinton; James, Jacqueline A.; Loughrey, Maurice B.; Hougton, Joseph P.; Boyle, David P.; Kelly, Paul; Maxwell, Perry; McCleary, David; Diamond, James; McArt, Darragh G.; Tunstall, Jonathon; Bankhead, Peter; Salto-Tellez, Manuel

2015-01-01

The discovery and clinical application of molecular biomarkers in solid tumors, increasingly relies on nucleic acid extraction from FFPE tissue sections and subsequent molecular profiling. This in turn requires the pathological review of haematoxylin & eosin (H&E) stained slides, to ensure sample quality, tumor DNA sufficiency by visually estimating the percentage tumor nuclei and tumor annotation for manual macrodissection. In this study on NSCLC, we demonstrate considerable variation in tumor nuclei percentage between pathologists, potentially undermining the precision of NSCLC molecular evaluation and emphasising the need for quantitative tumor evaluation. We subsequently describe the development and validation of a system called TissueMark for automated tumor annotation and percentage tumor nuclei measurement in NSCLC using computerized image analysis. Evaluation of 245 NSCLC slides showed precise automated tumor annotation of cases using Tissuemark, strong concordance with manually drawn boundaries and identical EGFR mutational status, following manual macrodissection from the image analysis generated tumor boundaries. Automated analysis of cell counts for % tumor measurements by Tissuemark showed reduced variability and significant correlation (p < 0.001) with benchmark tumor cell counts. This study demonstrates a robust image analysis technology that can facilitate the automated quantitative analysis of tissue samples for molecular profiling in discovery and diagnostics. PMID:26317646

Neuroprotective effects of Quercetin on radiation-induced brain injury in rats.

PubMed

Kale, Aydemir; Piskin, Özcan; Bas, Yilmaz; Aydin, Bengü Gülhan; Can, Murat; Elmas, Özlem; Büyükuysal, Çagatay

2018-04-24

Extensive research has been focused on radiation-induced brain injury. Animal and human studies have shown that flavonoids have remarkable toxicological profiles. This study aims to investigate the neuroprotective effects of quercetin in an experimental radiation-induced brain injury. A total of 32 adult male Wistar-Albino rats were randomly divided into four groups (control, quercetin, radiation, and radiation+quercetin groups, with eight rats in each group). Doses (50 mg/kg) of quercetin were administered to the animals in the quercetin and radiation+quercetin groups; radiation and radiation+quercetin groups were exposed to a dose of 20 Gy to the cranium region. Tissue samples, and biochemical levels of tissue injury markers in the four groups were compared. In all measured parameters of oxidative stress, administration of quercetin significantly demonstrated favorable effects. Both plasma and tissue levels of malondialdehyde and total antioxidant status significantly changed in favor of antioxidant activity. Histopathological evaluation of the tissues also demonstrated a significant decrease in cellular degeneration and infiltration parameters after quercetin administration. Quercetin demonstrated significant neuroprotection after radiation-induced brain injury. Further studies of neurological outcomes under different experimental settings are required in order to achieve conclusive results.

Silver halide fiber optic radiometry for temperature monitoring and control of tissues heated by microwave

NASA Astrophysics Data System (ADS)

Shenfeld, Ofer; Belotserkovsky, Edward; Goldwasser, Benad; Zur, Albert; Katzir, Abraham

1993-02-01

The heating of tissue by microwave radiation has attained a place of importance in various medical fields, such as the treatment of malignancies, urinary retention, and hypothermia. Accurate temperature measurements in these treated tissues is important for treatment planning and for the control of the heating process. It is also important to be able to measure spacial temperature distribution in the tissues because they are heated in a nonuniform way by the microwave radiation. Conventional temperature sensors used today are inaccurate in the presence of microwave radiation and require contact with the heated tissue. Fiber optic radiometry makes it possible to measure temperatures accurately in the presence of microwave radiation and does not require contact with the tissue. Accurate temperature measurements of tissues heated by microwave was obtained using a silver halide optic radiometer, enabling control of the heating process in other regions of the tissue samples. Temperature mappings of the heated tissues were performed and the nonuniform temperature distributions in these tissues was demonstrated.

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.

Multispectral fluorescence imaging of human ovarian and Fallopian tube tissue for early stage cancer detection

NASA Astrophysics Data System (ADS)

Tate, Tyler; Baggett, Brenda; Rice, Photini; Watson, Jennifer; Orsinger, Gabe; Nymeyer, Ariel C.; Welge, Weston A.; Keenan, Molly; Saboda, Kathylynn; Roe, Denise J.; Hatch, Kenneth; Chambers, Setsuko; Black, John; Utzinger, Urs; Barton, Jennifer

2015-03-01

With early detection, five year survival rates for ovarian cancer are over 90%, yet no effective early screening method exists. Emerging consensus suggests that perhaps over 50% of the most lethal form of the disease, high grade serous ovarian cancer, originates in the Fallopian tube. Cancer changes molecular concentrations of various endogenous fluorophores. Using specific excitation wavelengths and emissions bands on a Multispectral Fluorescence Imaging (MFI) system, spatial and spectral data over a wide field of view can be collected from endogenous fluorophores. Wavelength specific reflectance images provide additional information to normalize for tissue geometry and blood absorption. Ratiometric combination of the images may create high contrast between neighboring normal and abnormal tissue. Twenty-six women undergoing oophorectomy or debulking surgery consented the use of surgical discard tissue samples for MFI imaging. Forty-nine pieces of ovarian tissue and thirty-two pieces of Fallopian tube tissue were collected and imaged with excitation wavelengths between 280 nm and 550 nm. After imaging, each tissue sample was fixed, sectioned and HE stained for pathological evaluation. Comparison of mean intensity values between normal, benign, and cancerous tissue demonstrate a general trend of increased fluorescence of benign tissue and decreased fluorescence of cancerous tissue when compared to normal tissue. The predictive capabilities of the mean intensity measurements are tested using multinomial logistic regression and quadratic discriminant analysis. Adaption of the system for in vivo Fallopian tube and ovary endoscopic imaging is possible and is briefly described.

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.

Multispectral photoacoustic microscopy of lipids using a pulsed supercontinuum laser.

PubMed

Buma, Takashi; Conley, Nicole C; Choi, Sang Won

2018-01-01

We demonstrate optical resolution photoacoustic microscopy (OR-PAM) of lipid-rich tissue between 1050-1714 nm using a pulsed supercontinuum laser based on a large-mode-area photonic crystal fiber. OR-PAM experiments of lipid-rich samples show the expected optical absorption peaks near 1210 and 1720 nm. These results show that pulsed supercontinuum lasers are promising for OR-PAM applications such as label-free histology of lipid-rich tissue and imaging small animal models of disease.

OY-TES-1 expression and serum immunoreactivity in epithelial ovarian cancer.

PubMed

Tammela, Jonathan; Uenaka, Akiko; Ono, Toshiro; Noguchi, Yuji; Jungbluth, Achim A; Mhawech-Fauceglia, Paulette; Qian, Feng; Schneider, Sally; Sharma, Sameer; Driscoll, Deborah; Lele, Shashikant; Old, Lloyd J; Nakayama, Eiichi; Odunsi, Kunle

2006-10-01

OY-TES-1 is a novel target that belongs to the family of 'cancer/testis' (CT) antigens. Our goal was to examine the expression and immunogenicity of OY-TES-1 in epithelial ovarian cancer (EOC) to determine its potential as a target for vaccine therapy. OY-TES-1 expression was determined by one-step reverse transcriptase PCR on 100 EOC samples, 5 EOC cell lines, and a panel of normal tissues. Immunohistochemistry (IHC) was performed on the same panel of EOC tissues. Sera from a sub-group of patients were tested for OY-TES-1 antibody by ELISA. Thymus and leukocytes were weakly positive for OY-TES-1 while the remaining 5 normal tissues were negative. Expression of OY-TES-1 by either RT-PCR and/or IHC was demonstrable in 69/100 (69%) tumors. Humoral immunity to OY-TES-1 was demonstrated in 1/10 (10%) serum samples from patients whose tumors expressed the antigen. The median follow-up of the patient population was 34 months. There was no correlation between antigen expression and stage, grade, histology and survival. OY-TES-1 is expressed in 69% of patients with EOC, is absent from normal ovarian tissue, and a proportion of patients show evidence of a specific humoral immune response. These findings make OY-TES-1 an attractive target for antigen-specific immunotherapy in EOC.

Automated Liquid Microjunction Surface Sampling-HPLC-MS/MS Analysis of Drugs and Metabolites in Whole-Body Thin Tissue Sections

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

Kertesz, Vilmos; Van Berkel, Gary J

A fully automated liquid extraction-based surface sampling system utilizing a commercially available autosampler coupled to high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) detection is reported. Discrete spots selected for droplet-based sampling and automated sample queue generation for both the autosampler and MS were enabled by using in-house developed software. In addition, co-registration of spatially resolved sampling position and HPLC-MS information to generate heatmaps of compounds monitored for subsequent data analysis was also available in the software. The system was evaluated with whole-body thin tissue sections from propranolol dosed rat. The hands-free operation of the system was demonstrated by creating heatmapsmore » of the parent drug and its hydroxypropranolol glucuronide metabolites with 1 mm resolution in the areas of interest. The sample throughput was approximately 5 min/sample defined by the time needed for chromatographic separation. The spatial distributions of both the drug and its metabolites were consistent with previous studies employing other liquid extraction-based surface sampling methodologies.« less

Transgenic Zebrafish Reveal Tissue-Specific Differences in Estrogen Signaling in Response to Environmental Water Samples

PubMed Central

Iwanowicz, Luke R.; Hung, Alice L.; Blazer, Vicki S.; Halpern, Marnie E.

2014-01-01

Background: Environmental endocrine disruptors (EEDs) are exogenous chemicals that mimic endogenous hormones such as estrogens. Previous studies using a zebrafish transgenic reporter demonstrated that the EEDs bisphenol A and genistein preferentially activate estrogen receptors (ERs) in the larval heart compared with the liver. However, it was not known whether the transgenic zebrafish reporter was sensitive enough to detect estrogens from environmental samples, whether environmental estrogens would exhibit tissue-specific effects similar to those of BPA and genistein, or why some compounds preferentially target receptors in the heart. Methods: We tested surface water samples using a transgenic zebrafish reporter with tandem estrogen response elements driving green fluorescent protein expression (5xERE:GFP). Reporter activation was colocalized with tissue-specific expression of ER genes by RNA in situ hybridization. Results: We observed selective patterns of ER activation in transgenic fish exposed to river water samples from the Mid-Atlantic United States, with several samples preferentially activating receptors in embryonic and larval heart valves. We discovered that tissue specificity in ER activation was due to differences in the expression of ER subtypes. ERα was expressed in developing heart valves but not in the liver, whereas ERβ2 had the opposite profile. Accordingly, subtype-specific ER agonists activated the reporter in either the heart valves or the liver. Conclusion: The use of 5xERE:GFP transgenic zebrafish revealed an unexpected tissue-specific difference in the response to environmentally relevant estrogenic compounds. Exposure to estrogenic EEDs in utero was associated with adverse health effects, with the potentially unanticipated consequence of targeting developing heart valves. Citation: Gorelick DA, Iwanowicz LR, Hung AL, Blazer VS, Halpern ME. 2014. Transgenic zebrafish reveal tissue-specific differences in estrogen signaling in response to environmental water samples. Environ Health Perspect 122:356–362; http://dx.doi.org/10.1289/ehp.1307329 PMID:24425189

Epimicrobiota Associated with the Decay and Recovery of Orbicella Corals Exhibiting Dark Spot Syndrome

PubMed Central

Meyer, Julie L.; Rodgers, John M.; Dillard, Brian A.; Paul, Valerie J.; Teplitski, Max

2016-01-01

Dark Spot Syndrome (DSS) is one of the most common diseases of boulder corals in the Caribbean. It presents as sunken brown lesions in coral tissue, which can spread quickly over coral colonies. With this study, we tested the hypothesis that similar to other coral diseases, DSS is a dysbiosis characterized by global shifts in the coral microbiome. Because Black Band Disease (BBD) was sometimes found following DSS lesions, we also tested the hypothesis that DSS is a precursor of BBD. To track disease initiation and progression 24 coral colonies were tagged. Of them five Orbicella annularis corals and three O. faveolata corals exhibited DSS lesions at tagging. Microbiota of lesions and apparently healthy tissues from DSS-affected corals over the course of 18 months were collected. Final visual assessment showed that five of eight corals incurred substantial tissue loss while two corals remained stable and one appeared to recover from DSS lesions. Illumina sequencing of the V6 region of bacterial 16S rRNA genes demonstrated no significant differences in bacterial community composition associated with healthy tissue or DSS lesions. The epimicrobiomes of both healthy tissue and DSS lesions contained high relative abundances of Operational Taxonomic Units assigned to Halomonas, an unclassified gammaproteobacterial genus, Moritella, an unclassified Rhodobacteraceae genus, Renibacterium, Pseudomonas, and Acinetobacter. The relative abundance of bacterial taxa was not significantly different between samples when grouped by tissue type (healthy tissue vs. DSS lesion), coral species, collection month, or the overall outcome of DSS-affected corals (substantial tissue loss vs. stable/recovered). Two of the tagged corals with substantial tissue loss also developed BBD during the 18-month sampling period. The bacterial community of the BBD layer was distinct from both healthy tissue and DSS lesions, with high relative abundances of the presumed BBD pathogen Roseofilum reptotaenium and an unclassified Bacteroidales genus, similar to previous results. Roseofilum was detected in all samples from this study, with the highest relative abundance in healthy tissue from DSS-affected corals sampled in August, suggesting that while DSS is not a precursor to BBD, DSS-affected corals are in a weakened state and therefore more susceptible to additional infections. PMID:27375605

Effects of Ultrasound Frequency and Tissue Stiffness on the Histotripsy Intrinsic Threshold for Cavitation

PubMed Central

Vlaisavljevich, Eli; Lin, Kuang-Wei; Maxwell, Adam; Warnez, Matthew; Mancia, Lauren; Singh, Rahul; Putnam, Andrew J.; Fowlkes, Brian; Johnsen, Eric; Cain, Charles; Xu, Zhen

2015-01-01

Histotripsy is an ultrasound ablation method that depends on the initiation of a cavitation bubble cloud to fractionate soft tissue. Previous work has demonstrated a cavitation cloud can be formed by a single pulse with one high amplitude negative cycle, when the negative pressure amplitude directly exceeds a pressure threshold intrinsic to the medium. We hypothesize that the intrinsic threshold in water-based tissues is determined by the properties of the water inside the tissue and changes in tissue stiffness or ultrasound frequency will have a minimal impact on the histotripsy intrinsic threshold. To test this hypothesis, the histotripsy intrinsic threshold was investigated both experimentally and theoretically. The probability of cavitation was measured by subjecting tissue phantoms with adjustable mechanical properties and ex vivo tissues to a histotripsy pulse of 1–2 cycles produced by 345 kHz, 500 kHz, 1.5 MHz, and 3 MHz histotripsy transducers. Cavitation was detected and characterized by passive cavitation detection and high-speed photography, from which the probability of cavitation was measured vs. pressure amplitude. The results demonstrated that the intrinsic threshold (the negative pressure at which probability=0.5) is independent of stiffness for Young’s moduli (E) < 1 MPa with only a small increase (~2–3 MPa) in the intrinsic threshold for tendon (E=380 MPa). Additionally, results for all samples showed only a small increase of ~2–3 MPa when the frequency was increased from 345 kHz to 3 MHz. The intrinsic threshold was measured to be between 24.7–30.6 MPa for all samples and frequencies tested in this study. Overall, the results of this study indicate that the intrinsic threshold to initiate a histotripsy bubble cloud is not significantly impacted by tissue stiffness or ultrasound frequency in hundreds of kHz to MHz range. PMID:25766571

Automated assessment of breast tissue density in non-contrast 3D CT images without image segmentation based on a deep CNN

NASA Astrophysics Data System (ADS)

Zhou, Xiangrong; Kano, Takuya; Koyasu, Hiromi; Li, Shuo; Zhou, Xinxin; Hara, Takeshi; Matsuo, Masayuki; Fujita, Hiroshi

2017-03-01

This paper describes a novel approach for the automatic assessment of breast density in non-contrast three-dimensional computed tomography (3D CT) images. The proposed approach trains and uses a deep convolutional neural network (CNN) from scratch to classify breast tissue density directly from CT images without segmenting the anatomical structures, which creates a bottleneck in conventional approaches. Our scheme determines breast density in a 3D breast region by decomposing the 3D region into several radial 2D-sections from the nipple, and measuring the distribution of breast tissue densities on each 2D section from different orientations. The whole scheme is designed as a compact network without the need for post-processing and provides high robustness and computational efficiency in clinical settings. We applied this scheme to a dataset of 463 non-contrast CT scans obtained from 30- to 45-year-old-women in Japan. The density of breast tissue in each CT scan was assigned to one of four categories (glandular tissue within the breast <25%, 25%-50%, 50%-75%, and >75%) by a radiologist as ground truth. We used 405 CT scans for training a deep CNN and the remaining 58 CT scans for testing the performance. The experimental results demonstrated that the findings of the proposed approach and those of the radiologist were the same in 72% of the CT scans among the training samples and 76% among the testing samples. These results demonstrate the potential use of deep CNN for assessing breast tissue density in non-contrast 3D CT images.

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.

Human dental pulp stem cells derived from cryopreserved dental pulp tissues of vital extracted teeth with disease demonstrate hepatic-like differentiation.

PubMed

Chen, Y K; Huang, Anderson H C; Chan, Anthony W S; Lin, L M

2016-06-01

Reviewing the literature, hepatic differentiation of human dental pulp stem cells (hDPSCs) from cryopreserved dental pulp tissues of vital extracted teeth with disease has not been studied. This study is aimed to evaluate the hypothesis that hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease could possess potential hepatic differentiation. Forty vital extracted teeth with disease recruited for hDPSCs isolation, stem cell characterization and hepatic differentiation were randomly and equally divided into group A (liquid nitrogen-stored dental pulp tissues) and group B (freshly derived dental pulp tissues). Samples of hDPSCs isolated from groups A and B but without hepatic growth factors formed negative controls. A well-differentiated hepatocellular carcinoma cell line was employed as a positive control. All the isolated hDPSCs from groups A and B showed hepatic-like differentiation with morphological change from a spindle-shaped to a polygonal shape and normal karyotype. Differentiated hDPSCs and the positive control expressed hepatic metabolic function genes and liver-specific genes. Glycogen storage of differentiated hDPSCs was noted from day 7 of differentiation-medium culture. Positive immunofluorescence staining of low-density lipoprotein and albumin was observed from day 14 of differentiation-medium culture; urea production in the medium was noted from week 6. No hepatic differentiation was observed for any of the samples of the negative controls. We not only demonstrated the feasibility of hepatic-like differentiation of hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease but also indicated that the differentiated cells possessed normal karyotype and were functionally close to normal hepatic-like cells. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Complete validation of a unique digestion assay to detect Trichinella larvae in horse meat demonstrates the reliability of this assay for meeting food safety and trade requirements.

PubMed

Forbes, L B; Hill, D E; Parker, S; Tessaro, S V; Gamble, H R; Gajadhar, A A

2008-03-01

A tissue digestion assay using a double separatory funnel procedure for the detection of Trichinella larvae in horse meat was validated for application in food safety programs and trade. The assay consisted of a pepsin-HCl digestion step to release larvae from muscle tissue and two sequential sedimentation steps in separatory funnels to recover and concentrate larvae for detection with a stereomicroscope. With defined critical control points, the assay was conducted within a quality assurance system compliant with International Organization for Standardization-International Electrotechnical Commission (ISO/IEC) 17025 guidelines. Samples used in the validation were obtained from horses experimentally infected with Trichinella spiralis to obtain a range of muscle larvae densities. One-, 5-, and 10-g samples of infected tissue were combined with 99, 95, and 90 g, respectively, of known negative horse tissue to create a 100-g sample for testing. Samples of 5 and 10 g were more likely to be positive than were 1-g samples when larval densities were less than three larvae per gram (lpg). This difference is important because ingested meat with 1 lpg is considered the threshold for clinical disease in humans. Using a 5-g sample size, all samples containing 1.3 to 2 lpg were detected, and 60 to 100% of samples with infected horse meat containing 0.1 to 0.7 lpg were detected. In this study, the double separatory funnel digestion assay was efficient and reliable for its intended use in food safety and trade. This procedure is the only digestion assay for Trichinella in horse meat that has been validated as consistent and effective at critical levels of sensitivity.

Soft tissue adhesion of polished versus glazed lithium disilicate ceramic for dental applications.

PubMed

Brunot-Gohin, C; Duval, J-L; Azogui, E-E; Jannetta, R; Pezron, I; Laurent-Maquin, D; Gangloff, S C; Egles, C

2013-09-01

Ceramics are widely used materials for prosthesis, especially in dental fields. Despite multiple biomedical applications, little is known about ceramic surface modifications and the resulting cell behavior at its contact. The aim of this study is to evaluate the biological response of polished versus glazed surface treatments on lithium disilicate dental ceramic. We studied a lithium disilicate ceramic (IPS e.max(®) Press, Ivoclar Vivadent) with 3 different surface treatments: raw surface treatment, hand polished surface treatment, and glazed surface treatment (control samples are Thermanox(®), Nunc). In order to evaluate the possible modulation of cell response at the surface of ceramic, we compared polished versus glazed ceramics using an organotypic culture model of chicken epithelium. Our results show that the surface roughness is not modified as demonstrated by equivalent Ra measurements. On the contrary, the contact angle θ in water is very different between polished (84°) and glazed (33°) samples. The culture of epithelial tissues allowed a very precise assessment of histocompatibility of these interfaces and showed that polished samples increased cell adhesion and proliferation as compared to glazed samples. Lithium disilicate polished ceramic provided better adhesion and proliferation than lithium disilicate glazed ceramic. Taken together, our results demonstrate for the first time, how it is possible to use simple surface modifications to finely modulate the adhesion of tissues. Our results will help dental surgeons to choose the most appropriate surface treatment for a specific clinical application, in particular for the ceramic implant collar. Copyright © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Preservation of Multiple Mammalian Tissues to Maximize Science Return from Ground Based and Spaceflight Experiments.

PubMed

Choi, Sungshin; Ray, Hami E; Lai, San-Huei; Alwood, Joshua S; Globus, Ruth K

2016-01-01

Even with recent scientific advancements, challenges posed by limited resources and capabilities at the time of sample dissection continue to limit the collection of high quality tissues from experiments that can be conducted only infrequently and at high cost, such as in space. The resources and time it takes to harvest tissues post-euthanasia, and the methods and duration of long duration storage, potentially have negative impacts on sample quantity and quality, thereby limiting the scientific outcome that can be achieved. The goals of this study were to optimize methods for both sample recovery and science return from rodent experiments, with possible relevance to both ground based and spaceflight studies. The first objective was to determine the impacts of tissue harvest time post-euthanasia, preservation methods, and storage duration, focusing on RNA quality and enzyme activities in liver and spleen as indices of sample quality. The second objective was to develop methods that will maximize science return by dissecting multiple tissues after long duration storage in situ at -80°C. Tissues of C57Bl/6J mice were dissected and preserved at various time points post-euthanasia and stored at -80°C for up to 11 months. In some experiments, tissues were recovered from frozen carcasses which had been stored at -80°C up to 7 months. RNA quantity and quality was assessed by measuring RNA Integrity Number (RIN) values using an Agilent Bioanalyzer. Additionally, the quality of tissues was assessed by measuring activities of hepatic enzymes (catalase, glutathione reductase and GAPDH). Fresh tissues were collected up to one hour post-euthanasia, and stored up to 11 months at -80°C, with minimal adverse effects on the RNA quality of either livers or RNAlater-preserved spleens. Liver enzyme activities were similar to those of positive controls, with no significant effect observed at any time point. Tissues dissected from frozen carcasses that had been stored for up to 7 months at -80°C had variable results, depending on the specific tissue analyzed. RNA quality of liver, heart, and kidneys were minimally affected after 6-7 months of storage at -80°C, whereas RNA degradation was evident in tissues such as small intestine, bone, and bone marrow when they were collected from the carcasses frozen for 2.5 months. These results demonstrate that 1) the protocols developed for spaceflight experiments with on-orbit dissections support the retrieval of high quality samples for RNA expression and some protein analyses, despite delayed preservation post-euthanasia or prolonged storage, and 2) many additional tissues for gene expression analysis can be obtained by dissection even following prolonged storage of the tissue in situ at -80°C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to spaceflight experiments that entail on-orbit sample recovery by astronauts.

[Transciptome among Mexicans: a large scale methodology to analyze the genetics expression profile of simultaneous samples in muscle, adipose tissue and lymphocytes obtained from the same individual].

PubMed

Bastarrachea, Raúl A; López-Alvarenga, Juan Carlos; Kent, Jack W; Laviada-Molina, Hugo A; Cerda-Flores, Ricardo M; Calderón-Garcidueñas, Ana Laura; Torres-Salazar, Amada; Torres-Salazar, Amanda; Nava-González, Edna J; Solis-Pérez, Elizabeth; Gallegos-Cabrales, Esther C; Cole, Shelley A; Comuzzie, Anthony G

2008-01-01

We describe the methodology used to analyze multiple transcripts using microarray techniques in simultaneous biopsies of muscle, adipose tissue and lymphocytes obtained from the same individual as part of the standard protocol of the Genetics of Metabolic Diseases in Mexico: GEMM Family Study. We recruited 4 healthy male subjects with BM1 20-41, who signed an informed consent letter. Subjects participated in a clinical examination that included anthropometric and body composition measurements, muscle biopsies (vastus lateralis) subcutaneous fat biopsies anda blood draw. All samples provided sufficient amplified RNA for microarray analysis. Total RNA was extracted from the biopsy samples and amplified for analysis. Of the 48,687 transcript targets queried, 39.4% were detectable in a least one of the studied tissues. Leptin was not detectable in lymphocytes, weakly expressed in muscle, but overexpressed and highly correlated with BMI in subcutaneous fat. Another example was GLUT4, which was detectable only in muscle and not correlated with BMI. Expression level concordance was 0.7 (p< 0.001) for the three tissues studied. We demonstrated the feasibility of carrying out simultaneous analysis of gene expression in multiple tissues, concordance of genetic expression in different tissues, and obtained confidence that this method corroborates the expected biological relationships among LEPand GLUT4. TheGEMM study will provide a broad and valuable overview on metabolic diseases, including obesity and type 2 diabetes.

Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

PubMed

Chan, Pek-Lan; Rose, Ray J; Abdul Murad, Abdul Munir; Zainal, Zamri; Low, Eng-Ti Leslie; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

2014-01-01

The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses.

Application of non-lethal stable isotope analysis to assess feeding patterns of juvenile pallid sturgeon Scaphirhynchus albus: a comparison of tissue types and sample preservation methods

USGS Publications Warehouse

Andvik, R.T.; VanDeHey, J.A.; Fincel, M.J.; French, William E.; Bertrand, K.N.; Chipps, Steven R.; Klumb, Robert A.; Graeb, B.D.S.

2010-01-01

Traditional techniques for stable isotope analysis (SIA) generally require sacrificing animals to collect tissue samples; this can be problematic when studying diets of endangered species such as the pallid sturgeon Scaphirhynchus albus. Our objectives were to (i) determine if pectoral fin tissue (non-lethal) could be a substitute for muscle tissue (lethal) in SIA of juvenile pallid sturgeon, and (ii) evaluate the influence of preservation techniques on stable isotope values. In the laboratory, individual juvenile pallid sturgeon were held for up to 186 day and fed chironomids, fish, or a commercially available pellet diet. Significant, positive relationships (r² ≥ 0.8) were observed between fin and muscle tissues for both δ15N and δ13C; in all samples isotopes were enriched in fins compared to muscle tissue. Chironomid and fish based diets of juvenile pallid sturgeon were distinguishable for fast growing fish (0.3 mm day−1) using stable δ15N and δ13C isotopes. Frozen and preserved fin tissue δ15N isotopes were strongly related (r2 = 0.89) but δ13C isotopes were weakly related (r2 = 0.16). Therefore, freezing is recommended for preservation of fin clips to avoid the confounding effect of enrichment by ethanol. This study demonstrates the utility of a non-lethal technique to assess time integrated food habits of juvenile pallid sturgeon and should be applicable to other threatened or endangered species.

Association between Randall's Plaque and Calcifying Nanoparticles

NASA Technical Reports Server (NTRS)

Citfcioglu, Neva; Vejdani, Kaveh; Lee, Olivia; Mathew, Grace; Aho, Katja M.; Kajander, Olavi; McKay, David S.; Jones, Jeffrey A.; Feiveson, Alan H.; Stoller, Marshall L.

2007-01-01

Randall initially described calcified subepithelial papillary plaques, which he hypothesized as nidi for kidney stone formation. The discovery of calcifying nanoparticles (CNP) in many calcifying processes of human tissues has raised another hypothesis about their possible involvement in urinary stone formation. This research is the first attempt to investigate the potential association of these two hypotheses. We collected renal papilla and blood samples from 17 human patients who had undergone laparoscopic nephrectomy due to neoplasia. Immunohistochemical staining (IHS) was applied on the tissue samples using monoclonal antibody 8D10 (mAb) against CNP. Homogenized papillary tissues and serum samples were cultured for CNP. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) analysis were performed on fixed papillary samples. Randall's plaques were visible on gross inspection in 11 out of 17 collected samples. IHS was positive for CNP antigen in 8 of these 11 visually positive samples, but in only 1 of the remaining 6 samples. SEM revealed spherical apatite formations in 14 samples, all of which had calcium and phosphate peaks detected by EDS analysis. From this study, there was some evidence of a link between the presence of Randall's plaques and the detection of CNP, also referred to as nanobacteria. Although causality was not demonstrated, these results suggest that further studies with negative control samples should be made to explore the etiology of Randall's plaque formation, thus leading to a better understanding of the pathogenesis of stone formation.

Multimodal optical analysis discriminates freshly extracted human sample of gliomas, metastases and meningiomas from their appropriate controls

NASA Astrophysics Data System (ADS)

Zanello, Marc; Poulon, Fanny; Pallud, Johan; Varlet, Pascale; Hamzeh, H.; Abi Lahoud, Georges; Andreiuolo, Felipe; Ibrahim, Ali; Pages, Mélanie; Chretien, Fabrice; di Rocco, Federico; Dezamis, Edouard; Nataf, François; Turak, Baris; Devaux, Bertrand; Abi Haidar, Darine

2017-02-01

Delineating tumor margins as accurately as possible is of primordial importance in surgical oncology: extent of resection is associated with survival but respect of healthy surrounding tissue is necessary for preserved quality of life. The real-time analysis of the endogeneous fluorescence signal of brain tissues is a promising tool for defining margins of brain tumors. The present study aims to demonstrate the feasibility of multimodal optical analysis to discriminate fresh samples of gliomas, metastases and meningiomas from their appropriate controls. Tumor samples were studied on an optical fibered endoscope using spectral and fluorescence lifetime analysis and then on a multimodal set-up for acquiring spectral, one and two-photon fluorescence images, second harmonic generation signals and two-photon fluorescence lifetime datasets. The obtained data allowed us to differentiate healthy samples from tumor samples. These results confirmed the possible clinical relevance of this real-time multimodal optical analysis. This technique can be easily applied to neurosurgical procedures for a better delineation of surgical margins.

Post-sampling release of free fatty acids - effects of heat stabilization and methods of euthanasia.

PubMed

Jernerén, Fredrik; Söderquist, Marcus; Karlsson, Oskar

2015-01-01

The field of lipid research has made progress and it is now possible to study the lipidome of cells and organelles. A basic requirement of a successful lipid study is adequate pre-analytical sample handling, as some lipids can be unstable and postmortem changes can cause substantial accumulation of free fatty acids (FFAs). The aim of the present study was to investigate the effects of conductive heat stabilization and euthanasia methods on FFA levels in the rat brain and liver using liquid chromatography tandem mass spectrometry. The analysis of brain homogenates clearly demonstrated phospholipase activity and time-dependent post-sampling changes in the lipid pool of snap frozen non-stabilized tissue. There was a significant increase in FFAs already at 2min, which continued over time. Heat stabilization was shown to be an efficient method to reduce phospholipase activity and ex vivo lipolysis. Post-sampling effects due to tissue thawing and sample preparation induced a massive release of FFAs (up to 3700%) from non-stabilized liver and brain tissues compared to heat stabilized tissue. Furthermore, the choice of euthanasia method significantly influenced the levels of FFAs in the brain. The FFAs were decreased by 15-44% in the group of animals euthanized by pentobarbital injection compared with CO2 inhalation or decapitation. Our results highlight the importance of considering euthanasia methods and pre-analytical treatment in lipid analysis, factors which may otherwise interfere with the outcome of the experiments. Copyright © 2014 Elsevier Inc. All rights reserved.

Methylation of Werner syndrome protein is associated with the occurrence and development of invasive meningioma via the regulation of Myc and p53 expression.

PubMed

Li, Puxian; Hao, Shuyu; Bi, Zhiyong; Zhang, Junting; Wu, Zhen; Ren, Xiaohui

2015-08-01

The aim of the present study was to investigate the positive rate of Werner syndrome protein (WRN) methylation in meningioma patients, and further assess the association between WRN methylation and the occurrence of meningioma. A total of 56 consecutive meningioma patients and 26 healthy individuals were enrolled in the study. A methylation-specific polymerase chain reaction assay was performed to detect the positive rate of WRN methylation in the peripheral blood and tissue samples collected from the recruited subjects. In addition, western blot analysis was performed to determine the protein expression levels of WRN, Myc and p53 in the peripheral blood and tissue samples. The positive rate of WRN methylation in the peripheral blood of the meningioma group was increased when compared with the control group (P<0.05). In addition, the protein expression levels of WRN were significantly decreased in the peripheral blood and tissue samples collected from the individuals with a positive WRN methylation status (P<0.05), as compared with the samples without WRN methylation. Furthermore, the protein expression levels of Myc and p53 were increased in the peripheral blood and tissue samples that exhibited positive WRN methylation when compared with those without WRN methylation (P<0.05). Therefore, WRN methylation was demonstrated to be associated with the occurrence and development of invasive meningioma, possibly through the regulation of Myc and p53 expression.

Secreted Gaussia princeps luciferase as a reporter of Escherichia coli replication in a mouse tissue cage model of infection.

PubMed

Liu, Mingyu; Blinn, Christina; McLeod, Sarah M; Wiseman, John W; Newman, Joseph V; Fisher, Stewart L; Walkup, Grant K

2014-01-01

Measurement of bacterial burden in animal infection models is a key component for both bacterial pathogenesis studies and therapeutic agent research. The traditional quantification means for in vivo bacterial burden requires frequent animal sacrifice and enumerating colony forming units (CFU) recovered from infection loci. To address these issues, researchers have developed a variety of luciferase-expressing bacterial reporter strains to enable bacterial detection in living animals. To date, all such luciferase-based bacterial reporters are in cell-associated form. Production of luciferase-secreting recombinant bacteria could provide the advantage of reporting CFU from both infection loci themselves and remote sampling (eg. body fluid and plasma). Toward this end, we have genetically manipulated a pathogenic Escherichia coli (E. coli) strain, ATCC25922, to secrete the marine copepod Gaussia princeps luciferase (Gluc), and assessed the use of Gluc as both an in situ and ex situ reporter for bacterial burden in mouse tissue cage infections. The E. coli expressing Gluc demonstrates in vivo imaging of bacteria in a tissue cage model of infection. Furthermore, secreted Gluc activity and bacterial CFUs recovered from tissue cage fluid (TCF) are correlated along 18 days of infection. Importantly, secreted Gluc can also be detected in plasma samples and serve as an ex situ indicator for the established tissue cage infection, once high bacterial burdens are achieved. We have demonstrated that Gluc from marine eukaryotes can be stably expressed and secreted by pathogenic E. coli in vivo to enable a facile tool for longitudinal evaluation of persistent bacterial infection.

DNA methylation age is elevated in breast tissue of healthy women.

PubMed

Sehl, Mary E; Henry, Jill E; Storniolo, Anna Maria; Ganz, Patricia A; Horvath, Steve

2017-07-01

Limited evidence suggests that female breast tissue ages faster than other parts of the body according to an epigenetic biomarker of aging known as the "epigenetic clock." However, it is unknown whether breast tissue samples from healthy women show a similar accelerated aging effect relative to other tissues, and what could drive this acceleration. The goal of this study is to validate our initial finding of advanced DNA methylation (DNAm) age in breast tissue, by directly comparing it to that of peripheral blood tissue from the same individuals, and to do a preliminary assessment of hormonal factors that could explain the difference. We utilized n = 80 breast and 80 matching blood tissue samples collected from 40 healthy female participants of the Susan G. Komen Tissue Bank at the Indiana University Simon Cancer Center who donated these samples at two time points spaced at least a year apart. DNA methylation levels (Illumina 450K platform) were used to estimate the DNAm age. DNAm age was highly correlated with chronological age in both peripheral blood (r = 0.94, p < 0.0001) and breast tissues (r = 0.86, p < 0.0001). A measure of epigenetic age acceleration (age-adjusted DNAm Age) was substantially increased in breast relative to peripheral blood tissue (p = 1.6 × 10 -11 ). The difference between DNAm age of breast and blood decreased with advancing chronologic age (r = -0.53, p = 4.4 × 10 -4 ). Our data clearly demonstrate that female breast tissue has a higher epigenetic age than blood collected from the same subject. We also observe that the degree of elevation in breast diminishes with advancing age. Future larger studies will be needed to examine associations between epigenetic age acceleration and cumulative hormone exposure.

A mixture model-based approach to the clustering of microarray expression data.

PubMed

McLachlan, G J; Bean, R W; Peel, D

2002-03-01

This paper introduces the software EMMIX-GENE that has been developed for the specific purpose of a model-based approach to the clustering of microarray expression data, in particular, of tissue samples on a very large number of genes. The latter is a nonstandard problem in parametric cluster analysis because the dimension of the feature space (the number of genes) is typically much greater than the number of tissues. A feasible approach is provided by first selecting a subset of the genes relevant for the clustering of the tissue samples by fitting mixtures of t distributions to rank the genes in order of increasing size of the likelihood ratio statistic for the test of one versus two components in the mixture model. The imposition of a threshold on the likelihood ratio statistic used in conjunction with a threshold on the size of a cluster allows the selection of a relevant set of genes. However, even this reduced set of genes will usually be too large for a normal mixture model to be fitted directly to the tissues, and so the use of mixtures of factor analyzers is exploited to reduce effectively the dimension of the feature space of genes. The usefulness of the EMMIX-GENE approach for the clustering of tissue samples is demonstrated on two well-known data sets on colon and leukaemia tissues. For both data sets, relevant subsets of the genes are able to be selected that reveal interesting clusterings of the tissues that are either consistent with the external classification of the tissues or with background and biological knowledge of these sets. EMMIX-GENE is available at http://www.maths.uq.edu.au/~gjm/emmix-gene/

The Antimicrobial Peptide Lysozyme Is Induced after Multiple Trauma

PubMed Central

Klüter, Tim; Fitschen-Oestern, Stefanie; Lippross, Sebastian; Weuster, Matthias; Pufe, Thomas; Tohidnezhad, Mersedeh; Beyer, Andreas; Seekamp, Andreas; Varoga, Deike

2014-01-01

The antimicrobial peptide lysozyme is an important factor of innate immunity and exerts high potential of antibacterial activity. In the present study we evaluated the lysozyme expression in serum of multiple injured patients and subsequently analyzed their possible sources and signaling pathways. Expression of lysozyme was examined in blood samples of multiple trauma patients from the day of trauma until 14 days after trauma by ELISA. To investigate major sources of lysozyme, its expression and regulation in serum samples, different blood cells, and tissue samples were analysed by ELISA and real-time PCR. Neutrophils and hepatocytes were stimulated with cytokines and supernatant of Staphylococcus aureus. The present study demonstrates the induction and release of lysozyme in serum of multiple injured patients. The highest lysozyme expression of all tested cells and tissues was detected in neutrophils. Stimulation with trauma-related factors such as interleukin-6 and S. aureus induced lysozyme expression. Liver tissue samples of patients without trauma show little lysozyme expression compared to neutrophils. After stimulation with bacterial fragments, lysozyme expression of hepatocytes is upregulated significantly. Toll-like receptor 2, a classic receptor of Gram-positive bacterial protein, was detected as a possible target for lysozyme induction. PMID:25258475

Manufacturing of biodegradable polyurethane scaffolds based on polycaprolactone using a phase separation method: physical properties and in vitro assay

PubMed Central

Asefnejad, Azadeh; Khorasani, Mohammad Taghi; Behnamghader, Aliasghar; Farsadzadeh, Babak; Bonakdar, Shahin

2011-01-01

Background Biodegradable polyurethanes have found widespread use in soft tissue engineering due to their suitable mechanical properties and biocompatibility. Methods In this study, polyurethane samples were synthesized from polycaprolactone, hexamethylene diisocyanate, and a copolymer of 1,4-butanediol as a chain extender. Polyurethane scaffolds were fabricated by a combination of liquid–liquid phase separation and salt leaching techniques. The effect of the NCO:OH ratio on porosity content and pore morphology was investigated. Results Scanning electron micrographs demonstrated that the scaffolds had a regular distribution of interconnected pores, with pore diameters of 50–300 μm, and porosities of 64%–83%. It was observed that, by increasing the NCO:OH ratio, the average pore size, compressive strength, and compressive modulus increased. L929 fibroblast and chondrocytes were cultured on the scaffolds, and all samples exhibited suitable cell attachment and growth, with a high level of biocompatibility. Conclusion These biodegradable polyurethane scaffolds demonstrate potential for soft tissue engineering applications. PMID:22072874

Senescence-associated SIN3B promotes inflammation and pancreatic cancer progression

PubMed Central

Rielland, Maïté; Cantor, David J.; Graveline, Richard; Hajdu, Cristina; Mara, Lisa; de Diego Diaz, Beatriz; Miller, George; David, Gregory

2014-01-01

Pancreatic ductal adenocarcinoma (PDAC) is strikingly resistant to conventional therapeutic approaches. We previously demonstrated that the histone deacetylase–associated protein SIN3B is essential for oncogene-induced senescence in cultured cells. Here, using a mouse model of pancreatic cancer, we have demonstrated that SIN3B is required for activated KRAS-induced senescence in vivo. Surprisingly, impaired senescence as the result of genetic inactivation of Sin3B was associated with delayed PDAC progression and correlated with an impaired inflammatory response. In murine and human pancreatic cells and tissues, levels of SIN3B correlated with KRAS-induced production of IL-1α. Furthermore, evaluation of human pancreatic tissue and cancer cells revealed that Sin3B was decreased in control and PDAC samples, compared with samples from patients with pancreatic inflammation. These results indicate that senescence-associated inflammation positively correlates with PDAC progression and suggest that SIN3B has potential as a therapeutic target for inhibiting inflammation-driven tumorigenesis. PMID:24691445

Characterizing viscoelastic mechanical properties of highly compliant polymers and biological tissues using impact indentation.

PubMed

Mijailovic, Aleksandar S; Qing, Bo; Fortunato, Daniel; Van Vliet, Krystyn J

2018-04-15

Precise and accurate measurement of viscoelastic mechanical properties becomes increasingly challenging as sample stiffness decreases to elastic moduli <1 kPa, largely due to difficulties detecting initial contact with the compliant sample surface. This limitation is particularly relevant to characterization of biological soft tissues and compliant gels. Here, we employ impact indentation which, in contrast to shear rheology and conventional indentation, does not require contact detection a priori, and present a novel method to extract viscoelastic moduli and relaxation time constants directly from the impact response. We first validate our approach by using both impact indentation and shear rheology to characterize polydimethylsiloxane (PDMS) elastomers of stiffness ranging from 100 s of Pa to nearly 10 kPa. Assuming a linear viscoelastic constitutive model for the material, we find that the moduli and relaxation times obtained from fitting the impact response agree well with those obtained from fitting the rheological response. Next, we demonstrate our validated method on hydrated, biological soft tissues obtained from porcine brain, murine liver, and murine heart, and report the equilibrium shear moduli, instantaneous shear moduli, and relaxation time constants for each tissue. Together, our findings provide a new and straightforward approach capable of probing local mechanical properties of highly compliant viscoelastic materials with millimeter scale spatial resolution, mitigating complications involving contact detection or sample geometric constraints. Characterization and optimization of mechanical properties can be essential for the proper function of biomaterials in diverse applications. However, precise and accurate measurement of viscoelastic mechanical properties becomes increasingly difficult with increased compliance (particularly for elastic moduli <1 kPa), largely due to challenges detecting initial contact with the compliant sample surface and measuring response at short timescale or high frequency. By contrast, impact indentation has highly accurate contact detection and can be used to measure short timescale (glassy) response. Here, we demonstrate an experimental and analytical method that confers significant advantages over existing approaches to extract spatially resolved viscoelastic moduli and characteristic time constants of biological tissues (e.g., brain and heart) and engineered biomaterials. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Focused ion beam (FIB)/scanning electron microscopy (SEM) in tissue structural research.

PubMed

Leser, Vladka; Milani, Marziale; Tatti, Francesco; Tkalec, Ziva Pipan; Strus, Jasna; Drobne, Damjana

2010-10-01

The focused ion beam (FIB) and scanning electron microscope (SEM) are commonly used in material sciences for imaging and analysis of materials. Over the last decade, the combined FIB/SEM system has proven to be also applicable in the life sciences. We have examined the potential of the focused ion beam/scanning electron microscope system for the investigation of biological tissues of the model organism Porcellio scaber (Crustacea: Isopoda). Tissue from digestive glands was prepared as for conventional SEM or as for transmission electron microscopy (TEM). The samples were transferred into FIB/SEM for FIB milling and an imaging operation. FIB-milled regions were secondary electron imaged, back-scattered electron imaged, or energy dispersive X-ray (EDX) analyzed. Our results demonstrated that FIB/SEM enables simultaneous investigation of sample gross morphology, cell surface characteristics, and subsurface structures. The same FIB-exposed regions were analyzed by EDX to provide basic compositional data. When samples were prepared as for TEM, the information obtained with FIB/SEM is comparable, though at limited magnification, to that obtained from TEM. A combination of imaging, micro-manipulation, and compositional analysis appears of particular interest in the investigation of epithelial tissues, which are subjected to various endogenous and exogenous conditions affecting their structure and function. The FIB/SEM is a promising tool for an overall examination of epithelial tissue under normal, stressed, or pathological conditions.

Equilibrium sampling informs tissue residue and sediment remediation for pyrethroid insecticides in mariculture: A laboratory demonstration.

PubMed

Li, Juan-Ying; Shi, Wenxuan; Li, Zhenhua; Chen, Yiqin; Shao, Liu; Jin, Ling

2018-03-01

Mariculture product safety in relation to sediment quality has attracted increasing attention because of the accumulation of potentially hazardous chemicals, including pyrethroid insecticides, in sediment. Passive sampling has been widely used to assess the bioavailability of sediment-associated hydrophobic organic contaminants and predict their body residue in benthic organisms. Therefore, in this study, we introduced polydimethylsiloxane (PDMS) polymer as a biomimetic "chemometer" for freely-dissolved concentrations (C free ) to assess the efficacy of different carbon sorbents in reducing the bioavailability of pyrethroids in the process of sediment remediation. Black carbon (BC)-based materials (e.g., charcoal, biochar, and activated carbon) showed the advantageous sorption capacity over humic substance-based peat soil based on both C free and tissue residue in exposed clams. Of the tested BC-type materials, biochar appeared to be an ideal one in the remediation of pyrethroid-contaminated sediment. The predictive value of the PDMS chemometer approach to informing tissue residue was confirmed by a good agreement between the measured lipid-normalized concentrations of pyrethroids in clams and the lipid-based equilibrium concentrations calculated from C free via lipid-water partition coefficients. The quantitative inter-compartmental relationship underlying the laboratory system of sediment-pore water-PDMS-biota was also cross-validated by a mechanistically-based bioaccumulation model, thus confirming the validity of C free as a predictive intermediate to alert for tissue residue and guide sediment remediation. The present study revealed a great promise of sensing C free by polymer-based equilibrium sampling in predicting tissue residue of chemicals applied in mariculture against regulatory guidelines, and, in turn, informing remediation measures when needs arise. In situ demonstration is warranted in the future to ascertain the field applicability of this approach in real mariculture systems. Copyright © 2017 Elsevier B.V. All rights reserved.

The effects of Magnetic Resonance Imaging-guided High-Intensity Focused Ultrasound ablation on human cadaver breast tissue.

PubMed

Merckel, Laura G; Deckers, Roel; Baron, Paul; Bleys, Ronald L A W; van Diest, Paul J; Moonen, Chrit T W; Mali, Willem P Th M; van den Bosch, Maurice A A J; Bartels, Lambertus W

2013-10-05

Magnetic Resonance Imaging-guided High-Intensity Focused Ultrasound (MR-HIFU) is a promising technique for non-invasive breast tumor ablation. The purpose of this study was to investigate the effects of HIFU ablation and thermal exposure on ex vivo human breast tissue. HIFU ablations were performed in three unembalmed cadaveric breast specimens using a clinical MR-HIFU system. Sonications were performed in fibroglandular and adipose tissue. During HIFU ablation, time-resolved anatomical MR images were acquired to monitor macroscopic tissue changes. Furthermore, the breast tissue temperature was measured using a thermocouple to investigate heating and cooling under HIFU exposure. After HIFU ablation, breast tissue samples were excised and prepared for histopathological analysis. In addition, thermal exposure experiments were performed to distinguish between different levels of thermal damage using immunohistochemical staining. Irreversible macroscopic deformations up to 3.7 mm were observed upon HIFU ablation both in fibroglandular and in adipose tissue. No relationship was found between the sonication power or the maximum tissue temperature and the size of the deformations. Temperature measurements after HIFU ablation showed a slow decline in breast tissue temperature. Histopathological analysis of sonicated regions demonstrated ablated tissue and morphologically complete cell death. After thermal exposure, samples exposed to three different temperatures could readily be distinguished. In conclusion, the irreversible macroscopic tissue deformations in ex vivo human breast tissue observed during HIFU ablation suggest that it might be relevant to monitor tissue deformations during MR-HIFU treatments. Furthermore, the slow decrease in breast tissue temperature after HIFU ablation increases the risk of heat accumulation between successive sonications. Since cell death was inflicted after already 5 minutes at 75°C, MR-HIFU may find a place in non-invasive treatment of breast tumors. © 2013 Elsevier B.V. All rights reserved.

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.

Biological effects of two successive shock waves focused on liver tissues and melanoma cells.

PubMed

Benes, J; Sunka, P; Králová, J; Kaspar, J; Poucková, P

2007-01-01

A new generator of two successive shock waves focused to a common focal point has been developed. Cylindrical pressure waves created by multichannel electrical discharges on two cylindrical composite anodes are focused by a metallic parabolic reflector - cathode, and near the focus they are transformed to strong shock waves. Schlieren photos of the focal region have demonstrated that mutual interaction of the two waves results in generation of a large number of secondary short-wavelength shocks. Interaction of the focused shockwaves with liver tissues and cancer cell suspensions was investigated. Localized injury of rabbit liver induced by the shock waves was demonstrated by magnetic resonance imaging. Histological analysis of liver samples taken from the injured region revealed that the transition between the injured and the healthy tissues is sharp. Suspension of melanoma B16 cells was exposed and the number of the surviving cells rapidly decreased with increasing number of shocks and only 8 % of cells survived 350 shocks. Photographs of cells demonstrate that even small number of shocks results in perforation of cell membranes.

Increased epidermal laser fluence through simultaneous ultrasonic microporation

NASA Astrophysics Data System (ADS)

Whiteside, Paul J. D.; Chininis, Jeff A.; Schellenberg, Mason W.; Qian, Chenxi; Hunt, Heather K.

2016-03-01

Lasers have demonstrated widespread applicability in clinical dermatology as minimally invasive instruments that achieve photogenerated responses within tissue. However, before reaching its target, the incident light must first transmit through the surface layer of tissue, which is interspersed with chromophores (e.g. melanin) that preferentially absorb the light and may also generate negative tissue responses. These optical absorbers decrease the efficacy of the procedures. In order to ensure that the target receives a clinically relevant dose, most procedures simply increase the incident energy; however, this tends to exacerbate the negative complications of melanin absorption. Here, we present an alternative solution aimed at increasing epidermal energy uence while mitigating excess absorption by unintended targets. Our technique involves the combination of a waveguide-based contact transmission modality with simultaneous high-frequency ultrasonic pulsation, which alters the optical properties of the tissue through the agglomeration of dissolved gasses into micro-bubbles within the tissue. Doing so effectively creates optically transparent pathways for the light to transmit unobstructed through the tissue, resulting in an increase in forward scattering and a decrease in absorption. To demonstrate this, Q-switched nanosecond-pulsed laser light at 532nm was delivered into pig skin samples using custom glass waveguides clad in titanium and silver. Light transmission through the tissue was measured with a photodiode and integrating sphere for tissue with and without continuous ultrasonic pulsation at 510 kHz. The combination of these techniques has the potential to improve the efficiency of laser procedures while mitigating negative tissue effects caused by undesirable absorption.

Profiling of adrenocorticotropic hormone and arginine vasopressin in human pituitary gland and tumor thin tissue sections using droplet-based liquid-microjunction surface-sampling-HPLC–ESI-MS–MS

DOE PAGES

Kertesz, Vilmos; Calligaris, David; Feldman, Daniel R.; ...

2015-06-18

Described here are the results from the profiling of the proteins arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) from normal human pituitary gland and pituitary adenoma tissue sections using a fully automated droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system for spatially resolved sampling, HPLC separation, and mass spectral detection. Excellent correlation was found between the protein distribution data obtained with this droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system and those data obtained with matrix assisted laser desorption ionization (MALDI) chemical imaging analyses of serial sections of the same tissue. The protein distributions correlated with the visible anatomic pattern of the pituitary gland.more » AVP was most abundant in the posterior pituitary gland region (neurohypophysis) and ATCH was dominant in the anterior pituitary gland region (adenohypophysis). The relative amounts of AVP and ACTH sampled from a series of ACTH secreting and non-secreting pituitary adenomas correlated with histopathological evaluation. ACTH was readily detected at significantly higher levels in regions of ACTH secreting adenomas and in normal anterior adenohypophysis compared to non-secreting adenoma and neurohypophysis. AVP was mostly detected in normal neurohypophysis as anticipated. This work demonstrates that a fully automated droplet-based liquid microjunction surface sampling system coupled to HPLC-ESI-MS/MS can be readily used for spatially resolved sampling, separation, detection, and semi-quantitation of physiologically-relevant peptide and protein hormones, such as AVP and ACTH, directly from human tissue. In addition, the relative simplicity, rapidity and specificity of the current methodology support the potential of this basic technology with further advancement for assisting surgical decision-making.« less

Profiling of adrenocorticotropic hormone and arginine vasopressin in human pituitary gland and tumor 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; Calligaris, David; Feldman, Daniel R.

Described here are the results from the profiling of the proteins arginine vasopressin (AVP) and adrenocorticotropic hormone (ACTH) from normal human pituitary gland and pituitary adenoma tissue sections using a fully automated droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system for spatially resolved sampling, HPLC separation, and mass spectral detection. Excellent correlation was found between the protein distribution data obtained with this droplet-based liquid microjunction surface sampling-HPLC-ESI-MS/MS system and those data obtained with matrix assisted laser desorption ionization (MALDI) chemical imaging analyses of serial sections of the same tissue. The protein distributions correlated with the visible anatomic pattern of the pituitary gland.more » AVP was most abundant in the posterior pituitary gland region (neurohypophysis) and ATCH was dominant in the anterior pituitary gland region (adenohypophysis). The relative amounts of AVP and ACTH sampled from a series of ACTH secreting and non-secreting pituitary adenomas correlated with histopathological evaluation. ACTH was readily detected at significantly higher levels in regions of ACTH secreting adenomas and in normal anterior adenohypophysis compared to non-secreting adenoma and neurohypophysis. AVP was mostly detected in normal neurohypophysis as anticipated. This work demonstrates that a fully automated droplet-based liquid microjunction surface sampling system coupled to HPLC-ESI-MS/MS can be readily used for spatially resolved sampling, separation, detection, and semi-quantitation of physiologically-relevant peptide and protein hormones, such as AVP and ACTH, directly from human tissue. In addition, the relative simplicity, rapidity and specificity of the current methodology support the potential of this basic technology with further advancement for assisting surgical decision-making.« less

Non-destructive NIR-FT-Raman spectroscopy of plant and animal tissues, of food and works of art.

PubMed

Schrader, B; Schulz, H; Andreev, G N; Klump, H H; Sawatzki, J

2000-10-02

Just after the discovery of Raman spectroscopy in 1928, it became evident that fluorescence with a quantum yield of several orders of magnitude higher than that of the Raman effect was a great and apparently unbeatable competitor. Raman spectroscopy could therefore, in spite of many exciting advantages during the last 60 years, not be applied as an analytical routine method: for nearly every sample, fluorescing impurities had to be removed by distillation or crystallisation. Purification, however, is not possible for cells and tissues, since the removal of the fluorescing enzymes and coenzymes would destroy the cells. There is fortunately one alternative solution. When excited with the radiation of the Nd:YAG laser at 1064 nm Raman spectra are practically free of fluorescence. Raman spectra can now be recorded with minimal sample preparation. In order to facilitate non-destructive Raman spectroscopy of any sample, cells and tissues, food, textiles and works of art, a new entrance optics for Raman spectrometers is used. Typical results from several fields are demonstrated.

Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy

PubMed Central

Zhao, Yongxin; Bucur, Octavian; Irshad, Humayun; Chen, Fei; Weins, Astrid; Stancu, Andreea L.; Oh, Eun-Young; DiStasio, Marcello; Torous, Vanda; Glass, Benjamin; Stillman, Isaac E.; Schnitt, Stuart J.; Beck, Andrew H.; Boyden, Edward S.

2017-01-01

Expansion microscopy (ExM), a method for improving the resolution of light microscopy by physically expanding the specimen, has not been applied to clinical tissue samples. Here we report a clinically optimized form of ExM that supports nanoscale imaging of human tissue specimens that have been fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin (H&E), and/or fresh frozen. The method, which we call expansion pathology (ExPath), converts clinical samples into an ExM-compatible state, then applies an ExM protocol with protein anchoring and mechanical homogenization steps optimized for clinical samples. ExPath enables ~70 nm resolution imaging of diverse biomolecules in intact tissues using conventional diffraction-limited microscopes, and standard antibody and fluorescent DNA in situ hybridization reagents. We use ExPath for optical diagnosis of kidney minimal-change disease, which previously required electron microscopy (EM), and demonstrate high-fidelity computational discrimination between early breast neoplastic lesions that to date have challenged human judgment. ExPath may enable the routine use of nanoscale imaging in pathology and clinical research. PMID:28714966

Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy.

PubMed

Zhao, Yongxin; Bucur, Octavian; Irshad, Humayun; Chen, Fei; Weins, Astrid; Stancu, Andreea L; Oh, Eun-Young; DiStasio, Marcello; Torous, Vanda; Glass, Benjamin; Stillman, Isaac E; Schnitt, Stuart J; Beck, Andrew H; Boyden, Edward S

2017-08-01

Expansion microscopy (ExM), a method for improving the resolution of light microscopy by physically expanding a specimen, has not been applied to clinical tissue samples. Here we report a clinically optimized form of ExM that supports nanoscale imaging of human tissue specimens that have been fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin, and/or fresh frozen. The method, which we call expansion pathology (ExPath), converts clinical samples into an ExM-compatible state, then applies an ExM protocol with protein anchoring and mechanical homogenization steps optimized for clinical samples. ExPath enables ∼70-nm-resolution imaging of diverse biomolecules in intact tissues using conventional diffraction-limited microscopes and standard antibody and fluorescent DNA in situ hybridization reagents. We use ExPath for optical diagnosis of kidney minimal-change disease, a process that previously required electron microscopy, and we demonstrate high-fidelity computational discrimination between early breast neoplastic lesions for which pathologists often disagree in classification. ExPath may enable the routine use of nanoscale imaging in pathology and clinical research.

Embedding and Chemical Reactivation of Green Fluorescent Protein in the Whole Mouse Brain for Optical Micro-Imaging

PubMed Central

Gang, Yadong; Zhou, Hongfu; Jia, Yao; Liu, Ling; Liu, Xiuli; Rao, Gong; Li, Longhui; Wang, Xiaojun; Lv, Xiaohua; Xiong, Hanqing; Yang, Zhongqin; Luo, Qingming; Gong, Hui; Zeng, Shaoqun

2017-01-01

Resin embedding has been widely applied to fixing biological tissues for sectioning and imaging, but has long been regarded as incompatible with green fluorescent protein (GFP) labeled sample because it reduces fluorescence. Recently, it has been reported that resin-embedded GFP-labeled brain tissue can be imaged with high resolution. In this protocol, we describe an optimized protocol for resin embedding and chemical reactivation of fluorescent protein labeled mouse brain, we have used mice as experiment model, but the protocol should be applied to other species. This method involves whole brain embedding and chemical reactivation of the fluorescent signal in resin-embedded tissue. The whole brain embedding process takes a total of 7 days. The duration of chemical reactivation is ~2 min for penetrating 4 μm below the surface in the resin-embedded brain. This protocol provides an efficient way to prepare fluorescent protein labeled sample for high-resolution optical imaging. This kind of sample was demonstrated to be imaged by various optical micro-imaging methods. Fine structures labeled with GFP across a whole brain can be detected. PMID:28352214

Simulated remodeling of loaded collagen networks via strain-dependent enzymatic degradation and constant-rate fiber growth

PubMed Central

Hadi, M.F.; Sander, E.A.; Ruberti, J.W.; Barocas, V. H.

2011-01-01

Recent work has demonstrated that enzymatic degradation of collagen fibers exhibits strain-dependent kinetics. Conceptualizing how the strain dependence affects remodeling of collagenous tissues is vital to our understanding of collagen management in native and bioengineered tissues. As a first step towards this goal, the current study puts forward a multiscale model for enzymatic degradation and remodeling of collagen networks for two sample geometries we routinely use in experiments as model tissues. The multiscale model, driven by microstructural data from an enzymatic decay experiment, includes an exponential strain-dependent kinetic relation for degradation and constant growth. For a dogbone sample under uniaxial load, the model predicted that the distribution of fiber diameters would spread over the course of degradation because of variation in individual fiber load. In a cross-shaped sample, the central region, which experiences smaller, more isotropic loads, showed more decay and less spread in fiber diameter compared to the arms. There was also a slight shift in average orientation in different regions of the cruciform. PMID:22180691

[A study on pathology and immunoglobulin of orbital tissues from cases with Grave's ophthalmopathy].

PubMed

Zhou, X; Luo, Q; Xia, R

1999-07-01

To investigate pathological changes and the expression, localization of immunoglobulins (IgA & IgE) in orbital tissues from patients with Grave's ophthalmopathy. Orbital tissues were obtained at surgery from 19 patients with Grave's ophthalmopathy and 17 control subjects. Immunohistochemical studies were carried out by using anti-IgA, anti-IgE antibody and a streptavidin-biotin-peroxidase detection system. In addition, hematoxylin-eosin, alcian blue and Masson trichrome stains were employed to demonstrate the pathologic changes in the tissues. 12 of 17 samples from patients exhibited IgA positive staining for the connective tissue associated with the extraocular muscles and orbicularis oculi, however only 2 control muscles reacted minimally. IgE positive material was found in association with the majority of leukocytes and with muscle fibers in 13 patients, but only 4 control subjects displayed mild reactions. Significant expression of IgE was more often demonstrated in patients with eye disease of short course than in those of longer course. These results support the notion that the eye muscle fiber and fibroblast are important targets of the orbital autoimmune reaction in Graves' ophthalmopathy in which IgA and IgE both play important roles.

Dual-modal cancer detection based on optical pH sensing and Raman spectroscopy

NASA Astrophysics Data System (ADS)

Kim, Soogeun; Lee, Seung Ho; Min, Sun Young; Byun, Kyung Min; Lee, Soo Yeol

2017-10-01

A dual-modal approach using Raman spectroscopy and optical pH sensing was investigated to discriminate between normal and cancerous tissues. Raman spectroscopy has demonstrated the potential for in vivo cancer detection. However, Raman spectroscopy has suffered from strong fluorescence background of biological samples and subtle spectral differences between normal and disease tissues. To overcome those issues, pH sensing is adopted to Raman spectroscopy as a dual-modal approach. Based on the fact that the pH level in cancerous tissues is lower than that in normal tissues due to insufficient vasculature formation, the dual-modal approach combining the chemical information of Raman spectrum and the metabolic information of pH level can improve the specificity of cancer diagnosis. From human breast tissue samples, Raman spectra and pH levels are measured using fiber-optic-based Raman and pH probes, respectively. The pH sensing is based on the dependence of pH level on optical transmission spectrum. Multivariate statistical analysis is performed to evaluate the classification capability of the dual-modal method. The analytical results show that the dual-modal method based on Raman spectroscopy and optical pH sensing can improve the performance of cancer classification.

Growth hormone mRNA in mammary gland tumors of dogs and cats.

PubMed Central

Mol, J A; van Garderen, E; Selman, P J; Wolfswinkel, J; Rijinberk, A; Rutteman, G R

1995-01-01

We have shown recently that in the dog progestin administration results in mammary production of immunoreactive growth hormone (GH). At present we demonstrate the expression of the gene encoding GH in the mammary gland of dogs and cats using reverse-transcriptase PCR. GH mRNA was found in the great majority of normal mammary tissues as well as benign and malignant mammary tumors of the dog and was associated with the presence of immunoreactive GH in cryostat sections. The mammary PCR product proved to be identical to that of the pituitary. The highest expression levels were found after prolonged treatment with progestins. In carcinomas GH mRNA was also found in progesterone receptor-negative tissue samples, indicating that after malignant transformation GH gene expression may become progestin independent. GH mRNA was also present in mammary tissues of cats with progestin-induced fibroadenomatous changes. It is concluded that GH gene expression occurs in normal, hyperplastic, and neoplastic mammary tissue of the dog. The expression in normal tissue is stimulated by progestins and might mediate the progestin-stimulated development of canine mammary tumors. The demonstration of progestin-stimulated GH expression in mammary tissue of cats indicates that the phenomenon is more generalized among mammals. Images PMID:7738169

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.

Raman microspectroscopy of Hematoporphyrins. Imaging of the noncancerous and the cancerous human breast tissues with photosensitizers

NASA Astrophysics Data System (ADS)

Brozek-Pluska, B.; Kopec, M.

2016-12-01

Raman microspectroscopy combined with fluorescence were used to study the distribution of Hematoporphyrin (Hp) in noncancerous and cancerous breast tissues. The results demonstrate the ability of Raman spectroscopy to distinguish between noncancerous and cancerous human breast tissue and to identify differences in the distribution and photodegradation of Hematoporphyrin, which is a photosensitizer in photodynamic therapy (PDT), photodynamic diagnosis (PDD) and photoimmunotherapy (PIT) of cancer. Presented results show that Hematoporphyrin level in the noncancerous breast tissue is lower compared to the cancerous one. We have proved also that the Raman intensity of lipids and proteins doesn't change dramatically after laser light irradiation, which indicates that the PDT treatment destroys preferably cancer cells, in which the photosensitizer is accumulated. The specific subcellular localization of photosensitizer for breast tissues samples soaked with Hematoporphyrin was not observed.

Determination of sulfonamides in animal tissues by modified QuEChERS and liquid chromatography tandem mass spectrometry.

PubMed

Wen, Ching-Hsuan; Lin, Shu-Ling; Fuh, Ming-Ren

2017-03-01

In this study, the salting-out solvent extraction and dispersive solid-phase extraction (dSPE) clean-up steps in QuEChERS (quick, easy, cheap, effective, rugged, and safe) method were optimized to reduce matrix effect and efficiently extract target sulfonamides from a variety of edible animal tissues. The extracted sulfonamides were then analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). Good extraction recoveries (74.0-100.3% in five different sources of animal tissues; n=3) with acceptable matrix effect (<10%, except for liver samples) were obtained using the proposed method. For the first time, a commercial ND-lipids cartridge was used to remove hydrophobic matrix components from fat-rich animal tissues in the clean-up step of QuEChERS. In addition, good linearity (0.125-12.5ngg -1 ) was observed using matrix-matched calibration (in beef). Limits of detection (LODs) were estimated at 0.01-0.03ngg -1 in beef, pork, and chicken samples. For beef tripe and pig liver samples, the LODs were in the range of 0.02-0.04ngg -1 . Good intra-day/inter-day precision (1.0-10.5%/0.4-8.0%) and accuracy (95.2-107.2%/97.8-102.1%) were also achieved using the modified QuEChERS for sample pretreatment. The applicability of the modified QuEChERS-LC-MS/MS method was demonstrated by determining the occurrence of target sulfonamides in various edible animal tissues for potential food safety analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of articular cartilage by combining microscopic analysis with a fibril-reinforced finite-element model.

PubMed

Julkunen, Petro; Kiviranta, Panu; Wilson, Wouter; Jurvelin, Jukka S; Korhonen, Rami K

2007-01-01

Load-bearing characteristics of articular cartilage are impaired during tissue degeneration. Quantitative microscopy enables in vitro investigation of cartilage structure but determination of tissue functional properties necessitates experimental mechanical testing. The fibril-reinforced poroviscoelastic (FRPVE) model has been used successfully for estimation of cartilage mechanical properties. The model includes realistic collagen network architecture, as shown by microscopic imaging techniques. The aim of the present study was to investigate the relationships between the cartilage proteoglycan (PG) and collagen content as assessed by quantitative microscopic findings, and model-based mechanical parameters of the tissue. Site-specific variation of the collagen network moduli, PG matrix modulus and permeability was analyzed. Cylindrical cartilage samples (n=22) were harvested from various sites of the bovine knee and shoulder joints. Collagen orientation, as quantitated by polarized light microscopy, was incorporated into the finite-element model. Stepwise stress-relaxation experiments in unconfined compression were conducted for the samples, and sample-specific models were fitted to the experimental data in order to determine values of the model parameters. For comparison, Fourier transform infrared imaging and digital densitometry were used for the determination of collagen and PG content in the same samples, respectively. The initial and strain-dependent fibril network moduli as well as the initial permeability correlated significantly with the tissue collagen content. The equilibrium Young's modulus of the nonfibrillar matrix and the strain dependency of permeability were significantly associated with the tissue PG content. The present study demonstrates that modern quantitative microscopic methods in combination with the FRPVE model are feasible methods to characterize the structure-function relationships of articular cartilage.

Simple and robust image-based autofocusing for digital microscopy.

PubMed

Yazdanfar, Siavash; Kenny, Kevin B; Tasimi, Krenar; Corwin, Alex D; Dixon, Elizabeth L; Filkins, Robert J

2008-06-09

A simple image-based autofocusing scheme for digital microscopy is demonstrated that uses as few as two intermediate images to bring the sample into focus. The algorithm is adapted to a commercial inverted microscope and used to automate brightfield and fluorescence imaging of histopathology tissue sections.

Cryopreservation of Cell/Scaffold Tissue-Engineered Constructs

PubMed Central

Costa, Pedro F.; Dias, Ana F.; Reis, Rui L.

2012-01-01

The aim of this work was to study the effect of cryopreservation over the functionality of tissue-engineered constructs, analyzing the survival and viability of cells seeded, cultured, and cryopreserved onto 3D scaffolds. Further, it also evaluated the effect of cryopreservation over the properties of the scaffold material itself since these are critical for the engineering of most tissues and in particular, tissues such as bone. For this purpose, porous scaffolds, namely fiber meshes based on a starch and poly(caprolactone) blend were seeded with goat bone marrow stem cells (GBMSCs) and cryopreserved for 7 days. Discs of the same material seeded with GBMSCs were also used as controls. After this period, these samples were analyzed and compared to samples collected before the cryopreservation process. The obtained results demonstrate that it is possible to maintain cell viability and scaffolds properties upon cryopreservation of tissue-engineered constructs based on starch scaffolds and goat bone marrow mesenchymal cells using standard cryopreservation methods. In addition, the outcomes of this study suggest that the greater porosity and interconnectivity of scaffolds favor the retention of cellular content and cellular viability during cryopreservation processes, when compared with nonporous discs. These findings indicate that it might be possible to prepare off-the-shelf engineered tissue substitutes and preserve them to be immediately available upon request for patients' needs. PMID:22676448

On-the-spot lung cancer differential diagnosis by label-free, molecular vibrational imaging and knowledge-based classification

NASA Astrophysics Data System (ADS)

Gao, Liang; Li, Fuhai; Thrall, Michael J.; Yang, Yaliang; Xing, Jiong; Hammoudi, Ahmad A.; Zhao, Hong; Massoud, Yehia; Cagle, Philip T.; Fan, Yubo; Wong, Kelvin K.; Wang, Zhiyong; Wong, Stephen T. C.

2011-09-01

We report the development and application of a knowledge-based coherent anti-Stokes Raman scattering (CARS) microscopy system for label-free imaging, pattern recognition, and classification of cells and tissue structures for differentiating lung cancer from non-neoplastic lung tissues and identifying lung cancer subtypes. A total of 1014 CARS images were acquired from 92 fresh frozen lung tissue samples. The established pathological workup and diagnostic cellular were used as prior knowledge for establishment of a knowledge-based CARS system using a machine learning approach. This system functions to separate normal, non-neoplastic, and subtypes of lung cancer tissues based on extracted quantitative features describing fibrils and cell morphology. The knowledge-based CARS system showed the ability to distinguish lung cancer from normal and non-neoplastic lung tissue with 91% sensitivity and 92% specificity. Small cell carcinomas were distinguished from nonsmall cell carcinomas with 100% sensitivity and specificity. As an adjunct to submitting tissue samples to routine pathology, our novel system recognizes the patterns of fibril and cell morphology, enabling medical practitioners to perform differential diagnosis of lung lesions in mere minutes. The demonstration of the strategy is also a necessary step toward in vivo point-of-care diagnosis of precancerous and cancerous lung lesions with a fiber-based CARS microendoscope.

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.

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.

EGFR T790M mutation testing of non-small cell lung cancer tissue and blood samples artificially spiked with circulating cell-free tumor DNA: results of a round robin trial.

PubMed

Fassunke, Jana; Ihle, Michaela Angelika; Lenze, Dido; Lehmann, Annika; Hummel, Michael; Vollbrecht, Claudia; Penzel, Roland; Volckmar, Anna-Lena; Stenzinger, Albrecht; Endris, Volker; Jung, Andreas; Lehmann, Ulrich; Zeugner, Silke; Baretton, Gustavo; Kreipe, Hans; Schirmacher, Peter; Kirchner, Thomas; Dietel, Manfred; Büttner, Reinhard; Merkelbach-Bruse, Sabine

2017-10-01

The European Commision (EC) recently approved osimertinib for the treatment of adult patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) harboring EGFR T790M mutations. Besides tissue-based testing, blood samples containing cell-free circulating tumor DNA (ctDNA) can be used to interrogate T790M status. Herein, we describe the conditions and results of a round robin trial (RRT) for T790M mutation testing in NSCLC tissue specimens and peripheral blood samples spiked with cell line DNA mimicking tumor-derived ctDNA. The underlying objectives of this two-staged external quality assessment (EQA) approach were (a) to evaluate the accuracy of T790M mutations testing across multiple centers and (b) to investigate if a liquid biopsy-based testing for T790M mutations in spiked blood samples is feasible in routine diagnostic. Based on a successfully completed internal phase I RRT, an open RRT for EGFR T790M mutation testing in tumor tissue and blood samples was initiated. In total, 48 pathology centers participated in the EQA. Of these, 47 (97.9%) centers submitted their analyses within the pre-defined time frame and 44 (tissue), respectively, 40 (plasma) successfully passed the test. The overall success rates in the RRT phase II were 91.7% (tissue) and 83.3% (blood), respectively. Thirty-eight out of 48 participants (79.2%) successfully passed both parts of the RRT. The RRT for blood-based EGFR testing initiated in Germany is, to the best of our knowledge, the first of his kind in Europe. In summary, our results demonstrate that blood-based genotyping for EGFR resistance mutations can be successfully integrated in routine molecular diagnostics complementing the array of molecular methods already available at pathology centers in Germany.

Correcting the effect of refraction and dispersion of light in FT-IR spectroscopic imaging in transmission through thick infrared windows.

PubMed

Chan, K L Andrew; Kazarian, Sergei G

2013-01-15

Transmission mode is one of the most common sampling methods for FT-IR spectroscopic imaging because the spectra obtained generally have a reasonable signal-to-noise ratio. However, dispersion and refraction of infrared light occurs when samples are sandwiched between infrared windows or placed underneath a layer of liquid. Dispersion and refraction cause infrared light to focus with different focal lengths depending on the wavelength (wavenumber) of the light. As a result, images obtained are in focus only at a particular wavenumber while they are defocused at other wavenumber values. In this work, a solution to correct this spread of focus by means of adding a lens on top of the infrared transparent window, such that a pseudo hemisphere is formed, has been investigated. Through this lens (or pseudo hemisphere), refraction of light is removed and the light across the spectral range has the same focal depth. Furthermore, the lens acts as a solid immersion objective and an increase of both magnification and spatial resolution (by 1.4 times) is demonstrated. The spatial resolution was investigated using an USAF resolution target, showing that the Rayleigh criterion can be achieved, as well as a sample with a sharp polymer interface to indicate the spatial resolution that can be expected in real samples. The reported approach was used to obtain chemical images of cross sections of cancer tissue and hair samples sandwiched between infrared windows showing the versatility and applicability of the method. In addition to the improved spatial resolution, the results reported herein also demonstrate that the lens can reduce the effect of scattering near the edges of tissue samples. The advantages of the presented approach, obtaining FT-IR spectroscopic images in transmission mode with the same focus across all wavenumber values and simultaneous improvement in spatial resolution, will have wide implications ranging from studies of live cells to sorption of drugs into tissues.

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.

Quantitative Tissue Proteomics Analysis Reveals Versican as Potential Biomarker for Early-Stage Hepatocellular Carcinoma.

PubMed

Naboulsi, Wael; Megger, Dominik A; Bracht, Thilo; Kohl, Michael; Turewicz, Michael; Eisenacher, Martin; Voss, Don Marvin; Schlaak, Jörg F; Hoffmann, Andreas-Claudius; Weber, Frank; Baba, Hideo A; Meyer, Helmut E; Sitek, Barbara

2016-01-04

Hepatocellular carcinoma (HCC) is one of the most aggressive tumors, and the treatment outcome of this disease is improved when the cancer is diagnosed at an early stage. This requires biomarkers allowing an accurate and early tumor diagnosis. To identify potential markers for such applications, we analyzed a patient cohort consisting of 50 patients (50 HCC and 50 adjacent nontumorous tissue samples as controls) using two independent proteomics approaches. We performed label-free discovery analysis on 19 HCC and corresponding tissue samples. The data were analyzed considering events known to take place in early events of HCC development, such as abnormal regulation of Wnt/b-catenin and activation of receptor tyrosine kinases (RTKs). 31 proteins were selected for verification experiments. For this analysis, the second set of the patient cohort (31 HCC and corresponding tissue samples) was analyzed using selected (multiple) reaction monitoring (SRM/MRM). We present the overexpression of ATP-dependent RNA helicase (DDX39), Fibulin-5 (FBLN5), myristoylated alanine-rich C-kinase substrate (MARCKS), and Serpin H1 (SERPINH1) in HCC for the first time. We demonstrate Versican core protein (VCAN) to be significantly associated with well differentiated and low-stage HCC. We revealed for the first time the evidence of VCAN as a potential biomarker for early-HCC diagnosis.

Characterizing human vestibular sensory epithelia for experimental studies: new hair bundles on old tissue and implications for therapeutic interventions in ageing

PubMed Central

Taylor, Ruth R.; Jagger, Daniel J.; Saeed, Shakeel R.; Axon, Patrick; Donnelly, Neil; Tysome, James; Moffatt, David; Irving, Richard; Monksfield, Peter; Coulson, Chris; Freeman, Simon R.; Lloyd, Simon K.; Forge, Andrew

2015-01-01

Balance disequilibrium is a significant contributor to falls in the elderly. The most common cause of balance dysfunction is loss of sensory cells from the vestibular sensory epithelia of the inner ear. However, inaccessibility of inner ear tissue in humans severely restricts possibilities for experimental manipulation to develop therapies to ameliorate this loss. We provide a structural and functional analysis of human vestibular sensory epithelia harvested at trans-labyrinthine surgery. We demonstrate the viability of the tissue and labeling with specific markers of hair cell function and of ion homeostasis in the epithelium. Samples obtained from the oldest patients revealed a significant loss of hair cells across the tissue surface, but we found immature hair bundles present in epithelia harvested from patients >60 years of age. These results suggest that the environment of the human vestibular sensory epithelium could be responsive to stimulation of developmental pathways to enhance hair cell regeneration, as has been demonstrated successfully in the vestibular organs of adult mice. PMID:25818177

Characterizing human vestibular sensory epithelia for experimental studies: new hair bundles on old tissue and implications for therapeutic interventions in ageing.

PubMed

Taylor, Ruth R; Jagger, Daniel J; Saeed, Shakeel R; Axon, Patrick; Donnelly, Neil; Tysome, James; Moffatt, David; Irving, Richard; Monksfield, Peter; Coulson, Chris; Freeman, Simon R; Lloyd, Simon K; Forge, Andrew

2015-06-01

Balance disequilibrium is a significant contributor to falls in the elderly. The most common cause of balance dysfunction is loss of sensory cells from the vestibular sensory epithelia of the inner ear. However, inaccessibility of inner ear tissue in humans severely restricts possibilities for experimental manipulation to develop therapies to ameliorate this loss. We provide a structural and functional analysis of human vestibular sensory epithelia harvested at trans-labyrinthine surgery. We demonstrate the viability of the tissue and labeling with specific markers of hair cell function and of ion homeostasis in the epithelium. Samples obtained from the oldest patients revealed a significant loss of hair cells across the tissue surface, but we found immature hair bundles present in epithelia harvested from patients >60 years of age. These results suggest that the environment of the human vestibular sensory epithelium could be responsive to stimulation of developmental pathways to enhance hair cell regeneration, as has been demonstrated successfully in the vestibular organs of adult mice. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Influence of multiple scattering and absorption on the full scattering profile and the isobaric point in tissue

NASA Astrophysics Data System (ADS)

Duadi, Hamootal; Fixler, Dror

2015-05-01

Light reflectance and transmission from soft tissue has been utilized in noninvasive clinical measurement devices such as the photoplethysmograph (PPG) and reflectance pulse oximeter. Incident light on the skin travels into the underlying layers and is in part reflected back to the surface, in part transferred and in part absorbed. Most methods of near infrared (NIR) spectroscopy focus on the volume reflectance from a semi-infinite sample, while very few measure transmission. We have previously shown that examining the full scattering profile (angular distribution of exiting photons) provides more comprehensive information when measuring from a cylindrical tissue. Furthermore, an isobaric point was found which is not dependent on changes in the reduced scattering coefficient. The angle corresponding to this isobaric point depends on the tissue diameter. We investigated the role of multiple scattering and absorption on the full scattering profile of a cylindrical tissue. First, we define the range in which multiple scattering occurs for different tissue diameters. Next, we examine the role of the absorption coefficient in the attenuation of the full scattering profile. We demonstrate that the absorption linearly influences the intensity at each angle of the full scattering profile and, more importantly, the absorption does not change the position of the isobaric point. The findings of this work demonstrate a realistic model for optical tissue measurements such as NIR spectroscopy, PPG, and pulse oximetery.

Easy performance of 6-color confocal immunofluorescence with 4-laser line microscopes.

PubMed

Eissing, Nathalie; Heger, Lukas; Baranska, Anna; Cesnjevar, Robert; Büttner-Herold, Maike; Söder, Stephan; Hartmann, Arndt; Heidkamp, Gordon F; Dudziak, Diana

2014-09-01

Confocal laser scanning microscopy is an advanced technique for imaging tissue samples in vitro and in vivo at high optical resolution. The development of new fluorochrome variants do not only make it possible to perform multicolor flow cytometry of single cells, but in combination with high resolution laser scanning systems also to investigate the distribution of cells in lymphoid tissues by confocal immunofluorescence analyses, thus allowing the distinction of various cell populations directly in the tissue. Here, we provide a protocol for the visualization of at least six differently fluorochrome-labeled antibodies at the same time using a conventional confocal laser scanning microscope with four laser lines (405 nm, 488 nm, 555 nm, and 639 nm laser wavelength) in both murine and human tissue samples. We further demonstrate that compensation correction algorithms are not necessary to reduce spillover of fluorochromes into other channels when the used fluorochromes are combined according to their specific emission bands and the varying Stokes shift for co-excited fluorochromes with the same laser line. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification and quantitive analysis of calcium phosphate microparticles in intestinal tissue by nuclear microscopy

NASA Astrophysics Data System (ADS)

Gomez-Morilla, Inmaculada; Thoree, Vinay; Powell, Jonathan J.; Kirkby, Karen J.; Grime, Geoffrey W.

2006-08-01

Microscopic particles (0.5-2 μm diameter), rich in calcium and phosphorus, are found in the lumen of the mid-distal gut of all mammals investigated, including humans, and these may play a role in immuno-surveillance and immune regulation of antigens from food and symbiotic bacteria that are contained in the gut. Whether these particles can cross in to tissue of the intestinal mucosa is unclear. If so, characterising their morphology and chemical composition is an important task in elucidating their function. The analysis of calcium phosphate in biological tissues has been approached in several ways including optical microscopy, scanning electron microscopy and, most recently in this work, with nuclear microscopy. In this paper, we describe the use of microPIXE and microRBS to locate these particles and to determine, accurately, the ratio of phosphorus to calcium using the information on sample thickness obtained from RBS to allow the PIXE ratios to be corrected. A commercial sample of hydroxy apatite was used to demonstrate accuracy and precision of the technique. Then, in a pilot study on intestinal tissue of mice, we demonstrated the presence of calcium phosphate microparticles, consistent with confocal microscopy observations, and we identified the average molar P:Ca molar ratio as 1.0. Further work will confirm the exact chemical speciation of these particles and will examine the influence of differing calcium containing diets on the formation of these microparticles.

Study the effects of varying interference upon the optical properties of turbid samples using NIR spatial light modulation

NASA Astrophysics Data System (ADS)

Shaul, Oren; Fanrazi-Kahana, Michal; Meitav, Omri; Pinhasi, Gad A.; Abookasis, David

2018-03-01

Optical properties of biological tissues are valuable diagnostic parameters which can provide necessary information regarding tissue state during disease pathogenesis and therapy. However, different sources of interference, such as temperature changes may modify these properties, introducing confounding factors and artifacts to data, consequently skewing their interpretation and misinforming clinical decision-making. In the current study, we apply spatial light modulation, a type of diffuse reflectance hyperspectral imaging technique, to monitor the variation in optical properties of highly scattering turbid media in the presence varying levels of the following sources of interference: scattering concentration, temperature, and pressure. Spatial near-infrared (NIR) light modulation is a wide-field, non-contact emerging optical imaging platform capable of separating the effects of tissue scattering from those of absorption, thereby accurately estimating both parameters. With this technique, periodic NIR illumination patterns at alternately low and high spatial frequencies, at six discrete wavelengths between 690 to 970 nm, were sequentially projected upon the medium while a CCD camera collects the diffusely reflected light. Data analysis based assumptions is then performed off-line to recover the medium's optical properties. We conducted a series of experiments demonstrating the changes in absorption and reduced scattering coefficients of commercially available fresh milk and chicken breast tissue under different interference conditions. In addition, information on the refractive index was study under increased pressure. This work demonstrates the utility of NIR spatial light modulation to detect varying sources of interference upon the optical properties of biological samples.

Development of a wearable CMOS-based contact imaging system for real-time skin condition diagnosis

NASA Astrophysics Data System (ADS)

Petitdidier, Nils; Koenig, Anne; Gerbelot, Rémi; Gioux, Sylvain; Dinten, Jean-Marc

2017-07-01

Diffuse reflectance spectroscopy has been widely used in the field of biological tissue characterization with various modalities [1-5,6]. One of these modalities consists in measuring the spatially resolved diffuse reflectance (SRDR). In this technique, light is collected at multiple distances from the excitation point. The obtained reflectance decay curve is used to determine scattering and absorption properties of the tissue [7], which are directly related to tissue content and structure. Existing systems usually use fiber optics to collect light reflected from the tissue and transfer it to an optical sensor [1,6]. Such devices make it possible to perform SRDR measurements directly in contact with the tissue. However, they offer poor spatial sampling of the reflectance and low light collection efficiency. We propose to overcome these limitations by using a CMOS sensor placed in contact with the tissue to achieve light collection with high spatial sampling over several millimeters and with increased fill factor. Our objective in this paper is to demonstrate the potential of our instrument to determine the optical properties of tissues from SRDR measurements. We first describe the instrument and the employed methodology. Then, preliminary results obtained on optical phantoms are presented. Finally, the potential of our system for SRDR measurements is evaluated through comparison with a fiber-optic probe previously developed in our laboratory [6,8].

Hyperspectral imaging based on compressive sensing to determine cancer margins in human pancreatic tissue ex vivo

NASA Astrophysics Data System (ADS)

Peller, Joseph; Thompson, Kyle J.; Siddiqui, Imran; Martinie, John; Iannitti, David A.; Trammell, Susan R.

2017-02-01

Pancreatic cancer is the fourth leading cause of cancer death in the US. Currently, surgery is the only treatment that offers a chance of cure, however, accurately identifying tumor margins in real-time is difficult. Research has demonstrated that optical spectroscopy can be used to distinguish between healthy and diseased tissue. The design of a single-pixel imaging system for cancer detection is discussed. The system differentiates between healthy and diseased tissue based on differences in the optical reflectance spectra of these regions. In this study, pancreatic tissue samples from 6 patients undergoing Whipple procedures are imaged with the system (total number of tissue sample imaged was N=11). Regions of healthy and unhealthy tissue are determined based on SAM analysis of these spectral images. Hyperspectral imaging results are then compared to white light imaging and histological analysis. Cancerous regions were clearly visible in the hyperspectral images. Margins determined via spectral imaging were in good agreement with margins identified by histology, indicating that hyperspectral imaging system can differentiate between healthy and diseased tissue. After imaging the system was able to detect cancerous regions with a sensitivity of 74.50±5.89% and a specificity of 75.53±10.81%. Possible applications of this imaging system include determination of tumor margins during surgery/biopsy and assistance with cancer diagnosis and staging.

Oxygen consumption of keloids and hypertrophic scars.

PubMed

Ichioka, Shigeru; Ando, Taichi; Shibata, Masahiro; Sekiya, Naomi; Nakatsuka, Takashi

2008-02-01

The oxygen consumption of keloids and hypertrophic scars has never been quantitatively presented, although abnormal metabolic conditions must be associated with their pathophysiology. We invented an original measurement system equipped with a Clark oxygen electrode for ex vivo samples. The measurement of a mouse wound-healing model revealed immature repairing tissues consumed more oxygen than mature tissues. This finding is in accord with the current thinking and supported the validity of our measurement system. The analysis of fresh human samples clearly demonstrated the high oxygen consumption rate of keloid hypertrophic scars and the comparatively low consumption of mature scars. A high oxygen consuming potential, as well as insufficient oxygen diffusion, may possibly contribute to the pathophysiology of keloids and hypertrophic scars.

Validation of freezing tissues and cells for analysis of DNA strand break levels by comet assay

PubMed Central

Jackson, Petra

2013-01-01

The comet analysis of DNA strand break levels in tissues and cells has become a common method of screening for genotoxicity. The large majority of published studies have used fresh tissues and cells processed immediately after collection. However, we have used frozen tissues and cells for more than 10 years, and we believe that freezing samples improve efficiency of the method. We compared DNA strand break levels measured in fresh and frozen bronchoalveolar cells, and lung and liver tissues from mice exposed to the known mutagen methyl methanesulphonate (0, 25, 75, 112.5mg/kg). We used a high-throughput comet protocol with fully automated scoring of DNA strand break levels. The overall results from fresh and frozen samples were in agreement [R 2 = 0.93 for %DNA in tail (%TDNA) and R 2 = 0.78 for tail length (TL)]. A slightly increased %TDNA was observed in lung and liver tissue from vehicle controls; and TL was slightly reduced in bronchoalveolar lavage cells from the high-dose group. In our comet protocol, a small block of tissue designated for comet analysis is frozen immediately at tissue collection and kept deep frozen until rapidly homogenised and embedded in agarose. To demonstrate the feasibility of long-term freezing of samples, we analysed the day-to-day variation of our internal historical negative and positive comet assay controls collected over a 10-year period (1128 observations, 11 batches of frozen untreated and H2O2-treated A549 lung epithelial cells). The H2O2 treatment explained most of the variation 57–77% and the day-to-day variation was only 2–12%. The presented protocol allows analysis of samples collected over longer time span, at different locations, with reduced variation by reducing number of electrophoreses and is suitable for both toxicological and epidemiological studies. The use of frozen tissues; however, requires great care during preparation before analysis, with handling as a major risk factor. PMID:24136994

BMTC: --A Tool for Standardized Tissue Engineering on Ground and in Space ---

NASA Astrophysics Data System (ADS)

Kern, Peter; Kemmerle, Kurt; Jones, David

ESA is developing the BMTC (Biotechnology Mammalian Tissue Culture Facility) as ground demonstrator in order to: • establish a well characterised terrestrial platform for tissue engineer-ing under defined, reproducible conditions • prepare for future tissue engineering experiments in space using proven, well characterised, modular equipment. In the beginning the facility will be dedicated to support research of bone and cartilage growth under controlled mechanical and/or biochemical stimulation. Meanwhile, the industrial BMTC team has finalised the first model. The BMTC is highly automated system which provides standardized experiment hardware for tissue cultivation and stimulation under controlled conditions and the reproducible execution of the experiment according pre-programmed protocols. The BMTC consists of an incubator for the control of the experiment environment. Internally it offers all experiment relevant subsystems: • two Cultivation Units, each with eight Experiment Chamber Modules optical in-situ sensors for pO2 and pH • the Liquid Handling Device for medium exchange and sample taking • the handling devices for the internal transport of the experiment chamber modules to different experiment services • workstations for uni-axial loading of tissue samples; ZETOS (for bone tissue) / CHONDROS (for cartilage tissue) provision of reproducible displacement profiles measurement of the resulting forces computation of the visco-eleastic properties of the samples provision of flow induced shear stress fluorescence microscope • two different reactor types are included in the baseline flat reactor for 2D-and flat 3D-cultures with flow induced shear stress stimulation compatible with microscope cylindrical 3D-reactor for cultivation of vital bone and cartilage samples compatible with un-directional stimulation / analysis by ZETOS / CHONDROS. The modular, flexible design of the system allows the servicing and accommodation of a wide range of other experiment specific reactors. The functional principles and the essential features for controlled experiments will be reported. This facility complements the research done on ground on osteoporosis and the bone and muscle loss during bed rest studies during space flights. It is considered to become a new in-orbit research tool for tissue engineering and the verification of mechanical or pharmaceutical countermeasures.

Genotyping of Plant and Animal Samples without Prior DNA Purification

PubMed Central

Chum, Pak Y.; Haimes, Josh D.; André, Chas P.; Kuusisto, Pia K.; Kelley, Melissa L.

2012-01-01

The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors. PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination1, 2. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS)3,4 is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion3. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required2,5. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA for PCR analysis is purified from ear, tail or toe tissues6,7. However, with the Thermo Scientific Phire Animal Tissue Direct PCR Kit transgenic mice can be genotyped without prior DNA purification. In this protocol transgenic mouse genotyping is achieved directly from mouse ear tissues, as demonstrated here for a challenging example where only one primer set is used for amplification of two fragments differing greatly in size. PMID:23051689

Maximizing Science Return from Future Rodent Experiments on the International Space Station (ISS): Tissue Preservation

NASA Technical Reports Server (NTRS)

Choi, S. Y.; Lai, S.; Klotz, R.; Popova, Y.; Chakravarty, K.; Beegle, J. E.; Wigley, C. L.; Globus, R. K.

2014-01-01

To better understand how mammals adapt to long duration habitation in space, a system for performing rodent experiments on the ISS is under development. Rodent Research-1 is the first flight and will include validation of both on-orbit animal support and tissue preservation. To evaluate plans for on-orbit sample dissection and preservation, we simulated conditions for euthanasia, tissue dissection, and prolonged sample storage on the ISS, and we also developed methods for post-flight dissection and recovery of high quality RNA from multiple tissues following prolonged storage in situ for future science return. Livers and spleens from mice were harvested under conditions that simulated nominal, on-orbit euthanasia and dissection procedures including storage at minus 80 degrees Centigrade for 4 months. The RNA recovered was of high quality (RNA Integrity Number, RNA Integrity Number (RIN) greater than 8) and quantity, and the liver enzyme contents and activities (catalase, glutathione reductase, GAPDH) were similar to positive controls, which were collected under standard laboratory conditions. We also assessed the impact of possible delayed on-orbit dissection scenarios (off-nominal) by dissecting and preserving the spleen (RNA, later) and liver (fast-freezing) at various time points post-euthanasia (from 5 minutes up to 105 minutes). The RNA recovered was of high quality (spleen, RIN greater than 8; liver, RIN greater than 6) and liver enzyme activities were similar to positive controls at all time points, although an apparent decline in select enzyme activities was evident at 105 minutes. Additionally, various tissues were harvested from either intact or partially dissected, frozen carcasses after storage for approximately 2 months; most of the tissues (brain, heart, kidney, eye, adrenal glands and muscle) were of acceptable RNA quality for science return, whereas some tissues (small intestine, bone marrow and bones) were not. These data demonstrate: 1) The protocols developed for future flight experiments will support science return despite delayed preservation post-euthanasia or prolonged storage, and 2) High-quality RNA samples from many different tissues can be recovered by dissection following prolonged storage of the tissue in situ at minus 80 degrees Centigrade. These findings have relevance both to high-value, ground-based experiments when sample collection capability is severely constrained, and to future spaceflight experiments that entail on-orbit sample recovery by the ISS crew.

Microgravity

NASA Image and Video Library

2001-10-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens 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. Final samples from Mir and Earth appeared histologically cartilaginous throughout their entire cross sections (5-8 mm thick), with the exception of fibrous outer capsules. Constructs grown on Earth (A) appeared to have a more organized extracellular matrix with more uniform collagen orientation as compared with constructs grown on Mir (B), but the average collagen fiber diameter was similar in the two groups (22 +- 2 nm) and comparable to that previously reported for developing articular cartilage. Randomly oriented collagen in Mir samples would be consistent with previous reports that microgravity disrupts fibrillogenesis. These are transmission electron micrographs of constructs from Mir (A) and Earth (B) groups at magnifications of x3,500 and x120,000 (Inset). 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). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Credit: Proceedings of the National Academy of Sciences.

Ductal carcinoma of breast: nuclear grade as a predictor of S-phase fraction.

PubMed

Dabbs, D J

1993-06-01

Nuclear grade (NG) and S-phase fraction (SPF) are established independent prognostic variables for ductal breast carcinomas. Nuclear grade can be assigned by a pathologist in a simple fashion during histopathologic evaluation of the tumor, while SPF requires flow cytometric evaluation of tumor samples. This prospective study was undertaken to determine whether elevated SPF could be predicted from NG alone and how NG and SPF correlate with c-erbB-2 expression. Eighty-two breast carcinomas of ductal type were assigned an NG of low (grade 1 or grade 2) or high (grade 3). S-phase fraction was recorded initially from fresh-frozen tissue samples and was designated as either low SPF (below the value designated as the cutoff for elevated SPF) or high SPF (a value at or greater than the cutoff value). On fresh tissue the NG predicted the range of SPF (low or high) in 89% of cases. Four percent of the cases that did not correlate could definitely be attributed to sample error. The remaining 7% that did not correlate could have been due to sample error, specimen quality, or tumor heterogeneity, as demonstrated by reversal of SPF range as performed on paraffin blocks of tumor. Eighty-eight percent of the tumors positive for c-erbB-2 were NG 3 and 12% were NG 2. All c-erbB-2 tumors were aneuploid. This study demonstrates the importance of carefully assigning NGs on tissue and indicates the importance of reviewing flow cytometric data side by side with histopathologic parameters to detect discrepancies between these two modalities. Careful nuclear grading assignment can accurately predict the range of SPF.

Accelerated high-resolution photoacoustic tomography via compressed sensing

NASA Astrophysics Data System (ADS)

Arridge, Simon; Beard, Paul; Betcke, Marta; Cox, Ben; Huynh, Nam; Lucka, Felix; Ogunlade, Olumide; Zhang, Edward

2016-12-01

Current 3D photoacoustic tomography (PAT) systems offer either high image quality or high frame rates but are not able to deliver high spatial and temporal resolution simultaneously, which limits their ability to image dynamic processes in living tissue (4D PAT). A particular example is the planar Fabry-Pérot (FP) photoacoustic scanner, which yields high-resolution 3D images but takes several minutes to sequentially map the incident photoacoustic field on the 2D sensor plane, point-by-point. However, as the spatio-temporal complexity of many absorbing tissue structures is rather low, the data recorded in such a conventional, regularly sampled fashion is often highly redundant. We demonstrate that combining model-based, variational image reconstruction methods using spatial sparsity constraints with the development of novel PAT acquisition systems capable of sub-sampling the acoustic wave field can dramatically increase the acquisition speed while maintaining a good spatial resolution: first, we describe and model two general spatial sub-sampling schemes. Then, we discuss how to implement them using the FP interferometer and demonstrate the potential of these novel compressed sensing PAT devices through simulated data from a realistic numerical phantom and through measured data from a dynamic experimental phantom as well as from in vivo experiments. Our results show that images with good spatial resolution and contrast can be obtained from highly sub-sampled PAT data if variational image reconstruction techniques that describe the tissues structures with suitable sparsity-constraints are used. In particular, we examine the use of total variation (TV) regularization enhanced by Bregman iterations. These novel reconstruction strategies offer new opportunities to dramatically increase the acquisition speed of photoacoustic scanners that employ point-by-point sequential scanning as well as reducing the channel count of parallelized schemes that use detector arrays.

Serum immunoreactivity of cancer/testis antigen OY-TES-1 and its tissues expression in glioma.

PubMed

Li, Xisheng; Yan, Jun; Fan, Rong; Luo, Bin; Zhang, Qingmei; Lin, Yongda; Zhou, Sufang; Luo, Guorong; Xie, Xiaoxun; Xiao, Shaowen

2017-05-01

OY-TES-1 is a member of the cancer/testis antigen family that is expressed in healthy testis tissue and certain types of cancerous tissue. The present study aimed to analyze the expression pattern of OY-TES-1 and serum anti-OY-TES-1 antibody concentration in patients with glioma. OY-TES-1 mRNA was detected in 28/36 (78%) of glioma cases using conventional reverse transcription polymerase chain reaction (RT-PCR) analysis. RT-quantitative-PCR revealed that OY-TES-1 was expressed at a higher level in glioma tissues compared with normal adult tissues (with the exception of testis tissue). Anti-OY-TES-1 antibodies were present in the serum of 5/36 (14%) of patients with glioma, but absent in all the serum samples from 107 healthy donors. Immunohistochemical analysis demonstrated that OY-TES-1 protein was expressed in all glioma tissues from patients with anti-OY-TES-1 antibody seropositivity. These results suggest that OY-TES-1 is a novel candidate for glioma immunotherapy.

Imaging of zymogen granules in fully wet cells: evidence for restricted mechanism of granule growth.

PubMed

Hammel, Ilan; Anaby, Debbie

2007-09-01

The introduction of wet SEM imaging technology permits electron microscopy of wet samples. Samples are placed in sealed specimen capsules and are insulated from the vacuum in the SEM chamber by an impermeable, electron-transparent membrane. The complete insulation of the sample from the vacuum allows direct imaging of fully hydrated, whole-mount tissue. In the current work, we demonstrate direct inspection of thick pancreatic tissue slices (above 400 mum). In the case of scanning of the pancreatic surface, the boundaries of intracellular features are seen directly. Thus no unfolding is required to ascertain the actual particle size distribution based on the sizes of the sections. This method enabled us to investigate the true granule size distribution and confirm early studies of improved conformity to a Poisson-like distribution, suggesting that the homotypic granule growth results from a mechanism, which favors the addition of a single unit granule to mature granules.

Nondestructive mechanical characterization of developing biological tissues using inflation testing.

PubMed

Oomen, P J A; van Kelle, M A J; Oomens, C W J; Bouten, C V C; Loerakker, S

2017-10-01

One of the hallmarks of biological soft tissues is their capacity to grow and remodel in response to changes in their environment. Although it is well-accepted that these processes occur at least partly to maintain a mechanical homeostasis, it remains unclear which mechanical constituent(s) determine(s) mechanical homeostasis. In the current study a nondestructive mechanical test and a two-step inverse analysis method were developed and validated to nondestructively estimate the mechanical properties of biological tissue during tissue culture. Nondestructive mechanical testing was achieved by performing an inflation test on tissues that were cultured inside a bioreactor, while the tissue displacement and thickness were nondestructively measured using ultrasound. The material parameters were estimated by an inverse finite element scheme, which was preceded by an analytical estimation step to rapidly obtain an initial estimate that already approximated the final solution. The efficiency and accuracy of the two-step inverse method was demonstrated on virtual experiments of several material types with known parameters. PDMS samples were used to demonstrate the method's feasibility, where it was shown that the proposed method yielded similar results to tensile testing. Finally, the method was applied to estimate the material properties of tissue-engineered constructs. Via this method, the evolution of mechanical properties during tissue growth and remodeling can now be monitored in a well-controlled system. The outcomes can be used to determine various mechanical constituents and to assess their contribution to mechanical homeostasis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Low rank magnetic resonance fingerprinting.

PubMed

Mazor, Gal; Weizman, Lior; Tal, Assaf; Eldar, Yonina C

2016-08-01

Magnetic Resonance Fingerprinting (MRF) is a relatively new approach that provides quantitative MRI using randomized acquisition. Extraction of physical quantitative tissue values is preformed off-line, based on acquisition with varying parameters and a dictionary generated according to the Bloch equations. MRF uses hundreds of radio frequency (RF) excitation pulses for acquisition, and therefore high under-sampling ratio in the sampling domain (k-space) is required. This under-sampling causes spatial artifacts that hamper the ability to accurately estimate the quantitative tissue values. In this work, we introduce a new approach for quantitative MRI using MRF, called Low Rank MRF. We exploit the low rank property of the temporal domain, on top of the well-known sparsity of the MRF signal in the generated dictionary domain. We present an iterative scheme that consists of a gradient step followed by a low rank projection using the singular value decomposition. Experiments on real MRI data demonstrate superior results compared to conventional implementation of compressed sensing for MRF at 15% sampling ratio.

A novel approach to non-biased systematic random sampling: a stereologic estimate of Purkinje cells in the human cerebellum.

PubMed

Agashiwala, Rajiv M; Louis, Elan D; Hof, Patrick R; Perl, Daniel P

2008-10-21

Non-biased systematic sampling using the principles of stereology provides accurate quantitative estimates of objects within neuroanatomic structures. However, the basic principles of stereology are not optimally suited for counting objects that selectively exist within a limited but complex and convoluted portion of the sample, such as occurs when counting cerebellar Purkinje cells. In an effort to quantify Purkinje cells in association with certain neurodegenerative disorders, we developed a new method for stereologic sampling of the cerebellar cortex, involving calculating the volume of the cerebellar tissues, identifying and isolating the Purkinje cell layer and using this information to extrapolate non-biased systematic sampling data to estimate the total number of Purkinje cells in the tissues. Using this approach, we counted Purkinje cells in the right cerebella of four human male control specimens, aged 41, 67, 70 and 84 years, and estimated the total Purkinje cell number for the four entire cerebella to be 27.03, 19.74, 20.44 and 22.03 million cells, respectively. The precision of the method is seen when comparing the density of the cells within the tissue: 266,274, 173,166, 167,603 and 183,575 cells/cm3, respectively. Prior literature documents Purkinje cell counts ranging from 14.8 to 30.5 million cells. These data demonstrate the accuracy of our approach. Our novel approach, which offers an improvement over previous methodologies, is of value for quantitative work of this nature. This approach could be applied to morphometric studies of other similarly complex tissues as well.

A novel approach to non-biased systematic random sampling: A stereologic estimate of Purkinje cells in the human cerebellum

PubMed Central

Agashiwala, Rajiv M.; Louis, Elan D.; Hof, Patrick R.; Perl, Daniel P.

2010-01-01

Non-biased systematic sampling using the principles of stereology provides accurate quantitative estimates of objects within neuroanatomic structures. However, the basic principles of stereology are not optimally suited for counting objects that selectively exist within a limited but complex and convoluted portion of the sample, such as occurs when counting cerebellar Purkinje cells. In an effort to quantify Purkinje cells in association with certain neurodegenerative disorders, we developed a new method for stereologic sampling of the cerebellar cortex, involving calculating the volume of the cerebellar tissues, identifying and isolating the Purkinje cell layer and using this information to extrapolate non-biased systematic sampling data to estimate the total number of Purkinje cells in the tissues. Using this approach, we counted Purkinje cells in the right cerebella of four human male control specimens, aged 41, 67, 70 and 84 years, and estimated the total Purkinje cell number for the four entire cerebella to be 27.03, 19.74, 20.44 and 22.03 million cells, respectively. The precision of the method is seen when comparing the density of the cells within the tissue: 266,274, 173,166, 167,603 and 183,575 cells/cm3, respectively. Prior literature documents Purkinje cell counts ranging from 14.8 to 30.5 million cells. These data demonstrate the accuracy of our approach. Our novel approach, which offers an improvement over previous methodologies, is of value for quantitative work of this nature. This approach could be applied to morphometric studies of other similarly complex tissues as well. PMID:18725208

A postoperative anti-adhesion barrier based on photoinduced imine-crosslinking hydrogel with tissue-adhesive ability.

PubMed

Yang, Yunlong; Liu, Xiaolin; Li, Yan; Wang, Yang; Bao, Chunyan; Chen, Yunfeng; Lin, Qiuning; Zhu, Linyong

2017-10-15

Postoperative adhesion is a serious complication that can further lead to morbidity and/or mortality. Polymer anti-adhesion barrier material provides an effective precaution to reduce the probability of postoperative adhesion. Clinical application requires these materials to be easily handled, biocompatible, biodegradable, and most importantly tissue adherent to provide target sites with reliable isolation. However, currently there is nearly no polymer barrier material that can fully satisfy these requirements. In this study, based on the photoinduced imine-crosslinking (PIC) reaction, we had developed a photo-crosslinking hydrogel (CNG hydrogel) that composed of o-nitrobenzyl alcohol (NB) modified carboxymethyl cellulose (CMC-NB) and glycol chitosan (GC) as an anti-adhesion barrier material. Under light irradiation, CMC-NB generated aldehyde groups which subsequently reacted with amino groups distributed on GC or tissue surface to form a hydrogel barrier that covalently attached to tissue surface. Rheological analysis demonstrated that CNG hydrogel (30mg/mL polymer content) could be formed in 30s upon light irradiation. Tissue adhesive tests showed that the tissue adhesive strength of CNG hydrogel (30mg/mL) was about 8.32kPa-24.65kPa which increased with increasing CMC-NB content in CNG hydrogel. Toxicity evaluation by L929 cells demonstrated that CNG hydrogel was cytocompatible. Furthermore, sidewall defect-cecum abrasion model of rat was employed to evaluate the postoperative anti-adhesion efficacy of CNG hydrogel. And a significantly reduction of tissue adhesion (20% samples with low score adhesion) was found in CNG hydrogel treated group, compared with control group (100% samples with high score adhesion). In addition, CNG hydrogel could be degraded in nearly 14days and showed no side effect on wound healing. These findings indicated that CNG hydrogel can effectively expanded the clinical treatments of postoperative tissue adhesion. In this study, a tissue adhesive photo-crosslinking hydrogel (CNG) was developed based on photo-induced imine crosslinking reaction (PIC) for postoperative anti-adhesion. CNG hydrogel showed the features of easy and convenient operation, fast and controllable gelation, suitable gel strength, good biocompatibility, and most importantly strong tissue adhesiveness. Therefore, it shows very high performance to prevent postoperative tissue adhesion. Overall, our study provides a more suitable hydrogel barrier material that can overcome the shortcomings of current barriers for clinical postoperative anti-adhesion. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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.

Expression of plakophilin 3 in diffuse malignant pleural mesothelioma.

PubMed

Mašić, Silvija; Brčić, Luka; Krušlin, Božo; Šepac, Ana; Pigac, Biserka; Stančić-Rokotov, Dinko; Jakopović, Marko; Seiwerth, Sven

2018-05-03

Diffuse malignant pleural mesothelioma (DMPM) is the most common primary malignant pleural neoplasm still posing major diagnostic, prognostic and therapeutic challenges. Plakophilins are structural proteins considered to be important for cell stability and adhesion in both tumor and normal tissues. Plakophilin 3 is a protein present in desmosomes of stratified and simple epithelia of normal tissues with presence in malignant cells of various tumors where it participates in the process of tumorigenesis. The aim of this study was to investigate the expression of plakophilin 3 protein in DMPM, but also to study its prognostic significance and relation to histologically accessible parameters of aggressive growth. Archival samples of tissue with established diagnosis of DMPM and samples of normal pleural tissue were used. Tumor samples were classified into three histological types of DMPM (epithelioid, sarcomatoid and biphasic). Additional subclassification of epithelioid mesotheliomas into nine patterns based on the prevalent histological component of the tumor was then performed. After immunohistochemical staining, cytoplasmic and membrane immunopositivity of tumor cells was assesed by scoring the intensity of the staining from 0 (no staining) to 4 (very strong staining). Prognostic value and expression of plakophilin 3 with consideration to histologically estimated aggression in tumor growth were then statistically analyzed using non- parametric tests. The results demonstrated higher level of plakophilin 3 expression in tumor samples with histologically more aggressive tumor growth, but no significant prognostic value. According to our study, plakophilin 3 appears to be involved in tumor invasion in malignant mesothelioma.

UV-laser-based microscopic dissection of tree rings - a novel sampling tool for δ(13) C and δ(18) O studies.

PubMed

Schollaen, Karina; Heinrich, Ingo; Helle, Gerhard

2014-02-01

UV-laser-based microscopic systems were utilized to dissect and sample organic tissue for stable isotope measurements from thin wood cross-sections. We tested UV-laser-based microscopic tissue dissection in practice for high-resolution isotopic analyses (δ(13) C/δ(18) O) on thin cross-sections from different tree species. The method allows serial isolation of tissue of any shape and from millimetre down to micrometre scales. On-screen pre-defined areas of interest were automatically dissected and collected for mass spectrometric analysis. Three examples of high-resolution isotopic analyses revealed that: in comparison to δ(13) C of xylem cells, woody ray parenchyma of deciduous trees have the same year-to-year variability, but reveal offsets that are opposite in sign depending on whether wholewood or cellulose is considered; high-resolution tree-ring δ(18) O profiles of Indonesian teak reflect monsoonal rainfall patterns and are sensitive to rainfall extremes caused by ENSO; and seasonal moisture signals in intra-tree-ring δ(18) O of white pine are weighted by nonlinear intra-annual growth dynamics. The applications demonstrate that the use of UV-laser-based microscopic dissection allows for sampling plant tissue at ultrahigh resolution and unprecedented precision. This new technique facilitates sampling for stable isotope analysis of anatomical plant traits like combined tree eco-physiological, wood anatomical and dendroclimatological studies. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Rapid Mueller matrix polarimetry imaging based on four photoelastic modulators with no moving parts (Conference Presentation)

NASA Astrophysics Data System (ADS)

Gribble, Adam; Alali, Sanaz; Vitkin, Alex

2016-03-01

Polarized light has many applications in biomedical imaging. The interaction of a biological sample with polarized light reveals information about its composition, both structural and functional. For example, the polarimetry-derived metric of linear retardance (birefringence) is dependent on tissue structural organization (anisotropy) and can be used to diagnose myocardial infarct; circular birefringence (optical rotation) can measure glucose concentrations. The most comprehensive type of polarimetry analysis is to measure the Mueller matrix, a polarization transfer function that completely describes how a sample interacts with polarized light. To derive this 4x4 matrix it is necessary to observe how a tissue interacts with different polarizations. A well-suited approach for tissue polarimetry is to use photoelastic modulators (PEMs), which dynamically modulate the polarization of light. Previously, we have demonstrated a rapid time-gated Stokes imaging system that is capable of characterizing the state of polarized light (the Stokes vector) over a large field, after interacting with any turbid media. This was accomplished by synchronizing CCD camera acquisition times relative to two PEMs using a field-programmable gate array (FPGA). Here, we extend this technology to four PEMs, yielding a polarimetry system that is capable of rapidly measuring the complete sample Mueller matrix over a large field of view, with no moving parts and no beam steering. We describe the calibration procedure and evaluate the accuracy of the measurements. Results are shown for tissue-mimicking phantoms, as well as initial biological samples.

Activation of RAS family genes in urothelial carcinoma.

PubMed

Boulalas, I; Zaravinos, A; Karyotis, I; Delakas, D; Spandidos, D A

2009-05-01

Bladder cancer is the fifth most common malignancy in men in Western society. We determined RAS codon 12 and 13 point mutations and evaluated mRNA expression levels in transitional cell carcinoma cases. Samples from 30 human bladder cancers and 30 normal tissues were analyzed by polymerase chain reaction/restriction fragment length polymorphism and direct sequencing to determine the occurrence of mutations in codons 12 and 13 of RAS family genes. Moreover, we used real-time reverse transcriptase-polymerase chain reaction to evaluate the expression profile of RAS genes in bladder cancer specimens compared to that in adjacent normal tissues. Overall H-RAS mutations in codon 12 were observed in 9 tumor samples (30%). Two of the 9 patients (22%) had invasive bladder cancer and 7 (77%) had noninvasive bladder cancer. One H-RAS mutation (11%) was homozygous and the remaining 89% were heterozygous. All samples were WT for K and N-RAS oncogenes. Moreover, 23 of 30 samples (77%) showed over expression in at least 1 RAS family gene compared to adjacent normal tissue. K and N-RAS had the highest levels of over expression in bladder cancer specimens (50%), whereas 27% of transitional cell carcinomas demonstrated H-RAS over expression relative to paired normal tissues. Our results underline the importance of H-RAS activation in human bladder cancer by codon 12 mutations. Moreover, they provide evidence that increased expression of all 3 RAS genes is a common event in bladder cancer that is associated with disease development.

FIB-SEM imaging of carbon nanotubes in mouse lung tissue.

PubMed

Købler, Carsten; Saber, Anne Thoustrup; Jacobsen, Nicklas Raun; Wallin, Håkan; Vogel, Ulla; Qvortrup, Klaus; Mølhave, Kristian

2014-06-01

Ultrastructural characterisation is important for understanding carbon nanotube (CNT) toxicity and how the CNTs interact with cells and tissues. The standard method for this involves using transmission electron microscopy (TEM). However, in particular, the sample preparation, using a microtome to cut thin sample sections for TEM, can be challenging for investigation of regions with agglomerations of large and stiff CNTs because the CNTs cut with difficulty. As a consequence, the sectioning diamond knife may be damaged and the uncut CNTs are left protruding from the embedded block surface excluding them from TEM analysis. To provide an alternative to ultramicrotomy and subsequent TEM imaging, we studied focused ion beam scanning electron microscopy (FIB-SEM) of CNTs in the lungs of mice, and we evaluated the applicability of the method compared to TEM. FIB-SEM can provide serial section volume imaging not easily obtained with TEM, but it is time-consuming to locate CNTs in the tissue. We demonstrate that protruding CNTs after ultramicrotomy can be used to locate the region of interest, and we present FIB-SEM images of CNTs in lung tissue. FIB-SEM imaging was applied to lung tissue from mice which had been intratracheally instilled with two different multiwalled CNTs; one being short and thin, and the other longer and thicker. FIB-SEM was found to be most suitable for detection of the large CNTs (Ø ca. 70 nm), and to be well suited for studying CNT agglomerates in biological samples which is challenging using standard TEM techniques.

THE PASSAGE OF MARKED IONS INTO TISSUES AND FLUIDS OF THE REPRODUCTIVE TRACT OF PREGNANT RABBITS

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

Bennett, J.P.; Boursnell, J.C.; Lutwak-Mann, C.

1959-10-31

Rapid changes were demonstrated in the uptake of labeled ions both in the developing embryo and in the endometrium, mesodermal placental folds, and other closely associated tissues and fluids following the intravenous injection of labeled ions in pregnant rabbits. Phosphorus-32, sulfur-85, sodium-24, iodine- 131, and potassium-42 were used as tracers. A number of new techniques were developed to obtain, weigh, and handle the extremely small samples. The influence of exogenous materials on the early development of fetuses is discussed briefly. (C.R.)

The detection of phosphorus in the tissue of bomb victims in Gaza.

PubMed

Thomsen, Jørgen L; Worm-Leonhard, Martin

2010-01-01

One of the authors went on a fact finding mission to Gaza immediately after the termination of the Israeli invasion. One of the allegations towards the invading Israeli army was the use of phosphorus bombs. The author was able to take samples of tissue from burn victims. Attempts were made to demonstrate the presence of phosphorus in the burn wounds. Using histology and Raman spectroscopy it was found with a high degree of probability that phosphorus was present, although it was not proven with absolute certainty.

Imaging of oxygenation in 3D tissue models with multi-modal phosphorescent probes

NASA Astrophysics Data System (ADS)

Papkovsky, Dmitri B.; Dmitriev, Ruslan I.; Borisov, Sergei

2015-03-01

Cell-penetrating phosphorescence based probes allow real-time, high-resolution imaging of O2 concentration in respiring cells and 3D tissue models. We have developed a panel of such probes, small molecule and nanoparticle structures, which have different spectral characteristics, cell penetrating and tissue staining behavior. The probes are compatible with conventional live cell imaging platforms and can be used in different detection modalities, including ratiometric intensity and PLIM (Phosphorescence Lifetime IMaging) under one- or two-photon excitation. Analytical performance of these probes and utility of the O2 imaging method have been demonstrated with different types of samples: 2D cell cultures, multi-cellular spheroids from cancer cell lines and primary neurons, excised slices from mouse brain, colon and bladder tissue, and live animals. They are particularly useful for hypoxia research, ex-vivo studies of tissue physiology, cell metabolism, cancer, inflammation, and multiplexing with many conventional fluorophors and markers of cellular function.

Raman microspectroscopy of Hematoporphyrins. Imaging of the noncancerous and the cancerous human breast tissues with photosensitizers.

PubMed

Brozek-Pluska, B; Kopec, M

2016-12-05

Raman microspectroscopy combined with fluorescence were used to study the distribution of Hematoporphyrin (Hp) in noncancerous and cancerous breast tissues. The results demonstrate the ability of Raman spectroscopy to distinguish between noncancerous and cancerous human breast tissue and to identify differences in the distribution and photodegradation of Hematoporphyrin, which is a photosensitizer in photodynamic therapy (PDT), photodynamic diagnosis (PDD) and photoimmunotherapy (PIT) of cancer. Presented results show that Hematoporphyrin level in the noncancerous breast tissue is lower compared to the cancerous one. We have proved also that the Raman intensity of lipids and proteins doesn't change dramatically after laser light irradiation, which indicates that the PDT treatment destroys preferably cancer cells, in which the photosensitizer is accumulated. The specific subcellular localization of photosensitizer for breast tissues samples soaked with Hematoporphyrin was not observed. Copyright © 2016 Elsevier B.V. All rights reserved.

Iodine-131 dose-dependent gene expression: alterations in both normal and tumour thyroid tissues of post-Chernobyl thyroid cancers.

PubMed

Abend, M; Pfeiffer, R M; Ruf, C; Hatch, M; Bogdanova, T I; Tronko, M D; Hartmann, J; Meineke, V; Mabuchi, K; Brenner, A V

2013-10-15

A strong, consistent association between childhood irradiation and subsequent thyroid cancer provides an excellent model for studying radiation carcinogenesis. We evaluated gene expression in 63 paired RNA specimens from frozen normal and tumour thyroid tissues with individual iodine-131 (I-131) doses (0.008-8.6 Gy, no unirradiated controls) received from Chernobyl fallout during childhood (Ukrainian-American cohort). Approximately half of these randomly selected samples (32 tumour/normal tissue RNA specimens) were hybridised on 64 whole-genome microarrays (Agilent, 4 × 44 K). Associations between I-131 dose and gene expression were assessed separately in normal and tumour tissues using Kruskal-Wallis and linear trend tests. Of 155 genes significantly associated with I-131 after Bonferroni correction and with ≥2-fold increase per dose category, we selected 95 genes. On the remaining 31 RNA samples these genes were used for validation purposes using qRT-PCR. Expression of eight genes (ABCC3, C1orf9, C6orf62, FGFR1OP2, HEY2, NDOR1, STAT3, and UCP3) in normal tissue and six genes (ANKRD46, CD47, HNRNPH1, NDOR1, SCEL, and SERPINA1) in tumour tissue was significantly associated with I-131. PANTHER/DAVID pathway analyses demonstrated significant over-representation of genes coding for nucleic acid binding in normal and tumour tissues, and for p53, EGF, and FGF signalling pathways in tumour tissue. The multistep process of radiation carcinogenesis begins in histologically normal thyroid tissue and may involve dose-dependent gene expression changes.

Validation of a Real Time PCR for Classical Swine Fever Diagnosis

PubMed Central

Dias, Natanael Lamas; Fonseca Júnior, Antônio Augusto; Oliveira, Anapolino Macedo; Sales, Érica Bravo; Alves, Bruna Rios Coelho; Dorella, Fernanda Alves

2014-01-01

The viral disease classical swine fever (CSF), caused by a Pestivirus, is one of the major causes of economic losses for pig farming. The aim of this work was to validate a RT-qPCR using Taqman for detection of CSF in swine tissues. The parameters for the validation followed the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (OIE) and the guide ABNT NBR ISO/IEC 17025:2005. The analysis of the 5′NTR region of CSF virus was performed in 145 samples from 29 infected pigs and in 240 samples from 80 pigs originated in the Brazilian CSF-free zone. The tissues tested were spleen, kidney, blood, tonsils, and lymph nodes. Sequencing of the positive samples for 5′NTR region was performed to evaluate the specificity of the RT-qPCR. Tests performed for the RT-qPCR validation demonstrated that the PCR assay was efficient in detecting RNA from CSF virus in all materials from different tissues of infected animals. Furthermore, RNA from CSF virus was not detected in samples of swine originated from the Brazilian CSF-free zone. Hence, it is concluded that RT-qPCR can be used as a complementary diagnostic for CSF. PMID:24818039

Validation of a real time PCR for classical Swine Fever diagnosis.

PubMed

Dias, Natanael Lamas; Fonseca Júnior, Antônio Augusto; Oliveira, Anapolino Macedo; Sales, Erica Bravo; Alves, Bruna Rios Coelho; Dorella, Fernanda Alves; Camargos, Marcelo Fernandes

2014-01-01

The viral disease classical swine fever (CSF), caused by a Pestivirus, is one of the major causes of economic losses for pig farming. The aim of this work was to validate a RT-qPCR using Taqman for detection of CSF in swine tissues. The parameters for the validation followed the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (OIE) and the guide ABNT NBR ISO/IEC 17025:2005. The analysis of the 5'NTR region of CSF virus was performed in 145 samples from 29 infected pigs and in 240 samples from 80 pigs originated in the Brazilian CSF-free zone. The tissues tested were spleen, kidney, blood, tonsils, and lymph nodes. Sequencing of the positive samples for 5'NTR region was performed to evaluate the specificity of the RT-qPCR. Tests performed for the RT-qPCR validation demonstrated that the PCR assay was efficient in detecting RNA from CSF virus in all materials from different tissues of infected animals. Furthermore, RNA from CSF virus was not detected in samples of swine originated from the Brazilian CSF-free zone. Hence, it is concluded that RT-qPCR can be used as a complementary diagnostic for CSF.

Molecular genotyping of Echinococcus granulosus using formalin-fixed paraffin-embedded preparations from human isolates in unusual tissue sites.

PubMed

Hizem, A; M'rad, S; Oudni-M'rad, M; Mestiri, S; Hammedi, F; Mezhoud, H; Zakhama, A; Mokni, M; Babba, H

2016-07-01

Cystic echinococcosis (CE) caused by Echinococcus granulosus remains a serious problem worldwide for issues relating to public health and the economy. The most predominantly affected sites are the liver and the lungs, but other organs such as the heart, the spleen and the peritoneum can also be infected. Access to cysts from uncommon sites has limited genomic and molecular investigations. In the present study, genotypes of E. granulosus sensu lato were identified from formalin-fixed paraffin-embedded tissues (FF-PETs) implicated in human CE. Tissue samples were obtained from 57 patients with histologically confirmed CE. DNA samples were analysed using Egss 1 polymerase chain reaction (PCR) specific to the mitochondrial 12S rRNA gene of E. granulosus sensu stricto. All cysts were typed as E. granulosus sensu stricto with up to 35% of the liver and 16.6% of lungs being the most frequently infected, and up to 48.4% of samples being from rare sites. No correlation was found between cyst site and either the gender or the age of patients. This study demonstrates the possibility of exploiting atypical cysts using FF-PET samples and highlights the predominance of E. granulosus sensu stricto species in the Tunisian population, even in unusual infection sites.

Automatic and adaptive heterogeneous refractive index compensation for light-sheet microscopy.

PubMed

Ryan, Duncan P; Gould, Elizabeth A; Seedorf, Gregory J; Masihzadeh, Omid; Abman, Steven H; Vijayaraghavan, Sukumar; Macklin, Wendy B; Restrepo, Diego; Shepherd, Douglas P

2017-09-20

Optical tissue clearing has revolutionized researchers' ability to perform fluorescent measurements of molecules, cells, and structures within intact tissue. One common complication to all optically cleared tissue is a spatially heterogeneous refractive index, leading to light scattering and first-order defocus. We designed C-DSLM (cleared tissue digital scanned light-sheet microscopy) as a low-cost method intended to automatically generate in-focus images of cleared tissue. We demonstrate the flexibility and power of C-DSLM by quantifying fluorescent features in tissue from multiple animal models using refractive index matched and mismatched microscope objectives. This includes a unique measurement of myelin tracks within intact tissue using an endogenous fluorescent reporter where typical clearing approaches render such structures difficult to image. For all measurements, we provide independent verification using standard serial tissue sectioning and quantification methods. Paired with advancements in volumetric image processing, C-DSLM provides a robust methodology to quantify sub-micron features within large tissue sections.Optical clearing of tissue has enabled optical imaging deeper into tissue due to significantly reduced light scattering. Here, Ryan et al. tackle first-order defocus, an artefact of a non-uniform refractive index, extending light-sheet microscopy to partially cleared samples.

The potential influence of CO2, as an agent for euthanasia, on the pharmacokinetics of basic compounds in rodents.

PubMed

Angus, Derek W; Baker, James A; Mason, Rona; Martin, Iain J

2008-02-01

Rodent tissue distribution and pharmacokinetic studies were performed on basic compounds Org A and Org B in support of central nervous system drug discovery programs. A consistent observation from these studies was that drug concentrations in plasma obtained by cardiac puncture after CO(2) euthanasia were markedly higher compared with those from other sampling methods (serial sampling, isoflurane anesthesia, or cervical dislocation). Further investigations demonstrated that CO(2) euthanasia led to a reduction in blood pH in both rats and mice, which was not observed with the other sampling methods. The use of CO(2) euthanasia resulted in a decrease in the brain/plasma ratio of Org B, largely as a result of increased plasma concentrations. The pharmacokinetics of a basic drug, raloxifene, in rat were also influenced by sampling technique. CO(2) euthanasia before sampling, resulted in a 2- to 3-fold increase in the area under the drug concentration-time curve, a decrease in plasma clearance, and a decrease in the steady-state volume of distribution compared with isoflurane anesthesia. It is proposed that a decrease in the pH of blood relative to that of other tissues, as a consequence of CO(2) exposure, results in a redistribution of basic compounds out of the tissues, leading to higher concentrations in plasma.

Influence of experimental conditions on data variability in the liver comet assay.

PubMed

Guérard, M; Marchand, C; Plappert-Helbig, U

2014-03-01

The in vivo comet assay has increasingly been used for regulatory genotoxicity testing in recent years. While it has been demonstrated that the experimental execution of the assay, for example, electrophoresis or scoring, can have a strong impact on the results; little is known on how initial steps, that is, from tissue sampling during necropsy up to slide preparation, can influence the comet assay results. Therefore, we investigated which of the multitude of steps in processing the liver for the comet assay are most critical. All together eight parameters were assessed by using liver samples of untreated animals. In addition, two of those parameters (temperature and storage time of liver before embedding into agarose) were further investigated in animals given a single oral dose of ethyl methanesulfonate at dose levels of 50, 100, and 200 mg/kg, 3 hr prior to necropsy. The results showed that sample cooling emerged as the predominant influence factor, whereas variations in other elements of the procedure (e.g., size of the liver piece sampled, time needed to process the liver tissue post-mortem, agarose temperature, or time of lysis) seem to be of little relevance. Storing of liver samples of up to 6 hr under cooled conditions did not cause an increase in tail intensity. In contrast, storing the tissue at room temperature, resulted in a considerable time-dependent increase in comet parameters. Copyright © 2013 Wiley Periodicals, Inc.

Development and Testing of a Method for Validating Chemical Inactivation of Ebola Virus.

PubMed

Alfson, Kendra J; Griffiths, Anthony

2018-03-13

Complete inactivation of infectious Ebola virus (EBOV) is required before a sample may be removed from a Biosafety Level 4 laboratory. The United States Federal Select Agent Program regulations require that procedures used to demonstrate chemical inactivation must be validated in-house to confirm complete inactivation. The objective of this study was to develop a method for validating chemical inactivation of EBOV and then demonstrate the effectiveness of several commonly-used inactivation methods. Samples containing infectious EBOV ( Zaire ebolavirus ) in different matrices were treated, and the sample was diluted to limit the cytopathic effect of the inactivant. The presence of infectious virus was determined by assessing the cytopathic effect in Vero E6 cells. Crucially, this method did not result in a loss of infectivity in control samples, and we were able to detect less than five infectious units of EBOV ( Zaire ebolavirus ). We found that TRIzol LS reagent and RNA-Bee inactivated EBOV in serum; TRIzol LS reagent inactivated EBOV in clarified cell culture media; TRIzol reagent inactivated EBOV in tissue and infected Vero E6 cells; 10% neutral buffered formalin inactivated EBOV in tissue; and osmium tetroxide vapors inactivated EBOV on transmission electron microscopy grids. The methods described herein are easily performed and can be adapted to validate inactivation of viruses in various matrices and by various chemical methods.

Dissection and lateral mounting of zebrafish embryos: analysis of spinal cord development.

PubMed

Beck, Aaron P; Watt, Roland M; Bonner, Jennifer

2014-02-28

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

PubMed Central

Beck, Aaron P.; Watt, Roland M.; Bonner, Jennifer

2014-01-01

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue. PMID:24637734

Applications of two-photon fluorescence microscopy in deep-tissue imaging

NASA Astrophysics Data System (ADS)

Dong, Chen-Yuan; Yu, Betty; Hsu, Lily L.; Kaplan, Peter D.; Blankschstein, D.; Langer, Robert; So, Peter T. C.

2000-07-01

Based on the non-linear excitation of fluorescence molecules, two-photon fluorescence microscopy has become a significant new tool for biological imaging. The point-like excitation characteristic of this technique enhances image quality by the virtual elimination of off-focal fluorescence. Furthermore, sample photodamage is greatly reduced because fluorescence excitation is limited to the focal region. For deep tissue imaging, two-photon microscopy has the additional benefit in the greatly improved imaging depth penetration. Since the near- infrared laser sources used in two-photon microscopy scatter less than their UV/glue-green counterparts, in-depth imaging of highly scattering specimen can be greatly improved. In this work, we will present data characterizing both the imaging characteristics (point-spread-functions) and tissue samples (skin) images using this novel technology. In particular, we will demonstrate how blind deconvolution can be used further improve two-photon image quality and how this technique can be used to study mechanisms of chemically-enhanced, transdermal drug delivery.

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.

Detection and Heterogeneity of Herpesviruses Causing Pacheco's Disease in Parrots

PubMed Central

Tomaszewski, Elizabeth; Wilson, Van G.; Wigle, William L.; Phalen, David N.

2001-01-01

Pacheco's disease (PD) is a common, often fatal, disease of parrots. We cloned a virus isolate from a parrot that had characteristic lesions of PD. Three viral clones were partially sequenced, demonstrating that this virus was an alphaherpesvirus most closely related to the gallid herpesvirus 1. Five primer sets were developed from these sequences. The primer sets were used with PCR to screen tissues or tissue culture media suspected to contain viruses from 54 outbreaks of PD. The primer sets amplified DNA from all but one sample. Ten amplification patterns were detected, indicating that PD is caused by a genetically heterogeneous population of viruses. A single genetic variant (psittacid herpesvirus variant 1) amplified with all primer sets and was the most common virus variant (62.7%). A single primer set (23F) amplified DNA from all of the positive samples, suggesting that PCR could be used as a rapid postmortem assay for these viruses. PCR was found to be significantly more sensitive than tissue culture for the detection of psittacid herpesviruses. PMID:11158102

Accounting for tissue heterogeneity in infrared spectroscopic imaging for accurate diagnosis of thyroid carcinoma subtypes.

PubMed

Martinez-Marin, David; Sreedhar, Hari; Varma, Vishal K; Eloy, Catarina; Sobrinho-Simões, Manuel; Kajdacsy-Balla, André; Walsh, Michael J

2017-07-01

Fourier transform infrared (FT-IR) microscopy was used to image tissue samples from twenty patients diagnosed with thyroid carcinoma. The spectral data were then used to differentiate between follicular thyroid carcinoma and follicular variant of papillary thyroid carcinoma using principle component analysis coupled with linear discriminant analysis and a Naïve Bayesian classifier operating on a set of computed spectral metrics. Classification of patients' disease type was accomplished by using average spectra from a wide region containing follicular cells, colloid, and fibrosis; however, classification of disease state at the pixel level was only possible when the extracted spectra were limited to follicular epithelial cells in the samples, excluding the relatively uninformative areas of fibrosis. The results demonstrate the potential of FT-IR microscopy as a tool to assist in the difficult diagnosis of these subtypes of thyroid cancer, and also highlights the importance of selectively and separately analyzing spectral information from different features of a tissue of interest.

Novel Multiplex MethyLight Protocol for Detection of DNA Methylation in Patient Tissues and Bodily Fluids

PubMed Central

Olkhov-Mitsel, Ekaterina; Zdravic, Darko; Kron, Ken; van der Kwast, Theodorus; Fleshner, Neil; Bapat, Bharati

2014-01-01

Aberrant DNA methylation is a hallmark of cancer and is an important potential biomarker. Particularly, combined analysis of a panel of hypermethylated genes shows the most promising clinical performance. Herein, we developed, optimized and standardized a multiplex MethyLight assay to simultaneously detect hypermethylation of APC, HOXD3 and TGFB2 in DNA extracted from prostate cancer (PCa) cell lines, archival tissue specimens, and urine samples. We established that the assay is capable of discriminating between fully methylated and unmethylated alleles with 100% specificity and demonstrated the assay as highly accurate and reproducible as the singleplex approach. For proof of principle, we analyzed the methylation status of these genes in tissue and urine samples of PCa patients as well as PCa-free controls. These data show that the multiplex MethyLight assay offers a significant advantage when working with limited quantities of DNA and has potential applications in research and clinical settings. PMID:24651255

Methylation of tissue factor pathway inhibitor 2 as a prognostic biomarker for hepatocellular carcinoma after hepatectomy.

PubMed

Sun, Feng-Kai; Sun, Qi; Fan, Yu-Chen; Gao, Shuai; Zhao, Jing; Li, Feng; Jia, Yi-Bin; Liu, Chuan; Wang, Li-Yuan; Li, Xin-You; Ji, Xiang-Fen; Wang, Kai

2016-02-01

Methylation of tissue factor pathway inhibitor 2 (TFPI2) gene has been detected in hepatocellular carcinoma (HCC). However, the clinicopathologcial significance and prognostic value of TFPI2 methylation in HCC remains largely unknown. This study aimed to investigate the prognostic value of TFPI2 methylation in HCC after hepatectomy. Methylation status of TFPI2 gene was examined in 178 surgical specimens of HCC and 20 normal liver samples using methylation-specific polymerase chain reaction. Methylation of TFPI2 gene was detected in 44.9% (80 of 178) of primary HCC samples, 10.7% (19 of 178) of the corresponding non-tumorous liver samples, and 5.0% (1/20) of the normal liver samples. The mRNA concentrations of TFPI2 in primary HCC tissues were significantly lower than those in corresponding non-tumorous liver tissues and those in normal liver tissues. TFPI2 methylation was significantly associated with higher TNM stage. Patients with TFPI2 methylation demonstrated a significantly poorer prognosis than those without TFPI2 methylation for both overall survival and disease-free survival (P < 0.001, respectively). Multivariate analyses confirmed that TFPI2 methylation was an independent prognostic factor for both overall survival (P = 0.002) and disease-free survival (P = 0.000) in HCC after hepatectomy. Moreover, TFPI2 methylation was found to be the only independent predictor for early tumor recurrence of HCC after resection based on multivariate analysis (P = 0.002). Methylation of TFPI2 predicts high risk of advanced tumor stage, early tumor recurrence, and poor prognosis, and it could be a potential prognostic biomarker in patients with HCC after hepatectomy. © 2015 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Design and Validation of a Compressive Tissue Stimulator with High-Throughput Capacity and Real-Time Modulus Measurement Capability

PubMed Central

Salvetti, David J.; Pino, Christopher J.; Manuel, Steven G.; Dallmeyer, Ian; Rangarajan, Sanjeet V.; Meyer, Tobias; Kotov, Misha

2012-01-01

Mechanical stimulation has been shown to impact the properties of engineered hyaline cartilage constructs and is relevant for engineering of cartilage and osteochondral tissues. Most mechanical stimulators developed to date emphasize precision over adaptability to standard tissue culture equipment and protocols. The realization of mechanical characteristics in engineered constructs approaching native cartilage requires the optimization of complex variables (type of stimulus, regimen, and bimolecular signals). We have proposed and validated a stimulator design that focuses on high construct capacity, compatibility with tissue culture plastic ware, and regimen adaptability to maximize throughput. This design utilizes thin force sensors in lieu of a load cell and a linear encoder to verify position. The implementation of an individual force sensor for each sample enables the measurement of Young's modulus while stimulating the sample. Removable and interchangeable Teflon plungers mounted using neodymium magnets contact each sample. Variations in plunger height and design can vary the strain and force type on individual samples. This allows for the evaluation of a myriad of culture conditions and regimens simultaneously. The system was validated using contact accuracy, and Young's modulus measurements range as key parameters. Contact accuracy for the system was excellent within 1.16% error of the construct height in comparison to measurements made with a micrometer. Biomaterials ranging from bioceramics (cancellous bone, 123 MPa) to soft gels (1% agarose, 20 KPa) can be measured without any modification to the device. The accuracy of measurements in conjunction with the wide range of moduli tested demonstrate the unique characteristics of the device and the feasibility of using this device in mapping real-time changes to Young's modulus of tissue constructs (cartilage, bone) through the developmental phases in ex vivo culture conditions. PMID:21988089

Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

PubMed Central

Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

2014-01-01

Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses. PMID:24927412

Quantitative frequency-domain fluorescence spectroscopy in tissues and tissue-like media

NASA Astrophysics Data System (ADS)

Cerussi, Albert Edward

1999-09-01

In the never-ending quest for improved medical technology at lower cost, modern near-infrared optical spectroscopy offers the possibility of inexpensive technology for quantitative and non-invasive diagnoses. Hemoglobin is the dominant chromophore in the 700-900 nm spectral region and as such it allows for the optical assessment of hemoglobin concentration and tissue oxygenation by absorption spectroscopy. However, there are many other important physiologically relevant compounds or physiological states that cannot be effectively sensed via optical methods because of poor optical contrast. In such cases, contrast enhancements are required. Fluorescence spectroscopy is an attractive component of optical tissue spectroscopy. Exogenous fluorophores, as well as some endogenous ones, may furnish the desperately needed sensitivity and specificity that is lacking in near-infrared optical tissue spectroscopy. The main focus of this thesis was to investigate the generation and propagation of fluorescence photons inside tissues and tissue-like media (i.e., scattering dominated media). The standard concepts of fluorescence spectroscopy have been incorporated into a diffusion-based picture that is sometimes referred to as photon migration. The novelty of this work lies in the successful quantitative recovery of fluorescence lifetimes, absolute fluorescence quantum yields, fluorophore concentrations, emission spectra, and both scattering and absorption coefficients at the emission wavelength from a tissue-like medium. All of these parameters are sensitive to the fluorophore local environment and hence are indicators of the tissue's physiological state. One application demonstrating the capabilities of frequency-domain lifetime spectroscopy in tissue-like media is a study of the binding of ethidium bromide to bovine leukocytes in fresh milk. Ethidium bromide is a fluorescent dye that is commonly used to label DNA, and hence visualize chromosomes in cells. The lifetime of ethidium bromide increases by an order of magnitude upon binding to DNA. In this thesis, I demonstrated that the fluorescence photon migration model is capable of accurately determining the somatic cell count (SCC) in a milk sample. Although meant as a demonstration of fluorescence tissue spectroscopy, this specific problem has important implications for the dairy industry's warfare against subclinical mastitis (i.e., mammary gland inflammation), since the SCC is often used as an indication of bovine infection.

Computational adaptive optics for broadband optical interferometric tomography of biological tissue.

PubMed

Adie, Steven G; Graf, Benedikt W; Ahmad, Adeel; Carney, P Scott; Boppart, Stephen A

2012-05-08

Aberrations in optical microscopy reduce image resolution and contrast, and can limit imaging depth when focusing into biological samples. Static correction of aberrations may be achieved through appropriate lens design, but this approach does not offer the flexibility of simultaneously correcting aberrations for all imaging depths, nor the adaptability to correct for sample-specific aberrations for high-quality tomographic optical imaging. Incorporation of adaptive optics (AO) methods have demonstrated considerable improvement in optical image contrast and resolution in noninterferometric microscopy techniques, as well as in optical coherence tomography. Here we present a method to correct aberrations in a tomogram rather than the beam of a broadband optical interferometry system. Based on Fourier optics principles, we correct aberrations of a virtual pupil using Zernike polynomials. When used in conjunction with the computed imaging method interferometric synthetic aperture microscopy, this computational AO enables object reconstruction (within the single scattering limit) with ideal focal-plane resolution at all depths. Tomographic reconstructions of tissue phantoms containing subresolution titanium-dioxide particles and of ex vivo rat lung tissue demonstrate aberration correction in datasets acquired with a highly astigmatic illumination beam. These results also demonstrate that imaging with an aberrated astigmatic beam provides the advantage of a more uniform depth-dependent signal compared to imaging with a standard gaussian beam. With further work, computational AO could enable the replacement of complicated and expensive optical hardware components with algorithms implemented on a standard desktop computer, making high-resolution 3D interferometric tomography accessible to a wider group of users and nonspecialists.

TCF-1 participates in the occurrence of dedifferentiated chondrosarcoma.

PubMed

Xu, Xiaolong; Tang, Xiaodong; Guo, Wei; Yang, Kang; Ren, Tingting

2016-10-01

The present study demonstrated that T cell factor 1 (TCF-1) protein, a component of the canonical Wnt/β-catenin signaling pathway, can regulate the expression of runt-related transcription factor 2 (runx2) gene and Sry-related HMG box 9 (sox9) gene, which may participate in the differentiation of chondrosarcoma. Dedifferentiated chondrosarcoma (DDCS) is a special variant of conventional chondrosarcoma (CCS), associated with poor survival and high metastasis rate. However, little is known about the mechanism of its occurrence; thus, no effective treatment is available except surgery. Earlier, high expression of runx2 and low expression of sox9 were found in DDCS compared with CCS. Using Western blot to detect clinical tissue samples (including 8 CCS samples and 8 DDCS samples) and immunohistochemistry to detect 85 different-grade chondrosarcoma specimens, a high expression of TCF-1 in DDCS tissues was found compared with CCS tissues. This difference in expression was related to patients' prognosis. Results of luciferase, chromatin immunoprecipitation, and gel electrophoresis mobility shift assays demonstrated that TCF-1 protein could bind to the promoter of runx2 gene directly and sox9 gene indirectly. Hence, it could regulate expression of runx2 gene positively and sox9 gene negatively. Furthermore, in vitro and in vivo experiments showed that TCF-1 protein was closely related to the phenotype and aggressiveness of chondrosarcoma. In conclusion, this study proved that TCF-1 participates in the dedifferentiation of DDCS, which may be mediated by runx2 gene and sox9 gene. Also, TCF-1 can be of important prognostic value and a promising therapeutic target for DDCS patients.

Estimating risk at a Superfund site using passive sampling devices as biological surrogates in human health risk models.

PubMed

Allan, Sarah E; Sower, Gregory J; Anderson, Kim A

2011-10-01

Passive sampling devices (PSDs) sequester the freely dissolved fraction of lipophilic contaminants, mimicking passive chemical uptake and accumulation by biomembranes and lipid tissues. Public Health Assessments that inform the public about health risks from exposure to contaminants through consumption of resident fish are generally based on tissue data, which can be difficult to obtain and requires destructive sampling. The purpose of this study is to apply PSD data in a Public Health Assessment to demonstrate that PSDs can be used as a biological surrogate to evaluate potential human health risks and elucidate spatio-temporal variations in risk. PSDs were used to measure polycyclic aromatic hydrocarbons (PAHs) in the Willamette River; upriver, downriver and within the Portland Harbor Superfund megasite for 3 years during wet and dry seasons. Based on an existing Public Health Assessment for this area, concentrations of PAHs in PSDs were substituted for fish tissue concentrations. PSD measured PAH concentrations captured the magnitude, range and variability of PAH concentrations reported for fish/shellfish from Portland Harbor. Using PSD results in place of fish data revealed an unacceptable risk level for cancer in all seasons but no unacceptable risk for non-cancer endpoints. Estimated cancer risk varied by several orders of magnitude based on season and location. Sites near coal tar contamination demonstrated the highest risk, particularly during the dry season and remediation activities. Incorporating PSD data into Public Health Assessments provides specific spatial and temporal contaminant exposure information that can assist public health professionals in evaluating human health risks. Copyright © 2011 Elsevier Ltd. All rights reserved.

Estimating risk at a Superfund site using passive sampling devices as biological surrogates in human health risk models

PubMed Central

Allan, Sarah E.; Sower, Gregory J.; Anderson, Kim A.

2013-01-01

Passive sampling devices (PSDs) sequester the freely dissolved fraction of lipophilic contaminants, mimicking passive chemical uptake and accumulation by biomembranes and lipid tissues. Public Health Assessments that inform the public about health risks from exposure to contaminants through consumption of resident fish are generally based on tissue data, which can be difficulties to obtain and requires destructive sampling. The purpose of this study is to apply PSD data in a Public Health Assessment to demonstrate that PSDs can be used as a biological surrogate to evaluate potential human health risks and elucidate spatio-temporal variations in risk. PSDs were used to measure polycyclic aromatic hydrocarbons (PAHs) in the Willamette River; upriver, downriver and within the Portland Harbor Superfund megasite for three years during wet and dry seasons. Based on an existing Public Health Assessment for this area, concentrations of PAHs in PSDs were substituted for fish tissue concentrations. PSD measured PAH concentrations captured the magnitude, range and variability of PAH concentrations reported for fish/shellfish from Portland Harbor. Using PSD results in place of fish data revealed an unacceptable risk level for cancer in all seasons but no unacceptable risk for non-cancer endpoints. Estimated cancer risk varied by several orders of magnitude based on season and location. Sites near coal tar contamination demonstrated the highest risk, particularly during the dry season and remediation activities. Incorporating PSD data into Public Health Assessments provides specific spatial and temporal contaminant exposure information that can assist public health professionals in evaluating human health risks. PMID:21741671

Effective cancer laser-therapy design through the integration of nanotechnology and computational treatment planning models

NASA Astrophysics Data System (ADS)

Fisher, Jessica W.; Rylander, Marissa Nichole

2008-02-01

Laser therapies can provide a minimally invasive treatment alternative to surgical resection of tumors. However, the effectiveness of these therapies is limited due to nonspecific heating of target tissue which often leads to healthy tissue injury and extended treatment durations. These therapies can be further compromised due to heat shock protein (HSP) induction in tumor regions where non-lethal temperature elevation occurs, thereby imparting enhanced tumor cell viability and resistance to subsequent chemotherapy and radiation treatments. Introducing multi-walled nanotubes (MWNT) into target tissue prior to laser irradiation increases heating selectivity permitting more precise thermal energy delivery to the tumor region and enhances thermal deposition thereby increasing tumor injury and reducing HSP expression induction. This study investigated the impact of MWNT inclusion in untreated and laser irradiated monolayer cell culture and cell phantom model. Cell viability remained high for all samples with MWNT inclusion and cells integrated into alginate phantoms, demonstrating the non-toxic nature of both MWNTs and alginate phantom models. Following, laser irradiation samples with MWNT inclusion exhibited dramatic temperature elevations and decreased cell viability compared to samples without MWNT. In the cell monolayer studies, laser irradiation of samples with MWNT inclusion experienced up-regulated HSP27, 70 and 90 expression as compared to laser only or untreated samples due to greater temperature increases albeit below the threshold for cell death. Further tuning of laser parameters will permit effective cell killing and down-regulation of HSP. Due to optimal tuning of laser parameters and inclusion of MWNT in phantom models, extensive temperature elevations and cell death occurred, demonstrating MWNT-mediated laser therapy as a viable therapy option when parameters are optimized. In conclusion, MWNT-mediated laser therapies show great promise for effective tumor destruction, but require determination of appropriate MWNT characteristics and laser parameters for maximum tumor destruction.

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.

Application of Real-Time Fluorescent PCR for Quantitative Assessment of Neospora caninum Infections in Organotypic Slice Cultures of Rat Central Nervous System Tissue

PubMed Central

Müller, Norbert; Vonlaufen, Nathalie; Gianinazzi, Christian; Leib, Stephen L.; Hemphill, Andrew

2002-01-01

The previously described Nc5-specific PCR test for the diagnosis of Neospora caninum infections was used to develop a quantitative PCR assay which allows the determination of infection intensities within different experimental and diagnostic sample groups. The quantitative PCR was performed by using a dual fluorescent hybridization probe system and the LightCycler Instrument for online detection of amplified DNA. This assay was successfully applied for demonstrating the parasite proliferation kinetics in organotypic slice cultures of rat brain which were infected in vitro with N. caninum tachyzoites. This PCR-based method of parasite quantitation with organotypic brain tissue samples can be regarded as a novel ex vivo approach for exploring different aspects of cerebral N. caninum infection. PMID:11773124

Solid hemoglobin-polymer phantoms for evaluation of biophotonic systems.

PubMed

Jang, Hyounguk; Pfefer, T Joshua; Chen, Yu

2015-09-15

Stable tissue phantoms that incorporate the spectral absorption properties of hemoglobin would benefit a wide range of biophotonic technologies. Toward this end, we have developed and validated a novel polymer material incorporating hemoglobin. Our solid hemoglobin-polymer (SHP) material is fabricated by mixing liquid silicone base with a hemoglobin solution, followed by sonication and low temperature curing. The optical properties of samples were determined over 450-1000 nm using the inverse adding-doubling method and the Beer-Lambert law. Measurements indicated SHP optical stability over four months. Near-infrared spectroscopy and hyperspectral imaging measurements of SHP samples were performed to demonstrate the utility of this approach. SHP materials have the potential to improve tissue-simulating phantoms used for development, evaluation, and standardization of optical devices for oximetry and other applications.

Electrochemical imaging of cells and tissues

PubMed Central

Lin, Tzu-En; Rapino, Stefania; Girault, Hubert H.

2018-01-01

The technological and experimental progress in electrochemical imaging of biological specimens is discussed with a view on potential applications for skin cancer diagnostics, reproductive medicine and microbial testing. The electrochemical analysis of single cell activity inside cell cultures, 3D cellular aggregates and microtissues is based on the selective detection of electroactive species involved in biological functions. Electrochemical imaging strategies, based on nano/micrometric probes scanning over the sample and sensor array chips, respectively, can be made sensitive and selective without being affected by optical interference as many other microscopy techniques. The recent developments in microfabrication, electronics and cell culturing/tissue engineering have evolved in affordable and fast-sampling electrochemical imaging platforms. We believe that the topics discussed herein demonstrate the applicability of electrochemical imaging devices in many areas related to cellular functions. PMID:29899947

RCL2, a New Fixative, Preserves Morphology and Nucleic Acid Integrity in Paraffin-Embedded Breast Carcinoma and Microdissected Breast Tumor Cells

PubMed Central

Delfour, Christophe; Roger, Pascal; Bret, Caroline; Berthe, Marie-Laurence; Rochaix, Philippe; Kalfa, Nicolas; Raynaud, Pierre; Bibeau, Frédéric; Maudelonde, Thierry; Boulle, Nathalie

2006-01-01

Methacarn and RCL2, a new noncrosslinking fixative, were compared to formalin-fixed or frozen tissue samples of the same invasive breast carcinoma and were evaluated for their effects on tissue morphology and immunohistochemistry as well as DNA and RNA integrity. The histomorphology of methacarn- or RCL2-fixed paraffin-embedded tumors was similar to that observed with the matched formalin-fixed tissues. Immunohistochemistry using various antibodies showed comparable results with either fixative, leading to accurate breast tumor diagnosis and determination of estrogen and progesterone receptors, and HER2 status. Methacarn and RCL2 fixation preserved DNA integrity as demonstrated by successful amplification and sequencing of large DNA amplicons. Similarly, high-quality RNA could be extracted from methacarn- or RCL2-fixed paraffin-embedded MCF-7 cells, whole breast tumor tissues, or microdissected breast tumor cells, as assessed by electropherogram profiles and real-time reverse transcriptase-polymerase chain reaction quantification of various genes. Moreover, tissue morphology and RNA integrity were preserved after 8 months of storage. Altogether, these results indicate that methacarn, as previously shown, and RCL2, a promising new fixative, have great potential for performing both morphological and molecular analyses on the same fixed tissue sample, even after laser-capture microdissection, and can open new doors for investigating small target lesions such as premalignant breast lesions. PMID:16645201

The correlation of in vivo and ex vivo tissue dielectric properties to validate electromagnetic breast imaging: initial clinical experience

PubMed Central

Halter, Ryan J; Zhou, Tian; Meaney, Paul M; Hartov, Alex; Barth, Richard J; Rosenkranz, Kari M; Wells, Wendy A; Kogel, Christine A; Borsic, Andrea; Rizzo, Elizabeth J; Paulsen, Keith D

2009-01-01

Electromagnetic (EM) breast imaging provides low-cost, safe and potentially a more specific modality for cancer detection than conventional imaging systems. A primary difficulty in validating these EM imaging modalities is that the true dielectric property values of the particular breast being imaged are not readily available on an individual subject basis. Here, we describe our initial experience in seeking to correlate tomographic EM imaging studies with discrete point spectroscopy measurements of the dielectric properties of breast tissue. The protocol we have developed involves measurement of in vivo tissue properties during partial and full mastectomy procedures in the operating room (OR) followed by ex vivo tissue property recordings in the same locations in the excised tissue specimens in the pathology laboratory immediately after resection. We have successfully applied all of the elements of this validation protocol in a series of six women with cancer diagnoses. Conductivity and permittivity gauged from ex vivo samples over the frequency range 100 Hz–8.5 GHz are found to be similar to those reported in the literature. A decrease in both conductivity and permittivity is observed when these properties are gauged from ex vivo samples instead of in vivo. We present these results in addition to a case study demonstrating how discrete point spectroscopy measurements of the tissue can be correlated and used to validate EM imaging studies. PMID:19491436

Development of solid-phase microextraction coupled with liquid chromatography for analysis of tramadol in brain tissue using its molecularly imprinted polymer.

PubMed

Habibi-Khorasani, Monireh; Mohammadpour, Amir Hooshang; Mohajeri, Seyed Ahmad

2017-02-01

In this work, performance of a molecularly imprinted polymer (MIP) as a selective solid-phase microextraction sorbent for the extraction and enrichment of tramadol in aqueous solution and rabbit brain tissue, is described. Binding properties of MIPs were studied in comparison with their nonimprinted polymer (NIP). Ten milligrams of the optimized MIP was then evaluated as a sorbent, for preconcentration, in molecularly imprinted solid-phase microextraction (MISPME) of tramadol from aqueous solution and rabbit brain tissue. The analytical method was calibrated in the range of 0.004 ppm (4 ng mL -1 ) and 10 ppm (10 μg mL -1 ) in aqueous media and in the ranges of 0.01 and 10 ppm in rabbit brain tissue, respectively. The results indicated significantly higher binding affinity of MIPs to tramadol, in comparison with NIP. The MISPME procedure was developed and optimized with a recovery of 81.12-107.54% in aqueous solution and 76.16-91.20% in rabbit brain tissue. The inter- and intra-day variation values were <8.24 and 5.06%, respectively. Finally the calibrated method was applied for determination of tramadol in real rabbit brain tissue samples after administration of a lethal dose. Our data demonstrated the potential of MISPME for rapid, sensitive and cost-effective sample analysis. Copyright © 2016 John Wiley & Sons, Ltd.

Vandetanib-eluting Radiopaque Beads: In vivo Pharmacokinetics, Safety and Toxicity Evaluation following Swine Liver Embolization.

PubMed

Denys, Alban; Czuczman, Peter; Grey, David; Bascal, Zainab; Whomsley, Rhys; Kilpatrick, Hugh; Lewis, Andrew L

2017-01-01

To evaluate the plasma and tissue pharmacokinetics, safety and toxicity following intra-arterial hepatic artery administration of Vandetanib (VTB)-eluting Radiopaque Beads (VERB) in healthy swine. In a first phase, healthy swine were treated with hepatic intra-arterial administration of VERB at target dose loading strengths of 36 mg/mL (VERB36), 72 mg/mL (VERB72) and 120 mg/mL (VERB120). Blood and tissue samples were taken and analysed for VTB and metabolites to determine pharmacokinetic parameters for the different dose forms over 30 days. In a second phase, animals were treated with unloaded radiopaque beads or high dose VTB loaded beads (VERB100, 100 mg/mL). Tissue samples from embolized and non-embolized areas of the liver were evaluated at necropsy (30 and 90 days) for determination of VTB and metabolite levels and tissue pathology. Imaging was performed prior to sacrifice using multi-detector computed tomography (MDCT) and imaging findings correlated with pathological changes in the tissue and location of the radiopaque beads. The peak plasma levels of VTB (C max ) released from the various doses of VERB ranged between 6.19-17.3 ng/mL indicating a low systemic burst release. The plasma profile of VTB was consistent with a distribution phase up to 6 h after administration followed by elimination with a half-life of 20-23 h. The AUC of VTB and its major metabolite N-desmethyl vandetanib (NDM VTB) was approximately linear with the dose strength of VERB. VTB plasma levels were at or below limits of detection two weeks after administration. In liver samples, VTB and NDM VTB were present in treated sections at 30 days after administration at levels above the in vitro IC 50 for biological effectiveness. At 90 days both analytes were still present in treated liver but were near or below the limit of quantification in untreated liver sections, demonstrating sustained release from the VERB. Comparison of the reduction of the liver lobe size and associated tissue changes suggested a more effective embolization with VERB compared to the beads without drug. Hepatic intra-arterial administration of VERB results in a low systemic exposure and enables sustained delivery of VTB to target tissues following embolization. Changes in the liver tissue are consistent with an effective embolization and this study has demonstrated that VERB100 is well tolerated with no obvious systemic toxicity.

Pulmonary interstitial fibrosis with evidence of aflatoxin B1 in lung tissue

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

Dvorackova, I.; Pichova, V.

Three cases of pulmonary interstitial fibrosis, two in agricultural workers and one in a textile worker, are reported. In lung samples of all three patients the presence of aflatoxin B1 was demonstrated by radioimmunoassay (RIA). A possible occupational risk of aflatoxin exposure via the respiratory tract is suggested.

Lack of Marburg Virus Transmission From Experimentally Infected to Susceptible In-Contact Egyptian Fruit Bats.

PubMed

Paweska, Janusz T; Jansen van Vuren, Petrus; Fenton, Karla A; Graves, Kerry; Grobbelaar, Antoinette A; Moolla, Naazneen; Leman, Patricia; Weyer, Jacqueline; Storm, Nadia; McCulloch, Stewart D; Scott, Terence P; Markotter, Wanda; Odendaal, Lieza; Clift, Sarah J; Geisbert, Thomas W; Hale, Martin J; Kemp, Alan

2015-10-01

Egyptian fruit bats (Rousettus aegyptiacus) were inoculated subcutaneously (n = 22) with Marburg virus (MARV). No deaths, overt signs of morbidity, or gross lesions was identified, but microscopic pathological changes were seen in the liver of infected bats. The virus was detected in 15 different tissues and plasma but only sporadically in mucosal swab samples, urine, and fecal samples. Neither seroconversion nor viremia could be demonstrated in any of the in-contact susceptible bats (n = 14) up to 42 days after exposure to infected bats. In bats rechallenged (n = 4) on day 48 after infection, there was no viremia, and the virus could not be isolated from any of the tissues tested. This study confirmed that infection profiles are consistent with MARV replication in a reservoir host but failed to demonstrate MARV transmission through direct physical contact or indirectly via air. Bats develop strong protective immunity after infection with MARV. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

120nm resolution in thick samples with structured illumination and adaptive optics

NASA Astrophysics Data System (ADS)

Thomas, Benjamin; Sloan, Megan; Wolstenholme, Adrian J.; Kner, Peter

2014-03-01

μLinear Structured Illumination Microscopy (SIM) provides a two-fold increase over the diffraction limited resolution. SIM produces excellent images with 120nm resolution in tissue culture cells in two and three dimensions. For SIM to work correctly, the point spread function (PSF) and optical transfer function (OTF) must be known, and, ideally, should be unaberrated. When imaging through thick samples, aberrations will be introduced into the optical system which will reduce the peak intensity and increase the width of the PSF. This will lead to reduced resolution and artifacts in SIM images. Adaptive optics can be used to correct the optical wavefront restoring the PSF to its unaberrated state, and AO has been used in several types of fluorescence microscopy. We demonstrate that AO can be used with SIM to achieve 120nm resolution through 25m of tissue by imaging through the full thickness of an adult C. elegans roundworm. The aberrations can be corrected over a 25μm × 45μm field of view with one wavefront correction setting, demonstrating that AO can be used effectively with widefield superresolution techniques.

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.

A quantitative and non-contact technique to characterise microstructural variations of skin tissues during photo-damaging process based on Mueller matrix polarimetry.

PubMed

Dong, Yang; He, Honghui; Sheng, Wei; Wu, Jian; Ma, Hui

2017-10-31

Skin tissue consists of collagen and elastic fibres, which are highly susceptible to damage when exposed to ultraviolet radiation (UVR), leading to skin aging and cancer. However, a lack of non-invasive detection methods makes determining the degree of UVR damage to skin in real time difficult. As one of the fundamental features of light, polarization can be used to develop imaging techniques capable of providing structural information about tissues. In particular, Mueller matrix polarimetry is suitable for detecting changes in collagen and elastic fibres. Here, we demonstrate a novel, quantitative, non-contact and in situ technique based on Mueller matrix polarimetry for monitoring the microstructural changes of skin tissues during UVR-induced photo-damaging. We measured the Mueller matrices of nude mouse skin samples, then analysed the transformed parameters to characterise microstructural changes during the skin photo-damaging and self-repairing processes. Comparisons between samples with and without the application of a sunscreen showed that the Mueller matrix-derived parameters are potential indicators for fibrous microstructure in skin tissues. Histological examination and Monte Carlo simulations confirmed the relationship between the Mueller matrix parameters and changes to fibrous structures. This technique paves the way for non-contact evaluation of skin structure in cosmetics and dermatological health.

Dual-modal cancer detection based on optical pH sensing and Raman spectroscopy.

PubMed

Kim, Soogeun; Lee, Seung Ho; Min, Sun Young; Byun, Kyung Min; Lee, Soo Yeol

2017-10-01

A dual-modal approach using Raman spectroscopy and optical pH sensing was investigated to discriminate between normal and cancerous tissues. Raman spectroscopy has demonstrated the potential for in vivo cancer detection. However, Raman spectroscopy has suffered from strong fluorescence background of biological samples and subtle spectral differences between normal and disease tissues. To overcome those issues, pH sensing is adopted to Raman spectroscopy as a dual-modal approach. Based on the fact that the pH level in cancerous tissues is lower than that in normal tissues due to insufficient vasculature formation, the dual-modal approach combining the chemical information of Raman spectrum and the metabolic information of pH level can improve the specificity of cancer diagnosis. From human breast tissue samples, Raman spectra and pH levels are measured using fiber-optic-based Raman and pH probes, respectively. The pH sensing is based on the dependence of pH level on optical transmission spectrum. Multivariate statistical analysis is performed to evaluate the classification capability of the dual-modal method. The analytical results show that the dual-modal method based on Raman spectroscopy and optical pH sensing can improve the performance of cancer classification. (2017) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

The direct analysis of drug distribution of rotigotine-loaded microspheres from tissue sections by LESA coupled with tandem mass spectrometry.

PubMed

Xu, Li-Xiao; Wang, Tian-Tian; Geng, Yin-Yin; Wang, Wen-Yan; Li, Yin; Duan, Xiao-Kun; Xu, Bin; Liu, Charles C; Liu, Wan-Hui

2017-09-01

The direct analysis of drug distribution of rotigotine-loaded microspheres (RoMS) from tissue sections by liquid extraction surface analysis (LESA) coupled with tandem mass spectrometry (MS/MS) was demonstrated. The RoMS distribution in rat tissues assessed by the ambient LESA-MS/MS approach without extensive or tedious sample pretreatment was compared with that obtained by a conventional liquid chromatography tandem mass spectrometry (LC-MS/MS) method in which organ excision and subsequent solvent extraction were commonly employed before analysis. Results obtained from the two were well correlated for a majority of the organs, such as muscle, liver, stomach, and hippocampus. The distribution of RoMS in the brain, however, was found to be mainly focused in the hippocampus and striatum regions as shown by the LESA-imaged profiles. The LESA approach we developed is sensitive enough, with an estimated LLOQ at 0.05 ng/mL of rotigotine in brain tissue, and information-rich with minimal sample preparation, suitable, and promising in assisting the development of new drug delivery systems for controlled drug release and protection. Graphical abstract Workflow for the LESA-MS/MS imaging of brain tissue section after intramuscular RoMS administration.

A comparative study of three tissue-cultured Dendrobium species and their wild correspondences by headspace gas chromatography-mass spectrometry combined with chemometric methods.

PubMed

Chen, Nai-Dong; You, Tao; Li, Jun; Bai, Li-Tao; Hao, Jing-Wen; Xu, Xiao-Yuan

2016-10-01

Plant tissue culture technique is widely used in the conservation and utilization of rare and endangered medicinal plants and it is crucial for tissue culture stocks to obtain the ability to produce similar bioactive components as their wild correspondences. In this paper, a headspace gas chromatography-mass spectrometry method combined with chemometric methods was applied to analyze and evaluate the volatile compounds in tissue-cultured and wild Dendrobium huoshanense Cheng and Tang, Dendrobium officinale Kimura et Migo and Dendrobium moniliforme (Linn.) Sw. In total, 63 volatile compounds were separated, with 53 being identified from the three Dendrobium spp. Different provenances of Dendrobiums had characteristic chemicals and showed remarkable quantity discrepancy of common compositions. The similarity evaluation disclosed that the accumulation of volatile compounds in Dendrobium samples might be affected by their provenance. Principal component analysis showed that the first three components explained 85.9% of data variance, demonstrating a good discrimination between samples. Gas chromatography-mass spectrometry techniques, combined with chemometrics, might be an effective strategy for identifying the species and their provenance, especially in the assessment of tissue-cultured Dendrobium quality for use in raw herbal medicines. Copyright © 2016. Published by Elsevier B.V.

The lipid-reactive oxygen species phenotype of breast cancer. Raman spectroscopy and mapping, PCA and PLSDA for invasive ductal carcinoma and invasive lobular carcinoma. Molecular tumorigenic mechanisms beyond Warburg effect.

PubMed

Surmacki, Jakub; Brozek-Pluska, Beata; Kordek, Radzislaw; Abramczyk, Halina

2015-04-07

Vibrational signatures of human breast tissue (invasive ductal carcinoma and invasive lobular carcinoma) were used to identify, characterize and discriminate structures in normal (noncancerous) and cancerous tissues by confocal Raman imaging, Raman spectroscopy and IR spectroscopy. The most important differences between normal and cancerous tissues were found in regions characteristic for vibrations of carotenoids, fatty acids, proteins, and interfacial water. Particular attention was paid to the role played by unsaturated fatty acids and their derivatives. K-means clustering and basis analysis followed by PCA and PLSDA is employed to analyze Raman spectroscopic maps of human breast tissue and for a statistical analysis of the samples (82 patients, 164 samples). Raman maps successfully identify regions of carotenoids, fatty acids, and proteins. The intensities, frequencies and profiles of the average Raman spectra differentiate the biochemical composition of normal and cancerous tissues. The paper demonstrates that Raman imaging has reached a clinically relevant level in regard to breast cancer diagnosis applications. The sensitivity and specificity obtained directly from PLSLD and cross validation are equal to 90.5% and 84.8% for calibration and 84.7% and 71.9% for cross-validation respectively.

Effect of hypoxia on tissue factor pathway inhibitor expression in breast cancer.

PubMed

Cui, X Y; Tinholt, M; Stavik, B; Dahm, A E A; Kanse, S; Jin, Y; Seidl, S; Sahlberg, K K; Iversen, N; Skretting, G; Sandset, P M

2016-02-01

ESSENTIALS: A hypoxic microenvironment is a common feature of tumors that may influence activation of coagulation. MCF-7 and SK-BR-3 breast cancer cells and breast cancer tissue samples were used. The results showed transcriptional repression of tissue factor pathway inhibitor expression in hypoxia. Hypoxia-inducible factor 1α may be a target for the therapy of cancer-related coagulation and thrombosis. Activation of coagulation is a common finding in patients with cancer, and is associated with an increased risk of venous thrombosis. As a hypoxic microenvironment is a common feature of solid tumors, we investigated the role of hypoxia in the regulation of tissue factor (TF) pathway inhibitor (TFPI) expression in breast cancer. To explore the transcriptional regulation of TFPI by hypoxia-inducible factor (HIF)-1α in breast cancer cells and their correlation in breast cancer tissues. MCF-7 and SK-BR-3 breast cancer cells were cultured in 1% oxygen or treated with cobalt chloride (CoCl2 ) to mimic hypoxia. Time-dependent and dose-dependent downregulation of TFPI mRNA (quantitative RT-PCR) and of free TFPI protein (ELISA) were observed in hypoxia. Western blotting showed parallel increases in the levels of HIF-1α protein and TF. HIF-1α inhibitor abolished or attenuated the hypoxia-induced downregulation of TFPI. Luciferase reporter assay showed that both hypoxia and HIF-1α overexpression caused strong repression of TFPI promoter activity. Subsequent chromatin immunoprecipitation and mutagenesis analysis demonstrated a functional hypoxia response element within the TFPI promoter, located at -1065 to -1060 relative to the transcriptional start point. In breast cancer tissue samples, gene expression analyses showed a positive correlation between the mRNA expression of TFPI and that of HIF-1α. This study demonstrates that HIF-1α is involved in the transcriptional regulation of the TFPI gene, and suggests that a hypoxic microenvironment inside a breast tumor may induce a procoagulant state in breast cancer patients. © 2015 International Society on Thrombosis and Haemostasis.

Freehand diffuse optical spectroscopy imaging for intraoperative identification of major venous and arterial vessels underlying peritoneal fat: an in vivo demonstration in a pig model

NASA Astrophysics Data System (ADS)

Piao, Daqing; Ramadan, Mohammad; Park, Aaron; Bartels, Kenneth E.; Patel, Sanjay G.

2017-10-01

Inadvertent injury to important anatomic structures is a significant risk in minimally invasive surgery (MIS) that potentially requires conversion to an open procedure, which results in increased morbidity and mortality. Surgeons operating minimal-invasively currently do not have an easy-to-use, real-time device to aid in intraoperative identification of important anatomic structures that underlie tissue planes. We demonstrate freehand diffuse optical spectroscopy (DOS) imaging for intraoperatively identifying major underlying veins and arteries. An applicator probe that can be affixed to and detached from an 8-mm laparoscopic instrument has been developed. The 10-mm DOS source-detector separation renders sampling of tissue heterogeneities a few millimeters deep. DOS spectra acquired consecutively during freehand movement of the applicator probe on the tissue surface are displayed as a temporal and spectral image to assist in spatially resolved identification of the underlying structures. Open surgery identifications of the vena cava and aorta underlying peritoneal fat of ˜4 mm in thickness using the applicator probe under room light were demonstrated repeatedly in multiple pigs in vivo.

1300 nm and 890 nm OCT images of oral cancer tissue engineered models and biopsy samples offer complimentary performance (Conference Presentation)

NASA Astrophysics Data System (ADS)

Boadi, Joseph; Byers, Robert A.; Fernandes, Jon; Mittar, Shweta; Hearnden, Vanessa; Lu, Zenghai; MacNeil, Sheila; Thornhill, Martin; Murdoch, Craig; Hunter, Keith D.; McKechnie, Alasdair; Matcher, Stephen J.

2016-02-01

OCT has demonstrated great potential to non-invasively detect oral epithelial cancers, potentially guiding biopsy and surgical resection. On non-ophthalmic tissues the preferred illumination wavelength is 1300 nm. Previous studies on skin have shown that useful image data can also be obtained at shorter wavelengths, with systems at 1060 nm and 820 nm offering reduced depth penetration but higher contrast. Here we apply a similar comparison to tissue engineered models of oral cancer and also to human biopsy samples, generally finding a similar trend. 1300 nm multi-beam OCT (Michelson Diagnostics EX1301) visualises stromal structures and surface keratin more clearly, providing useful image contrast down to around 1 mm. This system was compared with an ultra-high resolution home-built system operating at 890 nm (2.5 micron resolution vs 7.5 micron axial resolution for the EX1301). The UHR system reveals epithelial features more clearly, especially in the DOK pre-invasive cell line model and the biopsy samples. The relative effects of center wavelength vs axial resolution in generating the differential, wavelength-dependent contrast are assessed and the OCT biopsy images are compared with contemporary histology.

Tuberculosis prevalence in forensic autopsies.

PubMed

Ozsoy, Sait; Demirel, Birol; Albay, Ali; Kisa, Ozgul; Dinc, Ahmet H; Safali, Mukerrem

2010-03-01

According to the 2008 World Health Organization report, in 2006, 9.2 million new cases were determined, and 1.7 million people have lost their life due to tuberculosis (TB) in all around the world. In our country (Turkey), it is estimated that 35,000 to 40,000 people have TB disease annually. The Ministry of Health could just determine 18,500 of these cases, and only 6500 patient could be treated effectively. According to the Tuberculosis Dispensary records, the incidence for TB in Turkey is 28/100,000. It is aimed to determine the infection with Mycobacterium tuberculosis using acidoresistant bacilli microscopy, TB culture, and histopathological methods in tissue samples that were obtained from lungs of forensic cases whose autopsies had been performed in Council of Forensic Medicine Ankara Department Morgue Specialized Committee. A total of 3 tissue samples that were obtained from lungs of randomized 302 cases, were positive for TB in Löwenstein-Jensen medium. Granuloma with caseating necrosis was found in histopathological examination and acidoresistant (+) bacilli (1+, 2+, and 2+, respectively) in microscopically analysis were also demonstrated in this 3 tissue samples. For this reason, we think that autopsy workers have to be careful about tuberculosis during their autopsy working.

Analysis of Apoptosis in Ultraviolet-Induced Sea Cucumber (Stichopus japonicus) Melting Using Terminal Deoxynucleotidyl-Transferase-Mediated dUTP Nick End-Labeling Assay and Cleaved Caspase-3 Immunohistochemistry.

PubMed

Yang, Jing-Feng; Gao, Rong-Chun; Wu, Hai-Tao; Li, Peng-Fei; Hu, Xian-Shu; Zhou, Da-Yong; Zhu, Bei-Wei; Su, Yi-Cheng

2015-11-04

The sea cucumber body wall melting phenomenon occurs under certain circumstances, and the mechanism of this phenomenon remains unclear. This study investigated the apoptosis in the ultraviolet (UV)-induced sea cucumber melting phenomenon. Fresh sea cucumbers (Stichopus japonicus) were exposed to UV radiation for half an hour at an intensity of 0.056 mW/cm(2) and then held at room temperature for melting development. The samples were histologically processed into formalin-fixed paraffin-embedded tissues. The apoptosis of samples was analyzed with the terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) assay and cleaved caspase-3 immunohistochemistry. The emergence of TUNEL-positive cells speeds up between 0.5 and 2 h after UV irradiation. Cleaved caspase-3 positive cells were obviously detected in sample tissues immediately after the UV irradiation. These results demonstrated that sea cucumber melting induced by UV irradiation was triggered by the activation of caspase-3 followed by DNA fragmentation in sea cucumber tissue, which was attributed to apoptosis but was not a consequence of autolysis activity.

Monitoring of interaction of low-frequency electric field with biological tissues upon optical clearing with optical coherence tomography.

PubMed

Peña, Adrián F; Doronin, Alexander; Tuchin, Valery V; Meglinski, Igor

2014-08-01

The influence of a low-frequency electric field applied to soft biological tissues ex vivo at normal conditions and upon the topical application of optical clearing agents has been studied by optical coherence tomography (OCT). The electro-kinetic response of tissues has been observed and quantitatively evaluated by the double correlation OCT approach, utilizing consistent application of an adaptive Wiener filtering and Fourier domain correlation algorithm. The results show that fluctuations, induced by the electric field within the biological tissues are exponentially increased in time. We demonstrate that in comparison to impedance measurements and the mapping of the temperature profile at the surface of the tissue samples, the double correlation OCT approach is much more sensitive to the changes associated with the tissues' electro-kinetic response. We also found that topical application of the optical clearing agent reduces the tissues' electro-kinetic response and is cooling the tissue, thus reducing the temperature induced by the electric current by a few degrees. We anticipate that dcOCT approach can find a new application in bioelectrical impedance analysis and monitoring of the electric properties of biological tissues, including the resistivity of high water content tissues and its variations.

A novel method to detect unlabeled inorganic nanoparticles and submicron particles in tissue by sedimentation field-flow fractionation

PubMed Central

Deering, Cassandra E; Tadjiki, Soheyl; Assemi, Shoeleh; Miller, Jan D; Yost, Garold S; Veranth, John M

2008-01-01

A novel methodology to detect unlabeled inorganic nanoparticles was experimentally demonstrated using a mixture of nano-sized (70 nm) and submicron (250 nm) silicon dioxide particles added to mammalian tissue. The size and concentration of environmentally relevant inorganic particles in a tissue sample can be determined by a procedure consisting of matrix digestion, particle recovery by centrifugation, size separation by sedimentation field-flow fractionation (SdFFF), and detection by light scattering. Background Laboratory nanoparticles that have been labeled by fluorescence, radioactivity, or rare elements have provided important information regarding nanoparticle uptake and translocation, but most nanomaterials that are commercially produced for industrial and consumer applications do not contain a specific label. Methods Both nitric acid digestion and enzyme digestion were tested with liver and lung tissue as well as with cultured cells. Tissue processing with a mixture of protease enzymes is preferred because it is applicable to a wide range of particle compositions. Samples were visualized via fluorescence microscopy and transmission electron microscopy to validate the SdFFF results. We describe in detail the tissue preparation procedures and discuss method sensitivity compared to reported levels of nanoparticles in vivo. Conclusion Tissue digestion and SdFFF complement existing techniques by precisely identifying unlabeled metal oxide nanoparticles and unambiguously distinguishing nanoparticles (diameter<100 nm) from both soluble compounds and from larger particles of the same nominal elemental composition. This is an exciting capability that can facilitate epidemiological and toxicological research on natural and manufactured nanomaterials. PMID:19055780

Micro-mechanical properties of the tendon-to-bone attachment.

PubMed

Deymier, Alix C; An, Yiran; Boyle, John J; Schwartz, Andrea G; Birman, Victor; Genin, Guy M; Thomopoulos, Stavros; Barber, Asa H

2017-07-01

The tendon-to-bone attachment (enthesis) is a complex hierarchical tissue that connects stiff bone to compliant tendon. The attachment site at the micrometer scale exhibits gradients in mineral content and collagen orientation, which likely act to minimize stress concentrations. The physiological micromechanics of the attachment thus define resultant performance, but difficulties in sample preparation and mechanical testing at this scale have restricted understanding of structure-mechanical function. Here, microscale beams from entheses of wild type mice and mice with mineral defects were prepared using cryo-focused ion beam milling and pulled to failure using a modified atomic force microscopy system. Micromechanical behavior of tendon-to-bone structures, including elastic modulus, strength, resilience, and toughness, were obtained. Results demonstrated considerably higher mechanical performance at the micrometer length scale compared to the millimeter tissue length scale, describing enthesis material properties without the influence of higher order structural effects such as defects. Micromechanical investigation revealed a decrease in strength in entheses with mineral defects. To further examine structure-mechanical function relationships, local deformation behavior along the tendon-to-bone attachment was determined using local image correlation. A high compliance zone near the mineralized gradient of the attachment was clearly identified and highlighted the lack of correlation between mineral distribution and strain on the low-mineral end of the attachment. This compliant region is proposed to act as an energy absorbing component, limiting catastrophic failure within the tendon-to-bone attachment through higher local deformation. This understanding of tendon-to-bone micromechanics demonstrates the critical role of micrometer scale features in the mechanics of the tissue. The tendon-to-bone attachment (enthesis) is a complex hierarchical tissue with features at a numerous scales that dissipate stress concentrations between compliant tendon and stiff bone. At the micrometer scale, the enthesis exhibits gradients in collagen and mineral composition and organization. However, the physiological mechanics of the enthesis at this scale remained unknown due to difficulty in preparing and testing micrometer scale samples. This study is the first to measure the tensile mechanical properties of the enthesis at the micrometer scale. Results demonstrated considerably enhanced mechanical performance at the micrometer length scale compared to the millimeter tissue length scale and identified a high-compliance zone near the mineralized gradient of the attachment. This understanding of tendon-to-bone micromechanics demonstrates the critical role of micrometer scale features in the mechanics of the tissue. Copyright © 2017. Published by Elsevier Ltd.

Modulation of stress related protein genes in the bass (Epinephelus guaza) caught from the Gulf of Suez, the Red Sea, Egypt.

PubMed

Abdel-Gawad, Fagr Kh; Khalil, Wagdy K B

2013-10-01

Impact of chemical pollution on expression of stress protein genes in the bass Epinephelus guaga collected from several locations including Suez Oil Production Port (Floating port), Atakah Fishing Port, Adabiya Port and Tawfik Port in Suez Governorate, Egypt, was investigated. In the current study, levels of polycyclic aromatic hydrocarbons (PAHs) in water and fish samples collected from Suez Gulf were assessed. In addition, gills and liver tissues of caught bass fish were used to address the interaction between pollution status and the expression of stress-related genes (Hsp70a, Hsp70b, Hsp47, MT and CYP1A). Our analysis demonstrated that levels of PAHs in Floating and Tawfik ports were higher than those found in the Atakah Fishing Port and the Adabiya Port. In addition, MDA and PC contents were significantly higher in gills and liver tissues collected from Floating and Tawfik ports than those collected from Adabiya and Atakah ports. In correlation to the above results, all fish collected from the Floating and Tawfik ports presented a significant increase in Hsp-, MT- and CYP1A-mRNAs. On the other hand, fish samples collected from the Atakah Fishing and Adabiya ports showed no induction of the stress-related genes transcription in such tissues. In conclusion, the current research demonstrates that remarkable increase in PAH contaminants levels in Floating and Tawfik ports are correlated with the levels stress protein-related genes transcription in E. guaga gills and liver tissues. Copyright © 2013 Elsevier Inc. All rights reserved.

Hyperspectral imaging of endogenous fluorescent metabolic molecules to identify pain states in central nervous system tissue

NASA Astrophysics Data System (ADS)

Staikopoulos, Vasiliki; Gosnell, Martin E.; Anwer, Ayad G.; Mustafa, Sanam; Hutchinson, Mark R.; Goldys, Ewa M.

2016-12-01

Fluorescence-based bio-imaging methods have been extensively used to identify molecular changes occurring in biological samples in various pathological adaptations. Auto-fluorescence generated by endogenous fluorescent molecules within these samples can interfere with signal to background noise making positive antibody based fluorescent staining difficult to resolve. Hyperspectral imaging uses spectral and spatial imaging information for target detection and classification, and can be used to resolve changes in endogenous fluorescent molecules such as flavins, bound and free NADH and retinoids that are involved in cell metabolism. Hyperspectral auto-fluorescence imaging of spinal cord slices was used in this study to detect metabolic differences within pain processing regions of non-pain versus sciatic chronic constriction injury (CCI) animals, an established animal model of peripheral neuropathy. By using an endogenous source of contrast, subtle metabolic variations were detected between tissue samples, making it possible to distinguish between animals from non-injured and injured groups. Tissue maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant tissue regions with compromised mitochondrial function. Taken together, our results demonstrate that hyperspectral imaging provides a new non-invasive method to investigate central changes of peripheral neuropathic injury and other neurodegenerative disease models, and paves the way for novel cellular characterisation in health, disease and during treatment, with proper account of intrinsic cellular heterogeneity.

UGT2B17 and SULT1A1 gene copy number variation (CNV) detection by LabChip microfluidic technology.

PubMed

Gaedigk, Andrea; Gaedigk, Roger; Leeder, J Steven

2010-05-01

Gene copy number variations (CNVs) are increasingly recognized to play important roles in the expression of genes and hence on their respective enzymatic activities. This has been demonstrated for a number of drug metabolizing genes, such as UDP-glucuronosyltransferases 2B17 (UGT2B17) and sulfotransferase 1A1 (SULT1A1), which are subject to genetic heterogeneity, including CNV. Quantitative assays to assess gene copy number are therefore becoming an integral part of accurate genotype assessment and phenotype prediction. In this study, we evaluated a microfluidics-based system, the Bio-Rad Experion system, to determine the power and utility of this platform to detect UGT2B17 and SULT1A1 CNV in DNA samples derived from blood and tissue. UGT2B17 is known to present with 0, 1 or 2 and SULT1A1 with up to 5 gene copies. Distinct clustering (p<0.001) into copy number groups was achieved for both genes. DNA samples derived from blood exhibited less inter-run variability compared to DNA samples obtained from liver tissue. This variability may be caused by tissue-specific PCR inhibitors as it could be overcome by using DNA from another tissue, or after the DNA had undergone whole genome amplification. This method produced results comparable to those reported for other quantitative test platforms.

TFDP3 was expressed in coordination with E2F1 to inhibit E2F1-mediated apoptosis in prostate cancer.

PubMed

Ma, Yueyun; Xin, Yijuan; Li, Rui; Wang, Zhe; Yue, Qiaohong; Xiao, Fengjing; Hao, Xiaoke

2014-03-10

TFDP3 has been previously identified as an inhibitor of E2F molecules. It has been shown to suppress E2F1-induced apoptosis dependent P53 and to play a potential role in carcinogenesis. However, whether it indeed helps cancer cells tolerate apoptosis stress in cancer tissues remains unknown. TFDP3 expression was assessed by RT-PCR, in situ hybridization and immunohistochemistry in normal human tissues, cancer tissues and prostate cancer tissues. The association between TFDP3 and E2F1 in prostate cancer development was analyzed in various stages. Apoptosis was evaluated with annexin-V and propidium iodide staining and flow-cytometry. The results show that, in 96 samples of normal human tissues, TFDP3 could be detected in the cerebrum, esophagus, stomach, small intestine, bronchus, breast, ovary, uterus, and skin, but seldom in the lung, muscles, prostate, and liver. In addition, TFDP3 was highly expressed in numerous cancer tissues, such as brain-keratinous, lung squamous cell carcinoma, testicular seminoma, cervical carcinoma, skin squamous cell carcinoma, gastric adenocarcinoma, liver cancer, and prostate cancer. Moreover, TFDP3 was positive in 23 (62.2%) of 37 prostate cancer samples regardless of stage. Furthermore, immunohistochemistry results show that TFDP3 was always expressed in coordination with E2F1 at equivalent expression levels in prostate cancer tissues, and was highly expressed particularly in samples of high stage. When E2F1 was extrogenously expressed in LNCap cells, TFDP3 could be induced, and the apoptosis induced by E2F1 was significantly decreased. It was demonstrated that TFDP3 was a broadly expressed protein corresponding to E2F1 in human tissues, and suggested that TFDP3 is involved in prostate cancer cell survival by suppressing apoptosis induced by E2F1. Copyright © 2013 Elsevier B.V. All rights reserved.

Imaging and sensing based on dual-pulse nonlinear photoacoustic contrast: a preliminary study on fatty liver

NASA Astrophysics Data System (ADS)

Tian, Chao; Xie, Zhixing; Fabiilli, Mario; Wang, Xueding

2015-03-01

We developed a simple and effective contrast for tissue characterization based on the recently proposed dual-pulse nonlinear photoacoustic technology. The new contrast takes advantage of the temperature dependence of Grüneisen parameter of tissue and involves a dual-pulse laser excitation process. A short pulse first heats the sample and causes a temperature jump, which then leads to the change of Grüneisen parameter and amplitude of the photoacoustic signal of the second pulse. For different tissues, the induced rate or trend of change is expected to be different, which constitutes the basis of the new contrast. Preliminary phantom experiment in blood and lipid mixtures and in vitro experiment in fatty rat liver have demonstrated that the proposed contrast has the capability of fast characterization of lipid-rich and blood-rich tissues.

Quantitative assessment of submicron scale anisotropy in tissue multifractality by scattering Mueller matrix in the framework of Born approximation

NASA Astrophysics Data System (ADS)

Das, Nandan Kumar; Dey, Rajib; Chakraborty, Semanti; Panigrahi, Prasanta K.; Meglinski, Igor; Ghosh, Nirmalya

2018-04-01

A number of tissue-like disordered media exhibit local anisotropy of scattering in the scaling behavior. Scaling behavior contains wealth of fractal or multifractal properties. We demonstrate that the spatial dielectric fluctuations in a sample of biological tissue exhibit multifractal anisotropy. Multifractal anisotropy encoded in the wavelength variation of the light scattering Mueller matrix and manifesting as an intriguing spectral diattenuation effect. We developed an inverse method for the quantitative assessment of the multifractal anisotropy. The method is based on the processing of relevant Mueller matrix elements in Fourier domain by using Born approximation, followed by the multifractal analysis. The approach promises for probing subtle micro-structural changes in biological tissues associated with the cancer and precancer, as well as for non-destructive characterization of a wide range of scattering materials.

A cross-shelf gradient in δ15N stable isotope values of krill and pollock indicates seabird foraging patterns in the Bering Sea

NASA Astrophysics Data System (ADS)

Jones, Nathan M.; Hoover, Brian A.; Heppell, Scott A.; Kuletz, Kathy J.

2014-11-01

Concurrent measurements of predator and prey δ15N isotope values demonstrated that a cross-shelf isotopic gradient can propagate through a marine food web from forage species to top-tier predators and indicate foraging areas at a scale of tens of kilometers. We measured δ13C and δ15N in muscle tissues of thick-billed murres (Uria lomvia) and black-legged kittiwakes (Rissa tridactyla), and in whole body tissues of walleye pollock (Gadus chalcogrammus) and krill (Thysanoessa spp), sampled across the continental shelf break in the Bering Sea in 2008 and in 2009. We found significant basin-shelf differences at fine scales (<100 km) in δ15N among murres but not kittiwakes, and no such differences in δ13C in either seabird species at that scale. We then quantified the multi-trophic signal and spatial structure of a basin-shelf δ15Nitrogen gradient in the central and southern Bering Sea, and used it to contrast foraging patterns of thick-billed murres and kittiwakes on the open ocean. Seabird muscle δ15N values were compared to baselines created from measurements in krill and pollock tissues sampled concurrently throughout the study area. Krill, pollock, and murre tissues from northern, shallow, shelf habitat (<200 m) were enriched 1-2‰ in δ15N relative to samples taken from deeper habitats (>200 m) to the south and west. Krill δ15N baseline values predicted 35-42% of the variability in murre tissue values. Patterns between kittiwakes and prey were less coherent. The persistence of strong spatial autocorrelation among sample values, and a congruence of geospatial patterns in δ15N among murre and prey tissues, suggest that murres forage repeatedly in specific areas. Murre isotope values showed distinct geospatial stratification, coincident with the spatial distribution of three colonies: St. Paul, St. George, and Bogoslof. This suggests some degree of foraging habitat partitioning among colonies.

Using high resolution computed tomography to visualize the three dimensional structure and function of plant vasculature.

PubMed

McElrone, Andrew J; Choat, Brendan; Parkinson, Dilworth Y; MacDowell, Alastair A; Brodersen, Craig R

2013-04-05

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods. Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

PubMed Central

McElrone, Andrew J.; Choat, Brendan; Parkinson, Dilworth Y.; MacDowell, Alastair A.; Brodersen, Craig R.

2013-01-01

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods. Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function. PMID:23609036

Detection of Francisella tularensis within infected mouse tissues by using a hand-held PCR thermocycler.

PubMed

Emanuel, Peter A; Bell, Ryan; Dang, Jessica L; McClanahan, Rebecca; David, John C; Burgess, Robert J; Thompson, Joseph; Collins, Lisa; Hadfield, Ted

2003-02-01

The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5' hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. The fopA and tul4 genes of F. tularensis were amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field.

Detection of Francisella tularensis within Infected Mouse Tissues by Using a Hand-Held PCR Thermocycler

PubMed Central

Emanuel, Peter A.; Bell, Ryan; Dang, Jessica L.; McClanahan, Rebecca; David, John C.; Burgess, Robert J.; Thompson, Joseph; Collins, Lisa; Hadfield, Ted

2003-01-01

The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5′ hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. The fopA and tul4 genes of F. tularensis were amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field. PMID:12574268

Cobalt deposition in mineralized bone tissue after metal-on-metal hip resurfacing: Quantitative μ-X-ray-fluorescence analysis of implant material incorporation in periprosthetic tissue.

PubMed

Hahn, Michael; Busse, Björn; Procop, Mathias; Zustin, Jozef; Amling, Michael; Katzer, Alexander

2017-10-01

Most resurfacing systems are manufactured from cobalt-chromium alloys with metal-on-metal (MoM) bearing couples. Because the quantity of particulate metal and corrosion products which can be released into the periprosthetic milieu is greater in MoM bearings than in metal-on-polyethylene (MoP) bearings, it is hypothesized that the quantity and distribution of debris released by the MoM components induce a compositional change in the periprosthetic bone. To determine the validity of this claim, nondestructive µ-X-ray fluorescence analysis was carried out on undecalcified histological samples from 13 femoral heads which had undergone surface replacement. These samples were extracted from the patients after gradient time points due to required revision surgery. Samples from nonintervened femoral heads as well as from a MoP resurfaced implant served as controls. Light microscopy and µ-X-ray fluorescence analyses revealed that cobalt debris was found not only in the soft tissue around the prosthesis and the bone marrow, but also in the mineralized bone tissue. Mineralized bone exposed to surface replacements showed significant increases in cobalt concentrations in comparison with control specimens without an implant. A maximum cobalt concentration in mineralized hard tissue of up to 380 ppm was detected as early as 2 years after implantation. Values of this magnitude are not found in implants with a MoP surface bearing until a lifetime of more than 20 years. This study demonstrates that hip resurfacing implants with MoM bearings present a potential long-term health risk due to rapid cobalt ion accumulation in periprosthetic hard tissue. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1855-1862, 2017. © 2016 Wiley Periodicals, Inc.

Discrimination and similarity evaluation of tissue-cultured and wild Dendrobium species using Fourier transform infrared spectroscopy

NASA Astrophysics Data System (ADS)

Chen, Nai-dong; Chen, Han; Li, Jun; Sang, Mang-mang; Ding, Shen; Yu, Hao

2015-04-01

The FTIR method was applied to evaluate the similarity of tissue-cultured and wild Dendrobium huoshanense C.Z. Tang et S.J. Cheng, Dendrobium officinale Kimura et Migo and Dendrobium moniliforme (Linn.) Sw and discriminate different Dendrobium species, especially D. huoshanense and its main goldbrick Dendrobium henanense J.L. Lu et L.X. Gao. Despite the general pattern of the IR spectra, different intensities, shapes and peak positions were found in the IR spectra of these samples, especially in the range of 1800-600 cm-1, which could be used to discriminate them. The methanol, aqueous extracting procedure and the second derivative transformation obviously enlarged the tiny spectral differences among these samples. The similarity evaluation based on the IR spectra and the second derivative IR spectrum revealed that the similarity of the methanol extracts between tissue-cultured and wild Dendrobiums might be lower than that between different Dendrobium species. The similarities of the powders and aqueous extracts between tissue-cultured and wild Dendrobiums were higher than those between different Dendrobium species. The further principal component analysis showed that the first three components explained 99.7%, 87.7% and 85.1% of data variance for powder, methanol extract and aqueous extract, respectively, demonstrating a good discrimination between samples. Our research suggested that the variations of secondary metabolites between different origins of the investigated Dendrobiums might be higher than what we had supposed. Tissue culture techniques were widely used in the conversation of rare and endangered medicinal amedica, however, our study suggested that the chemical constituents of tissue-cultured plants might be quite different from their wild correspondences.

Distribution of enrofloxacin and its active metabolite, using an in vivo ultrafiltration sampling technique after the injection of enrofloxacin to pigs.

PubMed

Messenger, K M; Papich, M G; Blikslager, A T

2012-10-01

The objective of this study was to determine the pharmacokinetics (PK) of enrofloxacin in pigs and compare to the tissue interstitial fluid (ISF). Six healthy, young pigs were administered 7.5 mg/kg enrofloxacin subcutaneously (SC). Blood and ISF samples were collected from preplaced intravenous catheters and ultrafiltration sampling probes placed in three different tissue sites (intramuscular, subcutaneous, and intrapleural). Enrofloxacin concentrations were measured using high-pressure liquid chromatography with fluorescence detection, PK parameters were analyzed using a one-compartment model, and protein binding was determined using a microcentrifugation system. Concentrations of the active metabolite ciprofloxacin were negligible. The mean ± SD enrofloxacin plasma half-life, volume of distribution, clearance, and peak concentration were 26.6 ± 6.2 h (harmonic mean), 6.4 ± 1.2 L/kg, 0.18 ± 0.08 L/kg/h, and 1.1 ± 0.3 μg/mL, respectively. The half-life of enrofloxacin from the tissues was 23.6 h, and the maximum concentration was 1.26 μg/mL. Tissue penetration, as measured by a ratio of area-under-the-curve (AUC), was 139% (± 69%). Plasma protein binding was 31.1% and 37.13% for high and low concentrations, respectively. This study demonstrated that the concentration of biologically active enrofloxacin in tissues exceeds the concentration predicted by the unbound fraction of enrofloxacin in pig plasma. At a dose of 7.5 mg/kg SC, the high tissue concentrations and long half-life produce an AUC/MIC ratio sufficient for the pathogens that cause respiratory infections in pigs. © 2011 Blackwell Publishing Ltd.

Heart rate sensitive optical coherence angiography

NASA Astrophysics Data System (ADS)

Alvarez, Karl; Lopez-Tremoleda, Jordi; Donnan, Rob; Michael-Titus, Adina T.; Tomlins, Peter H.

2018-02-01

Optical coherence angiography (OCA) enables visualisation of three-dimensional micro-vasculature from optical coherence tomography data volumes. Typically, various statistical methods are used to discriminate static tissue from blood flow within vessels. In this paper, we introduce a new method that relies upon the beating heart frequency to isolate blood vessels from the surrounding tissue. Vascular blood flow is assumed to be more strongly modulated by the heart-beat compared to surrounding tissue and therefore short-time Fourier transform of sequential measurements can discriminate the two. Furthermore, it is demonstrated that adjacent B-Scans within an OCT data volume can provide the required sampling frequency. As such, the technique can be considered to be a spatially mapped variation of photoplethysmography (PPG), whereby each image voxel operates as a PPG detector. This principle is demonstrated using both a model system and in vivo for monitoring the vascular changes effected by traumatic brain injury in mice. In vivo measurements were acquired at an A-Scan rate of 10kHz to form a 500x500x512 (lateral x lateral x axial) pixel volume, enabling sequential sampling of the mouse heart rate in an expected range of 300-600 bpm. One of the advantages of this new OCA processing method is that it can be used in conjunction with existing algorithms as an additional filter for signal to noise enhancement.

OCT as a convenient tool to assess the quality and application of organotypic retinal samples

NASA Astrophysics Data System (ADS)

Gater, Rachel; Khoshnaw, Nicholas; Nguyen, Dan; El Haj, Alicia J.; Yang, Ying

2016-03-01

Eye diseases such as macular degeneration and glaucoma have profound consequences on the quality of human life. Without treatment, these diseases can lead to loss of sight. To develop better treatments for retinal diseases, including cell therapies and drug intervention, establishment of an efficient and reproducible 3D native retinal tissue system, enabled over a prolonged culture duration, will be valuable. The retina is a complex tissue, consisting of ten layers with a different density and cellular composition to each. Uniquely, as a light transmitting tissue, retinal refraction of light differs among the layers, forming a good basis to use optical coherence tomography (OCT) in assessing the layered structure of the retina and its change during the culture and treatments. In this study, we develop a new methodology to generate retinal organotypic tissues and compare two substrates: filter paper and collagen hydrogel, to culture the organotypic tissue. Freshly slaughtered pig eyes have been obtained for use in this study. The layered morphology of intact organotypic retinal tissue cultured on two different substrates has been examined by spectral domain OCT. The viability of the tissues has been examined by live/dead fluorescence dye kit to cross validate the OCT images. For the first time, it is demonstrated that the use of a collagen hydrogel supports the viability of retinal organotypic tissue, capable of prolonged culture up to 2 weeks. OCT is a convenient tool for appraising the quality and application of organotypic retinal samples and is important in the development of current organotypic models.

The MiAge Calculator: a DNA methylation-based mitotic age calculator of human tissue types.

PubMed

Youn, Ahrim; Wang, Shuang

2018-01-01

Cell division is important in human aging and cancer. The estimation of the number of cell divisions (mitotic age) of a given tissue type in individuals is of great interest as it allows not only the study of biological aging (using a new molecular aging target) but also the stratification of prospective cancer risk. Here, we introduce the MiAge Calculator, a mitotic age calculator based on a novel statistical framework, the MiAge model. MiAge is designed to quantitatively estimate mitotic age (total number of lifetime cell divisions) of a tissue using the stochastic replication errors accumulated in the epigenetic inheritance process during cell divisions. With the MiAge model, the MiAge Calculator was built using the training data of DNA methylation measures of 4,020 tumor and adjacent normal tissue samples from eight TCGA cancer types and was tested using the testing data of DNA methylation measures of 2,221 tumor and adjacent normal tissue samples of five other TCGA cancer types. We showed that within each of the thirteen cancer types studied, the estimated mitotic age is universally accelerated in tumor tissues compared to adjacent normal tissues. Across the thirteen cancer types, we showed that worse cancer survivals are associated with more accelerated mitotic age in tumor tissues. Importantly, we demonstrated the utility of mitotic age by showing that the integration of mitotic age and clinical information leads to improved survival prediction in six out of the thirteen cancer types studied. The MiAge Calculator is available at http://www.columbia.edu/∼sw2206/softwares.htm .

An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing.

PubMed

Amini, Parisa; Ettlin, Julia; Opitz, Lennart; Clementi, Elena; Malbon, Alexandra; Markkanen, Enni

2017-08-23

Formalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA-protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult. We excised matched cancer-associated stroma (CAS) and normal stroma from clinical specimen of FFPE canine mammary tumours using LCM, and compared the commonly used protease-based RNA isolation procedure with an adapted novel technique that additionally incorporates a focused ultrasonication step. We successfully adapted a protocol that uses focused ultrasonication to isolate RNA from small amounts of deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach was qualitatively at least equal if not superior compared to the old approach, as Cq values in RT-qPCR were on average 2.3-fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well. We present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen. The possibility to study gene expression signatures in specific small sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now become amenable for investigation.

Detection and Molecular Characterization of Gemycircularvirus from Environmental Samples in Brazil.

PubMed

da Silva Assis, Matheus Ribeiro; Vieira, Carmen Baur; Fioretti, Julia Monassa; Rocha, Mônica Simões; de Almeida, Pedro Ivo Neves; Miagostovich, Marize Pereira; Fumian, Tulio Machado

2016-12-01

Gemycircularvirus (GemyCV) is a group of viruses which has been recently proposed as a new viral genus detected in fecal and environmental samples around the world. GemyCVs have been detected in human blood, brain tissue, cerebrospinal fluid, and stool sample. In the present study, we demonstrate for the first time, through molecular detection and characterization, the presence of GemyCVs in environmental samples from Brazil. Our results show a percentage of positivity ranging from 69 (25/36) to 97 % (35/36) in river water samples collected in Manaus, Amazon region, and wastewater from a wastewater treatment plant located in Rio de Janeiro, respectively, revealing GemyCVs as an important environmental contaminant.

Measurement of drug and macromolecule diffusion across atherosclerotic rabbit aorta ex vivo by attenuated total reflection-Fourier transform infrared imaging

NASA Astrophysics Data System (ADS)

Palombo, Francesca; Danoux, Charlène B.; Weinberg, Peter D.; Kazarian, Sergei G.

2009-07-01

Diffusion of two model drugs-benzyl nicotinate and ibuprofen-and the plasma macromolecule albumin across atherosclerotic rabbit aorta was studied ex vivo by attenuated total reflection-Fourier transform infrared (ATR-FTIR) imaging. Solutions of these molecules were applied to the endothelial surface of histological sections of the aortic wall that were sandwiched between two impermeable surfaces. An array of spectra, each corresponding to a specific location in the section, was obtained at various times during solute diffusion into the wall and revealed the distribution of the solutes within the tissue. Benzyl nicotinate in Ringer's solution showed higher affinity for atherosclerotic plaque than for apparently healthy tissue. Transmural concentration profiles for albumin demonstrated its permeation across the section and were consistent with a relatively low distribution volume for the macromolecule in the middle of the wall. The ability of albumin to act as a drug carrier for ibuprofen, otherwise undetected within the tissue, was demonstrated by multivariate subtraction image analysis. In conclusion, ATR-FTIR imaging can be used to study transport processes in tissue samples with high spatial and temporal resolution and without the need to label the solutes under study.

Optimization of a Widefield Structured Illumination Microscope for Non-Destructive Assessment and Quantification of Nuclear Features in Tumor Margins of a Primary Mouse Model of Sarcoma

PubMed Central

Fu, Henry L.; Mueller, Jenna L.; Javid, Melodi P.; Mito, Jeffrey K.; Kirsch, David G.; Ramanujam, Nimmi; Brown, J. Quincy

2013-01-01

Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1×1.6 mm (3.4 mm2) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm−1 used in conjunction with a 4×0.1 NA objective ( = 0.165). This yielded an optical section thickness of 128 µm and an 8.9×contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy. PMID:23894357

Tissue-aware RNA-Seq processing and normalization for heterogeneous and sparse data.

PubMed

Paulson, Joseph N; Chen, Cho-Yi; Lopes-Ramos, Camila M; Kuijjer, Marieke L; Platig, John; Sonawane, Abhijeet R; Fagny, Maud; Glass, Kimberly; Quackenbush, John

2017-10-03

Although ultrahigh-throughput RNA-Sequencing has become the dominant technology for genome-wide transcriptional profiling, the vast majority of RNA-Seq studies typically profile only tens of samples, and most analytical pipelines are optimized for these smaller studies. However, projects are generating ever-larger data sets comprising RNA-Seq data from hundreds or thousands of samples, often collected at multiple centers and from diverse tissues. These complex data sets present significant analytical challenges due to batch and tissue effects, but provide the opportunity to revisit the assumptions and methods that we use to preprocess, normalize, and filter RNA-Seq data - critical first steps for any subsequent analysis. We find that analysis of large RNA-Seq data sets requires both careful quality control and the need to account for sparsity due to the heterogeneity intrinsic in multi-group studies. We developed Yet Another RNA Normalization software pipeline (YARN), that includes quality control and preprocessing, gene filtering, and normalization steps designed to facilitate downstream analysis of large, heterogeneous RNA-Seq data sets and we demonstrate its use with data from the Genotype-Tissue Expression (GTEx) project. An R package instantiating YARN is available at http://bioconductor.org/packages/yarn .

Estimation of the number of fluorescent end-members for quantitative analysis of multispectral FLIM data.

PubMed

Gutierrez-Navarro, Omar; Campos-Delgado, Daniel U; Arce-Santana, Edgar R; Maitland, Kristen C; Cheng, Shuna; Jabbour, Joey; Malik, Bilal; Cuenca, Rodrigo; Jo, Javier A

2014-05-19

Multispectral fluorescence lifetime imaging (m-FLIM) can potentially allow identifying the endogenous fluorophores present in biological tissue. Quantitative description of such data requires estimating the number of components in the sample, their characteristic fluorescent decays, and their relative contributions or abundances. Unfortunately, this inverse problem usually requires prior knowledge about the data, which is seldom available in biomedical applications. This work presents a new methodology to estimate the number of potential endogenous fluorophores present in biological tissue samples from time-domain m-FLIM data. Furthermore, a completely blind linear unmixing algorithm is proposed. The method was validated using both synthetic and experimental m-FLIM data. The experimental m-FLIM data include in-vivo measurements from healthy and cancerous hamster cheek-pouch epithelial tissue, and ex-vivo measurements from human coronary atherosclerotic plaques. The analysis of m-FLIM data from in-vivo hamster oral mucosa identified healthy from precancerous lesions, based on the relative concentration of their characteristic fluorophores. The algorithm also provided a better description of atherosclerotic plaques in term of their endogenous fluorophores. These results demonstrate the potential of this methodology to provide quantitative description of tissue biochemical composition.

Exploitation of FTA cartridges for the sampling, long-term storage, and DNA-based analyses of plant-parasitic nematodes.

PubMed

Marek, Martin; Zouhar, Miloslav; Douda, Ondřej; Maňasová, Marie; Ryšánek, Pavel

2014-03-01

The use of DNA-based analyses in molecular plant nematology research has dramatically increased over recent decades. Therefore, the development and adaptation of simple, robust, and cost-effective DNA purification procedures are required to address these contemporary challenges. The solid-phase-based approach developed by Flinders Technology Associates (FTA) has been shown to be a powerful technology for the preparation of DNA from different biological materials, including blood, saliva, plant tissues, and various human and plant microbial pathogens. In this work, we demonstrate, for the first time, that this FTA-based technology is a valuable, low-cost, and time-saving approach for the sampling, long-term archiving, and molecular analysis of plant-parasitic nematodes. Despite the complex structure and anatomical organization of the multicellular bodies of nematodes, we report the successful and reliable DNA-based analysis of nematode high-copy and low-copy genes using the FTA technology. This was achieved by applying nematodes to the FTA cards either in the form of a suspension of individuals, as intact or pestle-crushed nematodes, or by the direct mechanical printing of nematode-infested plant tissues. We further demonstrate that the FTA method is also suitable for the so-called "one-nematode-assay", in which the target DNA is typically analyzed from a single individual nematode. More surprisingly, a time-course experiment showed that nematode DNA can be detected specifically in the FTA-captured samples many years after initial sampling occurs. Collectively, our data clearly demonstrate the applicability and the robustness of this FTA-based approach for molecular research and diagnostics concerning phytonematodes; this research includes economically important species such as the stem nematode (Ditylenchus dipsaci), the sugar beet nematode (Heterodera schachtii), and the Northern root-knot nematode (Meloidogyne hapla).

Critical issues with the in vivo comet assay: A report of the comet assay working group in the 6th International Workshop on Genotoxicity Testing (IWGT).

PubMed

Speit, Günter; Kojima, Hajime; Burlinson, Brian; Collins, Andrew R; Kasper, Peter; Plappert-Helbig, Ulla; Uno, Yoshifumi; Vasquez, Marie; Beevers, Carol; De Boeck, Marlies; Escobar, Patricia A; Kitamoto, Sachiko; Pant, Kamala; Pfuhler, Stefan; Tanaka, Jin; Levy, Dan D

2015-05-01

As a part of the 6th IWGT, an expert working group on the comet assay evaluated critical topics related to the use of the in vivo comet assay in regulatory genotoxicity testing. The areas covered were: identification of the domain of applicability and regulatory acceptance, identification of critical parameters of the protocol and attempts to standardize the assay, experience with combination and integration with other in vivo studies, demonstration of laboratory proficiency, sensitivity and power of the protocol used, use of different tissues, freezing of samples, and choice of appropriate measures of cytotoxicity. The standard protocol detects various types of DNA lesions but it does not detect all types of DNA damage. Modifications of the standard protocol may be used to detect additional types of specific DNA damage (e.g., cross-links, bulky adducts, oxidized bases). In addition, the working group identified critical parameters that should be carefully controlled and described in detail in every published study protocol. In vivo comet assay results are more reliable if they were obtained in laboratories that have demonstrated proficiency. This includes demonstration of adequate response to vehicle controls and an adequate response to a positive control for each tissue being examined. There was a general agreement that freezing of samples is an option but more data are needed in order to establish generally accepted protocols. With regard to tissue toxicity, the working group concluded that cytotoxicity could be a confounder of comet results. It is recommended to look at multiple parameters such as histopathological observations, organ-specific clinical chemistry as well as indicators of tissue inflammation to decide whether compound-specific toxicity might influence the result. The expert working group concluded that the alkaline in vivo comet assay is a mature test for the evaluation of genotoxicity and can be recommended to regulatory agencies for use. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparison of the mucoadhesive properties of thiolated polyacrylic acid to thiolated polyallylamine.

PubMed

Duggan, Sarah; O'Donovan, Orla; Owens, Eleanor; Duggan, Elaine; Hughes, Helen; Cummins, Wayne

2016-02-10

Synthetic polymers, polyacrylic acid (PAA) and polyallylamine (PAAm), were thiolated using different methods of thiolation. Both polymers resulted in comparable thiol contents, thus allowing for the direct comparison of mucoadhesive and cohesive properties between the well-established thiolated PAA and the more novel thiolated PAAm. Thiolation of both polymers improved the swelling ability and the cohesive and mucoadhesive properties in comparison to unmodified control samples. In this study, it was shown that the swelling abilities of the thiolated PAAm sample were far greater than that of the thiolated PAA sample which, in turn, affected the drug release profile of the thiolated PAAm sample. Importantly, however, the mucoadhesive properties of thiolated PAAm were equivalent to that of the thiolated PAA sample as demonstrated by both the adhesion times on porcine intestinal tissue as measured by the rotating cylinder method and by rheological studies with a mucin solution. This study demonstrates the potential thiolated polyallylamine has as a mucoadhesive drug delivery device. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid molecular detection of invasive species in ballast and harbor water by integrating environmental DNA and light transmission spectroscopy.

PubMed

Egan, Scott P; Grey, Erin; Olds, Brett; Feder, Jeffery L; Ruggiero, Steven T; Tanner, Carol E; Lodge, David M

2015-04-07

Invasive species introduced via the ballast water of commercial ships cause enormous environmental and economic damage worldwide. Accurate monitoring for these often microscopic and morphologically indistinguishable species is challenging but critical for mitigating damages. We apply eDNA sampling, which involves the filtering and subsequent DNA extraction of microscopic bits of tissue suspended in water, to ballast and harbor water sampled during a commercial ship's 1400 km voyage through the North American Great Lakes. Using a lab-based gel electrophoresis assay and a rapid, field-ready light transmission spectroscopy (LTS) assay, we test for the presence of two invasive species: quagga (Dreissena bugensis) and zebra (D. polymorpha) mussels. Furthermore, we spiked a set of uninfested ballast and harbor samples with zebra mussel tissue to further test each assay's detection capabilities. In unmanipulated samples, zebra mussel was not detected, while quagga mussel was detected in all samples at a rate of 85% for the gel assay and 100% for the LTS assay. In the spiked experimental samples, both assays detected zebra mussel in 94% of spiked samples and 0% of negative controls. Overall, these results demonstrate that eDNA sampling is effective for monitoring ballast-mediated invasions and that LTS has the potential for rapid, field-based detection.

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.

An Optimized Small Tissue Handling System for Immunohistochemistry and In Situ Hybridization

PubMed Central

Anthony, Giovanni; Lee, Ju-Ahng

2016-01-01

Recent development in 3D printing technology has opened an exciting possibility for manufacturing 3D devices on one’s desktop. We used 3D modeling programs to design 3D models of a tissue-handling system and these models were “printed” in a stereolithography (SLA) 3D printer to create precision histology devices that are particularly useful to handle multiple samples with small dimensions in parallel. Our system has been successfully tested for in situ hybridization of zebrafish embryos. Some of the notable features include: (1) A conveniently transferrable chamber with 6 mesh-bottomed wells, each of which can hold dozens of zebrafish embryos. This design allows up to 6 different samples to be treated per chamber. (2) Each chamber sits in a well of a standard 6-well tissue culture plate. Thus, up to 36 different samples can be processed in tandem using a single 6 well plate. (3) Precisely fitting lids prevent solution evaporation and condensation, even at high temperatures for an extended period of time: i.e., overnight riboprobe hybridization. (4) Flat bottom mesh maximizes the consistent treatment of individual tissue samples. (5) A magnet-based lifter was created to handle up to 6 chambers (= 36 samples) in unison. (6) The largely transparent resin aids in convenient visual inspection both with eyes and using a stereomicroscope. (7) Surface engraved labeling enables an accurate tracking of different samples. (8) The dimension of wells and chambers minimizes the required amount of precious reagents. (9) Flexible parametric modeling enables an easy redesign of the 3D models to handle larger or more numerous samples. Precise dimensions of 3D models and demonstration of how we use our devices in whole mount in situ hybridization are presented. We also provide detailed information on the modeling software, 3D printing tips, as well as 3D files that can be used with any 3D printer. PMID:27489962

Study of melanin bleaching after immunohistochemistry of melanin-containing tissues.

PubMed

Shen, Hongwu; Wu, Wenqiao

2015-04-01

Melanin may interfere with immunohistochemical staining. The goal of this study was to investigate the effects of trichloroisocyanuric acid (TCCA) bleaching, potassium permanganate bleaching, and potassium dichromate bleaching on melanin, tissue antigen, and 3,3'-diaminobenzidine (DAB) using melanin-containing and melanin-free tissue samples. Our results demonstrated that all 3 bleaching methods efficiently bleached melanin and partially destroyed tissue antigen. In addition, potassium permanganate bleaching and potassium dichromate bleaching clearly destroyed DAB, whereas TCCA bleaching had no significant effect on DAB. Therefore, neither potassium permanganate nor potassium dichromate is an ideal solution, whereas TCCA might be an ideal solution for melanin bleaching after the immunohistochemical staining of melanin-containing tissues. After immunostaining followed by TCCA bleaching, the melanin could be completely removed in all 120 malignant melanoma tissue sections. Compared with the control, the DAB intensity was clear, and the tissue structure and cellular nuclei were well maintained. It is worth noting that TCCA should be freshly prepared before each experiment, and used within 2 hours of its preparation. In addition, sections should not be incubated with TCCA for over 30 minutes.

Serum immunoreactivity of cancer/testis antigen OY-TES-1 and its tissues expression in glioma

PubMed Central

Li, Xisheng; Yan, Jun; Fan, Rong; Luo, Bin; Zhang, Qingmei; Lin, Yongda; Zhou, Sufang; Luo, Guorong; Xie, Xiaoxun; Xiao, Shaowen

2017-01-01

OY-TES-1 is a member of the cancer/testis antigen family that is expressed in healthy testis tissue and certain types of cancerous tissue. The present study aimed to analyze the expression pattern of OY-TES-1 and serum anti-OY-TES-1 antibody concentration in patients with glioma. OY-TES-1 mRNA was detected in 28/36 (78%) of glioma cases using conventional reverse transcription polymerase chain reaction (RT-PCR) analysis. RT-quantitative-PCR revealed that OY-TES-1 was expressed at a higher level in glioma tissues compared with normal adult tissues (with the exception of testis tissue). Anti-OY-TES-1 antibodies were present in the serum of 5/36 (14%) of patients with glioma, but absent in all the serum samples from 107 healthy donors. Immunohistochemical analysis demonstrated that OY-TES-1 protein was expressed in all glioma tissues from patients with anti-OY-TES-1 antibody seropositivity. These results suggest that OY-TES-1 is a novel candidate for glioma immunotherapy. PMID:28529561

The Resin-Embedded Cornea Prepared Via Rapid Processing Protocol : A Good Histomorphometric Target for Clinical Investigation in Ophthalmology and Optometry

PubMed Central

Cheah, Pike See; Mohidin, Norhani; Mohd Ali, Bariah; Maung, Myint; Latif, Azian Abdul

2008-01-01

This study illustrates and quantifies the changes on corneal tissue between the paraffin-embedded and resin-embedded blocks and thus, selects a better target in investigational ophthalmology and optometry via light microscopy. Corneas of two cynomolgus monkeys (Macaca fascicularis) were used in this study. The formalin-fixed cornea was prepared in paraffin block via the conventional tissue processing protocol (4-day protocol) and stained with haematoxylin and eosin. The glutaraldehyde-fixed cornea was prepared in resin block via the rapid and modified tissue processing procedure (1.2-day protocol) and stained with toluidine blue. The paraffin-embedded sample exhibits various undesired tissue damage and artifact such as thinner epithelium (due to the substantial volumic extraction from the tissue), thicker stroma layer (due to the separation of lamellae and the presence of voids) and the distorted endothelium. In contrast, the resin-embedded corneal tissue has demonstrated satisfactory corneal ultrastructural preservation. The rapid and modified tissue processing method for preparing the resin-embedded is particularly beneficial to accelerate the microscopic evaluation in ophthalmology and optometry. PMID:22570589

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.

CpG Methylation Analysis of HPV16 in Laser Capture Microdissected Archival Tissue and Whole Tissue Sections from High Grade Anal Squamous Intraepithelial Lesions: A Potential Disease Biomarker

PubMed Central

Molano, Monica; Tabrizi, Sepehr N.; Garland, Suzanne M.; Roberts, Jennifer M.; Machalek, Dorothy A.; Phillips, Samuel; Chandler, David; Hillman, Richard J.; Grulich, Andrew E.; Jin, Fengyi; Poynten, I. Mary; Templeton, David J.; Cornall, Alyssa M.

2016-01-01

Incidence and mortality rates of anal cancer are increasing globally. More than 90% of anal squamous cell carcinomas (ASCC) are associated with human papillomavirus (HPV). Studies on HPV-related anogenital lesions have shown that patterns of methylation of viral and cellular DNA targets could potentially be developed as disease biomarkers. Lesion-specific DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues from existing or prospective patient cohorts may constitute a valuable resource for methylation analysis. However, low concentrations of DNA make these samples technically challenging to analyse using existing methods. We therefore set out to develop a sensitive and reproducible nested PCR-pyrosequencing based method to accurately quantify methylation at 10 CpG sites within the E2BS1, E2BS2,3,4 and Sp1 binding sites in the viral upstream regulatory region of HPV16 genome. Methylation analyses using primary and nested PCR-pyrosequencing on 52 FFPE tissue [26 paired whole tissue sections (WTS) and laser capture microdissected (LCM) tissues] from patients with anal squamous intraepithelial lesions was performed. Using nested PCR, methylation results were obtained for the E2BS1, E2BS2,3,4 and Sp1 binding sites in 86.4% of the WTS and 81.8% of the LCM samples. Methylation patterns were strongly correlated within median values of matched pairs of WTS and LCM sections, but overall methylation was higher in LCM samples at different CpG sites. High grade lesions showed low methylation levels in the E2BS1 and E2BS2 regions, with increased methylation detected in the E2BS,3,4/Sp1 regions, showing the highest methylation at CpG site 37. The method developed is highly sensitive in samples with low amounts of DNA and demonstrated to be suitable for archival samples. Our data shows a possible role of specific methylation in the HPV16 URR for detection of HSIL. PMID:27529629

CpG Methylation Analysis of HPV16 in Laser Capture Microdissected Archival Tissue and Whole Tissue Sections from High Grade Anal Squamous Intraepithelial Lesions: A Potential Disease Biomarker.

PubMed

Molano, Monica; Tabrizi, Sepehr N; Garland, Suzanne M; Roberts, Jennifer M; Machalek, Dorothy A; Phillips, Samuel; Chandler, David; Hillman, Richard J; Grulich, Andrew E; Jin, Fengyi; Poynten, I Mary; Templeton, David J; Cornall, Alyssa M

2016-01-01

Incidence and mortality rates of anal cancer are increasing globally. More than 90% of anal squamous cell carcinomas (ASCC) are associated with human papillomavirus (HPV). Studies on HPV-related anogenital lesions have shown that patterns of methylation of viral and cellular DNA targets could potentially be developed as disease biomarkers. Lesion-specific DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues from existing or prospective patient cohorts may constitute a valuable resource for methylation analysis. However, low concentrations of DNA make these samples technically challenging to analyse using existing methods. We therefore set out to develop a sensitive and reproducible nested PCR-pyrosequencing based method to accurately quantify methylation at 10 CpG sites within the E2BS1, E2BS2,3,4 and Sp1 binding sites in the viral upstream regulatory region of HPV16 genome. Methylation analyses using primary and nested PCR-pyrosequencing on 52 FFPE tissue [26 paired whole tissue sections (WTS) and laser capture microdissected (LCM) tissues] from patients with anal squamous intraepithelial lesions was performed. Using nested PCR, methylation results were obtained for the E2BS1, E2BS2,3,4 and Sp1 binding sites in 86.4% of the WTS and 81.8% of the LCM samples. Methylation patterns were strongly correlated within median values of matched pairs of WTS and LCM sections, but overall methylation was higher in LCM samples at different CpG sites. High grade lesions showed low methylation levels in the E2BS1 and E2BS2 regions, with increased methylation detected in the E2BS,3,4/Sp1 regions, showing the highest methylation at CpG site 37. The method developed is highly sensitive in samples with low amounts of DNA and demonstrated to be suitable for archival samples. Our data shows a possible role of specific methylation in the HPV16 URR for detection of HSIL.

Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas.

PubMed

Mohapatra, Gayatry; Engler, David A; Starbuck, Kristen D; Kim, James C; Bernay, Derek C; Scangas, George A; Rousseau, Audrey; Batchelor, Tracy T; Betensky, Rebecca A; Louis, David N

2011-04-01

Array comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA). Because diffuse malignant gliomas are often sampled by small biopsies, formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis; FFPE tissues are also needed to study the intratumoral heterogeneity that characterizes these neoplasms. In this paper, we present a combination of evaluations and technical advances that provide strong support for the ready use of oligonucleotide aCGH on FFPE diffuse gliomas. We first compared aCGH using bacterial artificial chromosome (BAC) arrays in 45 paired frozen and FFPE gliomas, and demonstrate a high concordance rate between FFPE and frozen DNA in an individual clone-level analysis of sensitivity and specificity, assuring that under certain array conditions, frozen and FFPE DNA can perform nearly identically. However, because oligonucleotide arrays offer advantages to BAC arrays in genomic coverage and practical availability, we next developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. To demonstrate utility in FFPE tissues, we applied this approach to biphasic anaplastic oligoastrocytomas and demonstrate CNA differences between DNA obtained from the two components. Therefore, BAC and oligonucleotide aCGH can be sensitive and specific tools for detecting CNAs in FFPE DNA, and novel labeling techniques enable the routine use of oligonucleotide arrays for FFPE DNA. In combination, these advances should facilitate genome-wide analysis of rare, small and/or histologically heterogeneous gliomas from FFPE tissues.

Molecular characterization of a novel gammaretrovirus in killer whales (Orcinus orca).

PubMed

Lamere, Sarah A; St Leger, Judy A; Schrenzel, Mark D; Anthony, Simon J; Rideout, Bruce A; Salomon, Daniel R

2009-12-01

There are currently no published data documenting the presence of retroviruses in cetaceans, though the occurrences of cancers and immunodeficiency states suggest the potential. We examined tissues from adult killer whales and detected a novel gammaretrovirus by degenerate PCR. Reverse transcription-PCR also demonstrated tissue and serum expression of retroviral mRNA. The full-length sequence of the provirus was obtained by PCR, and a TaqMan-based copy number assay did not demonstrate evidence of productive infection. PCR on blood samples from 11 healthy captive killer whales and tissues from 3 free-ranging animals detected the proviral DNA in all tissues examined from all animals. A survey of multiple cetacean species by PCR for gag, pol, and env sequences showed homologs of this virus in the DNA of eight species of delphinids, pygmy and dwarf sperm whales, and harbor porpoises, but not in beluga or fin whales. Analysis of the bottlenose dolphin genome revealed two full-length proviral sequences with 97.4% and 96.9% nucleotide identity to the killer whale gammaretrovirus. The results of single-cell PCR on killer whale sperm and Southern blotting are also consistent with the conclusion that the provirus is endogenous. We suggest that this gammaretrovirus entered the delphinoid ancestor's genome before the divergence of modern dolphins or that an exogenous variant existed following divergence that was ultimately endogenized. However, the transcriptional activity demonstrated in tissues and the nearly intact viral genome suggest a more recent integration into the killer whale genome, favoring the latter hypothesis. The proposed name for this retrovirus is killer whale endogenous retrovirus.

Maximizing Science Return from Future Rodent Experiments on the International Space Station (ISS): Tissue Preservation

NASA Technical Reports Server (NTRS)

Choi, S. Y.; Lai, S.; Klotz, R.; Popova, Y.; Chakravarty, K.; Beegle, J. E.; Wigley, C. L.; Globus, R. K.

2014-01-01

To better understand how mammals adapt to long duration habitation in space, a system for performing rodent experiments on the ISS is under development; Rodent Research-1 is the first flight and will include validation of both on-orbit animal support and tissue preservation. To evaluate plans for on-orbit sample dissection and preservation, we simulated conditions for euthanasia, tissue dissection, and prolonged sample storage on the ISS, and we also developed methods for post-flight dissection and recovery of high quality RNA from multiple tissues following prolonged storage in situ for future science. Mouse livers and spleens were harvested under conditions that simulated nominal, on-orbit euthanasia and dissection operations including storage at -80 C for 4 months. The RNA recovered was of high quality (RNA Integrity Number, RIN(is) greater than 8) and quantity, and the liver enzyme contents and activities (catalase, glutathione reductase, GAPDH) were similar to positive controls, which were collected under standard laboratory conditions. We also assessed the impact of possible delayed on-orbit dissection scenarios (off-nominal) by dissecting and preserving the spleen (RNAlater) and liver (fast-freezing) at various time points post-euthanasia (from 5 min up to 105 min). The RNA recovered was of high quality (spleen, RIN (is) greater than 8; liver, RIN (is) greater than 6) and liver enzyme activities were similar to positive controls at all time points, although an apparent decline in select enzyme activities was evident at the latest time (105 min). Additionally, various tissues were harvested from either intact or partially dissected, frozen carcasses after storage for approximately 2 months; most of the tissues (brain, heart, kidney, eye, adrenal glands and muscle) were of acceptable RNA quality for science return, whereas some tissues (small intestine, bone marrow and bones) were not. These data demonstrate: 1) The protocols developed for future flight experiments will support science return despite delayed preservation post-euthanasia or prolonged storage, and 2) Many additional tissues for gene expression analysis can be obtained by dissection following prolonged storage of the tissue in situ at -80 C. These findings have relevance both to high value, ground-based experiments when sample collection capability is severely constrained, and to all future spaceflight experiments that entail on-orbit sample recovery by the ISS crew.

Analysis of the distribution and expression of claudin-1 tight junction protein in the oral cavity.

PubMed

Ouban, Abderrahman; Ahmed, Atif

2015-07-01

Claudins are the main sealing proteins of the intercellular tight junctions and play an important role in cancer cell progression and dissemination. The authors have previously shown that overexpression of claudin-1 is associated with angiolymphatic and perineural invasion, consistent with aggressive tumor behavior and with advanced stage disease in oral squamous cell carcinomas (OSCCs). Our goal in this study was to examine claudin-1 expression in a tissue microarray of OSCCs taken from multiple sites within the oral cavity. This study examined and compared the expression of claudin-1 by immunohistochemistry in 60 tissue samples (49 OSCCs and 10 cases of non-neoplastic tissue, single core per case) were analyzed for claudin-1 expression by immunohistochemistry. The tumors included SCCs from the tongue (n=28), the cheek (n=9), gingival (n=4), lip (n=3), and oral cavity (n=5). Nonmalignant normal oral mucosa from the tongue (unmatched cases, n=2). Cancer adjacent tissue samples were taken from the tongue (n=6), gingival (n=2), and palate (n=1). This study demonstrates the expression of claudin-1 protein across a sample of OSCCs originating from multiple locations in the oral cavity. The highest expression of claudin-1 was observed in well-differentiated OSCCs, whereas poorly differentiated OSCCs exhibited mostly negative staining for claudin-1. In addition, we hereby report differential pattern of expression among tumors of different sites within the oral cavity, and between benign and cancerous samples. Our understanding of the exact function and role of claudin-1 in tumorigenesis is expanding exponentially.

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.

Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles.

PubMed

Peel, Trisha N; Dylla, Brenda L; Hughes, John G; Lynch, David T; Greenwood-Quaintance, Kerryl E; Cheng, Allen C; Mandrekar, Jayawant N; Patel, Robin

2016-01-05

Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs) is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM) in addition to applying the Infectious Diseases Society of America (IDSA) criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014) at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32%) met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively); this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003). The time to microorganism detection was shorter with BCBs than with standard media (P < 0.0001), with aerobic and anaerobic BCBs yielding positive results within a median of 21 and 23 h, respectively. Results of our study demonstrate that the semiautomated method of periprosthetic tissue culture in blood culture bottles is more sensitive than and as specific as agar and thioglycolate broth cultures and yields results faster. Prosthetic joint infections are a devastating complication of arthroplasty surgery. Despite this, current microbiological techniques to detect and diagnose infections are imperfect. This study examined a new approach to diagnosing infections, through the inoculation of tissue samples from around the prosthetic joint into blood culture bottles. This study demonstrated that, compared to current laboratory practices, this new technique increased the detection of infection. These findings are important for patient care to allow timely and accurate diagnosis of infection. Copyright © 2016 Peel et al.

Myofibroblastoma of the male breast: a rare entity with radiologic-pathologic correlation

PubMed Central

Comer, John D.; Cui, Xiaoyan; Eisen, Carolyn Sharyn; Abbey, Genevieve; Arleo, Elizabeth Kagan

2016-01-01

A 73-year old man with a history of multiple genitourinary malignancies was found to have a left retroareolar soft tissue mass on CT assessment of disease, and dedicated breast imaging was recommended. Diagnostic mammography and ultrasonography confirmed a solid mass, for which biopsy was recommended. Pathologic analysis demonstrated a spindle cell neoplasm with an immunoreactivity pattern consistent with myofibroblastoma. While this entity is benign, nonspecific imaging features necessitate tissue sampling for pathologic diagnosis, and, given pathologic rarity, open communication between the radiologist and pathologist is important to establish the correct diagnosis and to recommend appropriate management. PMID:27936420

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. The proposed principles represent a paradigm shift in microfluidic technology in three important ways: Microfluidic devices are to be directly integrated with, onto, or around tissue samples, in contrast to the conventional method of off-chip sample extraction followed by sample insertion in microfluidic devices. Architectural and operational principles of microfluidic devices are to be subordinated to suit specific tissue structure and needs, in contrast to the conventional method of building devices according to fluidic function alone and without regard to tissue structure. Sample acquisition from tissue is to be performed on-chip and is to be integrated with the diagnostic measurement within the same device, in contrast to the conventional method of off-chip sample prep and subsequent insertion into a diagnostic device. A more advanced form of tissue integration with microfluidics is tissue encapsulation, wherein the sample is completely encapsulated within a microfluidic device, to allow for full surface access. The immediate applications of these approaches lie with diagnostics of tissue slices and biopsy samples e.g. for cancer but the approaches would also be very useful in comparative genomics and other areas of fundamental research involving heterogeneous tissue samples.

qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

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

Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch; Gallati, Sabina, E-mail: sabina.gallati@insel.ch; Schaller, Andre, E-mail: andre.schaller@insel.ch

2012-07-06

Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serialmore » qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two genomes is demonstrated and which evaluates systematically the impact of DNA degradation on quantification of mtDNA copy number.« less

Application of solid-phase microextraction for in vivo laboratory and field sampling of pharmaceuticals in fish.

PubMed

Zhou, Simon Ningsun; Oakes, Ken D; Servos, Mark R; Pawliszyn, Janusz

2008-08-15

Previous field studies utilizing solid-phase microextraction (SPME) predominantly focused on volatile and semivolatile compounds in air or water. Earlier in vivo sampling studies utilizing SPME were limited to the liquid matrix (blood). The present study has expanded the SPME technique to semisolid tissues under laboratory and field conditions through the investigation of both theoretical and applied experimental approaches. Pre-equilibrium extraction and desorption were performed in vivo in two separate animals. Excellent linearity was found between the amounts extracted by SPME from the muscle of living fish and the waterborne concentrations of pharmaceuticals. A simple SPME method is also described to simultaneously determine free and total analyte concentrations in living tissue. The utility of in vivo SPME sampling was evaluated in wild fish collected from a number of different river locations under varying degrees of influence from municipal wastewater effluents. Diphenhydramine and diltiazem were detected in the muscle of fish downstream of a local wastewater treatment plant. Based on this study, SPME demonstrated several important advantages such as simplicity, sensitivity, and robustness under laboratory and in vivo field sampling conditions.

Determination of Monensin in Bovine Tissues: A Bridging Study Comparing the Bioautographic Method (FSIS CLG-MON) with a Liquid Chromatography-Tandem Mass Spectrometry Method (OMA 2011.24).

PubMed

Mizinga, Kemmy M; Burnett, Thomas J; Brunelle, Sharon L; Wallace, Michael A; Coleman, Mark R

2018-05-01

The U.S. Department of Agriculture, Food Safety Inspection Service regulatory method for monensin, Chemistry Laboratory Guidebook CLG-MON, is a semiquantitative bioautographic method adopted in 1991. Official Method of AnalysisSM (OMA) 2011.24, a modern quantitative and confirmatory LC-tandem MS method, uses no chlorinated solvents and has several advantages, including ease of use, ready availability of reagents and materials, shorter run-time, and higher throughput than CLG-MON. Therefore, a bridging study was conducted to support the replacement of method CLG-MON with OMA 2011.24 for regulatory use. Using fortified bovine tissue samples, CLG-MON yielded accuracies of 80-120% in 44 of the 56 samples tested (one sample had no result, six samples had accuracies of >120%, and five samples had accuracies of 40-160%), but the semiquantitative nature of CLG-MON prevented assessment of precision, whereas OMA 2011.24 had accuracies of 88-110% and RSDr of 0.00-15.6%. Incurred residue results corroborated these results, demonstrating improved accuracy (83.3-114%) and good precision (RSDr of 2.6-20.5%) for OMA 2011.24 compared with CLG-MON (accuracy generally within 80-150%, with exceptions). Furthermore, χ2 analysis revealed no statistically significant difference between the two methods. Thus, the microbiological activity of monensin correlated with the determination of monensin A in bovine tissues, and OMA 2011.24 provided improved accuracy and precision over CLG-MON.

Identification of muscle necrosis in the mdx mouse model of Duchenne muscular dystrophy using three-dimensional optical coherence tomography

NASA Astrophysics Data System (ADS)

Klyen, Blake R.; Shavlakadze, Thea; Radley-Crabb, Hannah G.; Grounds, Miranda D.; Sampson, David D.

2011-07-01

Three-dimensional optical coherence tomography (3D-OCT) was used to image the structure and pathology of skeletal muscle tissue from the treadmill-exercised mdx mouse model of human Duchenne muscular dystrophy. Optical coherence tomography (OCT) images of excised muscle samples were compared with co-registered hematoxylin and eosin-stained and Evans blue dye fluorescence histology. We show, for the first time, structural 3D-OCT images of skeletal muscle dystropathology well correlated with co-located histology. OCT could identify morphological features of interest and necrotic lesions within the muscle tissue samples based on intrinsic optical contrast. These findings demonstrate the utility of 3D-OCT for the evaluation of small-animal skeletal muscle morphology and pathology, particularly for studies of mouse models of muscular dystrophy.

Multi-elemental imaging of paraffin-embedded human samples by laser-induced breakdown spectroscopy

NASA Astrophysics Data System (ADS)

Moncayo, S.; Trichard, F.; Busser, B.; Sabatier-Vincent, M.; Pelascini, F.; Pinel, N.; Templier, I.; Charles, J.; Sancey, L.; Motto-Ros, V.

2017-07-01

Chemical elements play central roles for physiological homeostasis in human cells, and their dysregulation might lead to a certain number of pathologies. Novel imaging techniques that improve the work of pathologists for tissue analysis and diagnostics are continuously sought. We report the use of Laser-Induced Breakdown Spectroscopy (LIBS) to perform multi-elemental images of human paraffin-embedded skin samples on the entire biopsy scale in a complementary and compatible way with microscope histopathological examination. A specific instrumental configuration is proposed in order to detect most of the elements of medical interest (i.e. P, Al, Mg, Na, Zn, Si, Fe, and Cu). As an example of medical application, we selected and analysed skin biopsies, including healthy skin tissue, cutaneous metastasis of melanoma, Merkel-cell carcinoma and squamous cell carcinoma. Clear distinctions in the distribution of chemical elements are observed from the different samples investigated. This study demonstrates the high complementarity of LIBS elemental imaging with conventional histopathology, opening new opportunities for any medical application involving metals.

Measurement of the viscoelastic properties of the vocal folds.

PubMed

Wiikmann, Christian; da Silva, Marcelo Alves; Arêas, Elizabeth Pinheiro Gomes; Tsuji, Domingos Hiroshi; Sennes, Luiz Ubirajara

2009-06-01

Studies of the viscoelastic properties of the vocal folds are normally performed with rheometers that use parallel plates whose interplate space is usually arbitrarily assigned a fixed value. In tissues subject to variation of thickness between samples, fixed gaps could result in different compressions, compromising the comparison among them. We performed an experimental study to determine whether different compressions can lead to different results in measurements of dynamic viscosity (DV) of vocal fold samples. We measured the DV of vocal fold samples of 10 larynges of cadavers under 3 different compression levels, corresponding to 0.2, 0.5, and 10 N on an 8-mm-diameter parallel-plate rheometer. The DV directly varied with compression. We observed statistically significant differences between the results of 0.2 and 10 N (p = 0.0396) and 0.5 and 10 N (p = 0.0442). The study demonstrated that the level of compression influences the DV measure and suggests that a defined compression level should be used in rheometric studies of biological tissues.

An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

PubMed Central

Sterling, Catherine H.; Veksler-Lublinsky, Isana; Ambros, Victor

2015-01-01

The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids. PMID:25056322

Measuring Ultrasonic Backscatter in the Presence of Nonlinear Propagation

NASA Astrophysics Data System (ADS)

Stiles, Timothy; Guerrero, Quinton

2011-11-01

A goal of medical ultrasound is the formation of quantitative ultrasound images in which contrast is determined by acoustic or physical properties of tissue rather than relative echo amplitude. Such images could greatly enhance early detection of many diseases, including breast cancer and liver cirrhosis. Accurate determination of the ultrasonic backscatter coefficient from patients remains a difficult task. One reason for this difficulty is the inherent nonlinear propagation of ultrasound at high intensities used for medical imaging. The backscatter coefficient from several tissue-mimicking samples were measured using the planar reflector method. In this method, the power spectrum from a sample is compared to the power spectrum of an optically flat sample of quartz. The results should be independent of incident pressure amplitude. Results demonstrate that backscatter coefficients can vary by more than an order of magnitude when ultrasound pressure varies from 0.1 MPa to 1.5 MPa at 5.0 MHz. A new method that incorporates nonlinear propagation is proposed to explain these discrepancies.

Two-photon excited fluorescence microscopy application for ex vivo investigation of ocular fundus samples

NASA Astrophysics Data System (ADS)

Peters, Sven; Hammer, Martin; Schweitzer, Dietrich

2011-07-01

Two-photon excited fluorescence (TPEF) imaging of ocular tissue has recently become a promising tool in ophthalmology for diagnostic and research purposes. The feasibility and the advantages of TPEF imaging, namely deeper tissue penetration and improved high-resolution imaging of microstructures, have been demonstrated lately using human ocular samples. The autofluorescence properties of endogenous fluorophores in ocular fundus tissue are well known from spectrophotometric analysis. But fluorophores, especially when it comes to fluorescence lifetime, typically display a dependence of their fluorescence properties on local environmental parameters. Hence, a more detailed investigation of ocular fundus autofluorescence ideally in vivo is of utmost interest. The aim of this study is to determine space-resolved the stationary and time-resolved fluorescence properties of endogenous fluorophores in ex vivo porcine ocular fundus samples by means of two-photon excited fluorescence spectrum and lifetime imaging microscopy (FSIM/FLIM). By our first results, we characterized the autofluorescence of individual anatomical structures of porcine retina samples excited at 760 nm. The fluorescence properties of almost all investigated retinal layers are relatively homogenous. But as previously unknown, ganglion cell bodies show a significantly shorter fluorescence lifetime compared to the adjacent mueller cells. Since all retinal layers exhibit bi-exponential autofluorescence decays, we were able to achieve a more precise characterization of fluorescence properties of endogenous fluorophores compared to a present in vivo FLIM approach by confocal scanning laser ophthalmoscope (cSLO).

Mechanical properties and biocompatibility of porous titanium scaffolds for bone tissue engineering.

PubMed

Chen, Yunhui; Frith, Jessica Ellen; Dehghan-Manshadi, Ali; Attar, Hooyar; Kent, Damon; Soro, Nicolas Dominique Mathieu; Bermingham, Michael J; Dargusch, Matthew S

2017-11-01

Synthetic scaffolds are a highly promising new approach to replace both autografts and allografts to repair and remodel damaged bone tissue. Biocompatible porous titanium scaffold was manufactured through a powder metallurgy approach. Magnesium powder was used as space holder material which was compacted with titanium powder and removed during sintering. Evaluation of the porosity and mechanical properties showed a high level of compatibility with human cortical bone. Interconnectivity between pores is higher than 95% for porosity as low as 30%. The elastic moduli are 44.2GPa, 24.7GPa and 15.4GPa for 30%, 40% and 50% porosity samples which match well to that of natural bone (4-30GPa). The yield strengths for 30% and 40% porosity samples of 221.7MPa and 117MPa are superior to that of human cortical bone (130-180MPa). In-vitro cell culture tests on the scaffold samples using Human Mesenchymal Stem Cells (hMSCs) demonstrated their biocompatibility and indicated osseointegration potential. The scaffolds allowed cells to adhere and spread both on the surface and inside the pore structures. With increasing levels of porosity/interconnectivity, improved cell proliferation is obtained within the pores. It is concluded that samples with 30% porosity exhibit the best biocompatibility. The results suggest that porous titanium scaffolds generated using this manufacturing route have excellent potential for hard tissue engineering applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Activation of phagocytic cells by Staphylococcus epidermidis biofilms: effects of extracellular matrix proteins and the bacterial stress protein GroEL on netosis and MRP-14 release.

PubMed

Dapunt, Ulrike; Gaida, Matthias M; Meyle, Eva; Prior, Birgit; Hänsch, Gertrud M

2016-07-01

The recognition and phagocytosis of free-swimming (planktonic) bacteria by polymorphonuclear neutrophils have been investigated in depth. However, less is known about the neutrophil response towards bacterial biofilms. Our previous work demonstrated that neutrophils recognize activating entities within the extracellular polymeric substance (EPS) of biofilms (the bacterial heat shock protein GroEL) and that this process does not require opsonization. Aim of this study was to evaluate the release of DNA by neutrophils in response to biofilms, as well as the release of the inflammatory cytokine MRP-14. Neutrophils were stimulated with Staphylococcus epidermidis biofilms, planktonic bacteria, extracted EPS and GroEL. Release of DNA and of MRP-14 was evaluated. Furthermore, tissue samples from patients suffering from biofilm infections were collected and evaluated by histology. MRP-14 concentration in blood samples was measured. We were able to show that biofilms, the EPS and GroEL induce DNA release. MRP-14 was only released after stimulation with EPS, not GroEL. Histology of tissue samples revealed MRP-14 positive cells in association with neutrophil infiltration and MRP-14 concentration was elevated in blood samples of patients suffering from biofilm infections. Our data demonstrate that neutrophil-activating entities are present in the EPS and that GroEL induces DNA release by neutrophils. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Photoacoustic determination of glucose concentration in whole blood by a near-infrared laser diode

NASA Astrophysics Data System (ADS)

Zhao, Zuomin; Myllylae, Risto A.

2001-06-01

The near-infrared photoacoustic technique is recognized as a potential method for the non-invasive determination of human glucose, because near-infrared light can incident a few millimeters into human tissue, where it produces an acoustic wave capable of carrying information about the composition of the tissue. This paper demonstrates a photoacoustic glucose measurement in a blood sample as a step toward a non-invasive measurement. The experimental apparatus consists of a near-infrared laser diode operating with 4 micro joules pulse energy at 905 nm, a roller pump connected to a silicon plastic tube and a cuvette for circulating the blood sample. In addition, the apparatus comprises a PZT piezoelectric transducer integrated with a battery-powered preamplifier to receive the photoacoustic signal. During the experiment, a glucose solution is mixed into a human blood sample to change its concentration. Although the absorption coefficient of glucose is much smaller than that of blood in the near-infrared region, the osmotic and hydrophilic properties of glucose decrease the reduced scattering coefficient of blood caused by the dissolved glucose surrounding the blood cells. This changes the distribution of the absorbed optical energy in blood, which, in turn, produces a change in the photoacoustic signal. Our experiment demonstrates that signal amplitudes in fresh and stored blood samples in crease about 7% and 10%, respectively, when the glucose concentration reaches the upper limit of the physiological region (500 mg/dl).

Evaluating nephrotoxicity of high-molecular-weight organic compounds in drinking water from lignite aquifers

USGS Publications Warehouse

Bunnell, J.E.; Tatu, C.A.; Lerch, H.E.; Orem, W.H.; Pavlovic, N.

2007-01-01

High-molecular-weight organic compounds such as humic acids and/or fulvic acids that are naturally mobilized from lignite beds into untreated drinking-water supplies were suggested as one possible cause of Balkan endemic nephropathy (BEN) and cancer of the renal pelvis. A lab investigation was undertaken in order to assess the nephrotoxic potential of such organic compounds using an in vitro tissue culture model. Because of the infeasibility of exposing kidney tissue to low concentrations of organics for years in the lab, tangential flow ultrafiltration was employed to hyperconcentrate samples suitable for discerning effects in the short time frames necessitated by tissue culture systems. Effects on HK-2 kidney cells were measured using two different cell proliferation assays (MTT and alamarBlue). Results demonstrated that exposure of kidney tissue to high-molecular-weight organics produced excess cell death or proliferation depending on concentration and duration of exposure. Copyright ?? Taylor & Francis Group, LLC.

Combined autofluorescence and Raman spectroscopy method for skin tumor detection in visible and near infrared regions

NASA Astrophysics Data System (ADS)

Zakharov, V. P.; Bratchenko, I. A.; Artemyev, D. N.; Myakinin, O. O.; Khristoforova, Y. A.; Kozlov, S. V.; Moryatov, A. A.

2015-07-01

The combined application of Raman and autofluorescence spectroscopy in visible and near infrared regions for the analysis of malignant neoplasms of human skin was demonstrated. Ex vivo experiments were performed for 130 skin tissue samples: 28 malignant melanomas, 19 basal cell carcinomas, 15 benign tumors, 9 nevi and 59 normal tissues. Proposed method of Raman spectra analysis allows for malignant melanoma differentiating from other skin tissues with accuracy of 84% (sensitivity of 97%, specificity of 72%). Autofluorescence analysis in near infrared and visible regions helped us to increase the diagnostic accuracy by 5-10%. Registration of autofluorescence in near infrared region is realized in one optical unit with Raman spectroscopy. Thus, the proposed method of combined skin tissues study makes possible simultaneous large skin area study with autofluorescence spectra analysis and precise neoplasm type determination with Raman spectroscopy.

Extraction of the 3D local orientation of myocytes in human cardiac tissue using X-ray phase-contrast micro-tomography and multi-scale analysis.

PubMed

Varray, François; Mirea, Iulia; Langer, Max; Peyrin, Françoise; Fanton, Laurent; Magnin, Isabelle E

2017-05-01

This paper presents a methodology to access the 3D local myocyte arrangements in fresh human post-mortem heart samples. We investigated the cardiac micro-structure at a high and isotropic resolution of 3.5 µm in three dimensions using X-ray phase micro-tomography at the European Synchrotron Radiation Facility. We then processed the reconstructed volumes to extract the 3D local orientation of the myocytes using a multi-scale approach with no segmentation. We created a simplified 3D model of tissue sample made of simulated myocytes with known size and orientations, to evaluate our orientation extraction method. Afterwards, we applied it to 2D histological cuts and to eight 3D left ventricular (LV) cardiac tissue samples. Then, the variation of the helix angles, from the endocardium to the epicardium, was computed at several spatial resolutions ranging from 3.6 3  mm 3 to 112 3  µm 3 . We measure an increased range of 20° to 30° from the coarsest resolution level to the finest level in the experimental samples. This result is in line with the higher values measured from histology. The displayed tractography demonstrates a rather smooth evolution of the transmural helix angle in six LV samples and a sudden discontinuity of the helix angle in two septum samples. These measurements bring a new vision of the human heart architecture from macro- to micro-scale. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterization of Mycobacterium tuberculosis Complex DNAs from Egyptian Mummies by Spoligotyping

PubMed Central

Zink, Albert R.; Sola, Christophe; Reischl, Udo; Grabner, Waltraud; Rastogi, Nalin; Wolf, Hans; Nerlich, Andreas G.

2003-01-01

Bone and soft tissue samples from 85 ancient Egyptian mummies were analyzed for the presence of ancient Mycobacterium tuberculosis complex DNA (aDNA) and further characterized by spoligotyping. The specimens were obtained from individuals from different tomb complexes in Thebes West, Upper Egypt, which were used for upper social class burials between the Middle Kingdom (since ca. 2050 BC) and the Late Period (until ca. 500 BC). A total of 25 samples provided a specific positive signal for the amplification of a 123-bp fragment of the repetitive element IS6110, indicating the presence of M. tuberculosis DNA. Further PCR-based tests for the identification of subspecies failed due to lack of specific amplification products in the historic tissue samples. Of these 25 positive specimens, 12 could be successfully characterized by spoligotyping. The spoligotyping signatures were compared to those in an international database. They all show either an M. tuberculosis or an M. africanum pattern, but none revealed an M. bovis-specific pattern. The results from a Middle Kingdom tomb (used exclusively between ca. 2050 and 1650 BC) suggest that these samples bear an M. africanum-type specific spoligotyping signature. The samples from later periods provided patterns typical for M. tuberculosis. This study clearly demonstrates that spoligotyping can be applied to historic tissue samples. In addition, our results do not support the theory that M. tuberculosis originated from the M. bovis type but, rather, suggest that human M. tuberculosis may have originated from a precursor complex probably related to M. africanum. PMID:12517873

SABRE: a method for assessing the stability of gene modules in complex tissues and subject populations.

PubMed

Shannon, Casey P; Chen, Virginia; Takhar, Mandeep; Hollander, Zsuzsanna; Balshaw, Robert; McManus, Bruce M; Tebbutt, Scott J; Sin, Don D; Ng, Raymond T

2016-11-14

Gene network inference (GNI) algorithms can be used to identify sets of coordinately expressed genes, termed network modules from whole transcriptome gene expression data. The identification of such modules has become a popular approach to systems biology, with important applications in translational research. Although diverse computational and statistical approaches have been devised to identify such modules, their performance behavior is still not fully understood, particularly in complex human tissues. Given human heterogeneity, one important question is how the outputs of these computational methods are sensitive to the input sample set, or stability. A related question is how this sensitivity depends on the size of the sample set. We describe here the SABRE (Similarity Across Bootstrap RE-sampling) procedure for assessing the stability of gene network modules using a re-sampling strategy, introduce a novel criterion for identifying stable modules, and demonstrate the utility of this approach in a clinically-relevant cohort, using two different gene network module discovery algorithms. The stability of modules increased as sample size increased and stable modules were more likely to be replicated in larger sets of samples. Random modules derived from permutated gene expression data were consistently unstable, as assessed by SABRE, and provide a useful baseline value for our proposed stability criterion. Gene module sets identified by different algorithms varied with respect to their stability, as assessed by SABRE. Finally, stable modules were more readily annotated in various curated gene set databases. The SABRE procedure and proposed stability criterion may provide guidance when designing systems biology studies in complex human disease and tissues.

Effects of human oral mucosal tissue, saliva and oral microflora on intraoral metabolism and bioactivation of black raspberry anthocyanins

PubMed Central

Mallery, Susan R.; Budendorf, Deric E.; Larsen, Matthew P.; Pei, Ping; Tong, Meng; Holpuch, Andrew S.; Larsen, Peter E.; Stoner, Gary D.; Fields, Henry W.; Chan, Kenneth K.; Ling, Yonghua; Liu, Zhongfa

2011-01-01

Our oral cancer chemoprevention trial data implied that patient-specific differences in local retention and metabolism of freeze-dried black raspberries' (BRB) components affected therapeutic responsiveness. Subsequent studies have confirmed that anthocyanins are key contributors to BRB's chemopreventive effects. Consequently, functional assays, immunoblotting and immunohistochemical analyses to evaluate levels and distribution of BRB anthocyanin-relevant metabolic enzymes in human oral tissues were performed. LC-MS/MS analyses of time course saliva samples collected following BRB rinses were conducted to assess local pharmacokinetics and compare the capacities of three different BRB rinse formulations to provide sustained intraoral levels of anthocyanins. Protein profiles demonstrated the presence of key metabolic enzymes in all 15 oral mucosal tissues evaluated while immunohistochemistry confirmed these enzymes were distributed within surface oral epithelia and terminal salivary ducts. β-glucosidase assays confirmed that whole and microflora-reduced saliva can deglycosylate BRB anthocyanins, enabling generation of the bioactive aglycone, cyanidin. LC-MS/MS analyses demonstrated retention of parent anthocyanins and their functional, stable metabolite, protocatechuic acid, in saliva for up to 4 hours after rinsing. Furthermore, post-rinse saliva samples contained glucuronidated anthocyanin conjugates, consistent with intracellular uptake and Phase II conversion of BRB anthocyanins into forms amenable to local recycling. Our data demonstrate that comparable to the small intestine, the requisite hydrolytic, Phase II and efflux transporting enzymes necessary for local enteric recycling are present and functional in human oral mucosa. Notably, inter-patient differences in anthocyanin bioactivation and capacities for enteric recycling would impact treatment as retention of bioactivated chemopreventives at the target site would sustain therapeutic effectiveness. PMID:21558412

Morphometric study of uninvolved rectal mucosa 10 cm and 20 cm away from the malignant tumor.

PubMed

Despotović, Sanja Z; Milićević, Novica M; Milosević, Dragoslav P; Despotović, Nebojsa; Erceg, Predrag; Bojić, Bozidar; Bojić, Danijela; Svorcan, Petar; Mihajlović, Gordana; Dorđević, Jelena; Lalić, Ivana M; Milićević, Zivana

2014-02-01

Recently, many details of the interplay between tumor cells and tumor-associated stromal elements leading to the progression of malignant disease were elucidated. In contrast, little is known about the role of uninvolved stromal tissue in the remote surrounding of the malignant tumor. Therefore, we performed a computer-aided morphometric study of rectal mucosa in samples taken 10 cm and 20 cm away from the malignant tumor during endoscopic examination of 23 patients older than 60 years. The samples of rectal mucosa from 10 healthy persons of corresponding age subjected to diagnostic rectoscopy during active screening for asymptomatic cancer were used as control. All structural elements of the rectal mucosa were studied and the number of nucleated cells in the lamina propria per 0.1 mm² of tissue was assessed. Our study revealed a reduced number of cells in the lamina propria of the rectal mucosa 10 cm and 20 cm away from the tumor lesion in both male and female patients. The decreased mucosal height and increased crypt number were registered in female patients 10 cm away from the tumor. The connective tissue of lamina propria showed a disorderly organization: the collagen fibers were frail, loosely arranged and signs of tissue edema were present. Small blood vessels and capillaries were much more frequently seen than in healthy tissue. Our results demonstrate the complex interactions between the cancer and remote mucosal tissue of the affected organ.

Blood-based detection of RAS mutations to guide anti-EGFR therapy in colorectal cancer patients: concordance of results from circulating tumor DNA and tissue-based RAS testing.

PubMed

Schmiegel, Wolff; Scott, Rodney J; Dooley, Susan; Lewis, Wendy; Meldrum, Cliff J; Pockney, Peter; Draganic, Brian; Smith, Steve; Hewitt, Chelsee; Philimore, Hazel; Lucas, Amanda; Shi, Elva; Namdarian, Kateh; Chan, Timmy; Acosta, Danilo; Ping-Chang, Su; Tannapfel, Andrea; Reinacher-Schick, Anke; Uhl, Waldemar; Teschendorf, Christian; Wolters, Heiner; Stern, Josef; Viebahn, Richard; Friess, Helmut; Janssen, Klaus-Peter; Nitsche, Ulrich; Slotta-Huspenina, Julia; Pohl, Michael; Vangala, Deepak; Baraniskin, Alexander; Dockhorn-Dworniczak, Barbara; Hegewisch-Becker, Susanne; Ronga, Philippe; Edelstein, Daniel L; Jones, Frederick S; Hahn, Stephan; Fox, Stephen B

2017-02-01

An accurate blood-based RAS mutation assay to determine eligibility of metastatic colorectal cancer (mCRC) patients for anti-EGFR therapy would benefit clinical practice by better informing decisions to administer treatment independent of tissue availability. The objective of this study was to determine the level of concordance between plasma and tissue RAS mutation status in patients with mCRC to gauge whether blood-based RAS mutation testing is a viable alternative to standard-of-care RAS tumor testing. RAS testing was performed on plasma samples from newly diagnosed metastatic patients, or from recurrent mCRC patients using the highly sensitive digital PCR technology, BEAMing (beads, emulsions, amplification, and magnetics), and compared with DNA sequencing data of respective FFPE (formalin-fixed paraffin-embedded) tumor samples. Discordant tissue RAS results were re-examined by BEAMing, if possible. The prevalence of RAS mutations detected in plasma (51%) vs. tumor (53%) was similar, in accord with the known prevalence of RAS mutations observed in mCRC patient populations. The positive agreement between plasma and tumor RAS results was 90.4% (47/52), the negative agreement was 93.5% (43/46), and the overall agreement (concordance) was 91.8% (90/98). The high concordance of plasma and tissue results demonstrates that blood-based RAS mutation testing is a viable alternative to tissue-based RAS testing. © 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Stiffness mapping prostate biopsy samples using a tactile sensor.

PubMed

Peng, Qiyu; Omata, Sadao; Peehl, Donna M; Constantinou, Chris E

2011-01-01

Previous studies have demonstrated that the stiffness of cancerous cells reflects their pathological stage and progression rates, with increased cancerous cell stiffness associated with increased aggressiveness. Therefore, the elasticity of the cancerous cells has the potential to be used as an indicator of the cancer's aggressiveness. However, the sensitivity and resolution of current palpation and imaging techniques are not sufficient to detect small cancerous tissues. In previous studies, we developed a tactile-based device to map with high resolution the stiffness of a tissue section. The purpose of this study is to evaluate this device using different tissues (BPH, Cancer and PZ) collected from human prostates. The preliminary results show that the tactile device is sensitive enough to tell the differences of the stiffness of different tissues. The results also disclosed the factors (humidity, temperature and tissue degradation) which could dramatically affect the results of stiffness mapping. The tactile technology described in this paper has the potential to help disclose the underlying mechanical mechanisms that lead to increased stiffness in prostate tumors.

Optical pathology study of human abdominal aorta tissues using confocal micro resonance Raman spectroscopy

NASA Astrophysics Data System (ADS)

Liu, Cheng-hui; Boydston-White, Susie; Wang, Wubao; Sordillo, Laura A.; Shi, Lingyan; Weisberg, Arel; Tomaselli, Vincent P.; Sordillo, Peter P.; Alfano, Robert R.

2016-03-01

Resonance Raman (RR) spectroscopic technique has a high potential for label-free and in-situ detection of biomedical lesions in vivo. This study evaluates the ability of RR spectroscopy method as an optical histopathology tool to detect the atherosclerotic plaque states of abdominal aorta in vitro. This part demonstrates the RR spectral molecular fingerprint features from different sites of the atherosclerotic abdominal aortic wall tissues. Total 57 sites of five pieces aortic samples in intimal and adventitial wall from an autopsy specimen were examined using confocal micro Raman system of WITec 300R with excitation wavelength of 532nm. The preliminary RR spectral biomarkers of molecular fingerprints indicated that typical calcified atherosclerotic plaque (RR peak at 964cm-1) tissue; fibrolipid plaque (RR peaks at 1007, 1161, 1517 and 2888cm-1) tissue, lipid pool with the fatty precipitation cholesterol) with collagen type I (RR peaks at 864, 1452, 1658, 2888 and 2948cm-1) in the soft tissue were observed and investigated.

Analysis of the genetic phylogeny of multifocal prostate cancer identifies multiple independent clonal expansions in neoplastic and morphologically normal prostate tissue.

PubMed

Cooper, Colin S; Eeles, Rosalind; Wedge, David C; Van Loo, Peter; Gundem, Gunes; Alexandrov, Ludmil B; Kremeyer, Barbara; Butler, Adam; Lynch, Andrew G; Camacho, Niedzica; Massie, Charlie E; Kay, Jonathan; Luxton, Hayley J; Edwards, Sandra; Kote-Jarai, ZSofia; Dennis, Nening; Merson, Sue; Leongamornlert, Daniel; Zamora, Jorge; Corbishley, Cathy; Thomas, Sarah; Nik-Zainal, Serena; O'Meara, Sarah; Matthews, Lucy; Clark, Jeremy; Hurst, Rachel; Mithen, Richard; Bristow, Robert G; Boutros, Paul C; Fraser, Michael; Cooke, Susanna; Raine, Keiran; Jones, David; Menzies, Andrew; Stebbings, Lucy; Hinton, Jon; Teague, Jon; McLaren, Stuart; Mudie, Laura; Hardy, Claire; Anderson, Elizabeth; Joseph, Olivia; Goody, Victoria; Robinson, Ben; Maddison, Mark; Gamble, Stephen; Greenman, Christopher; Berney, Dan; Hazell, Steven; Livni, Naomi; Fisher, Cyril; Ogden, Christopher; Kumar, Pardeep; Thompson, Alan; Woodhouse, Christopher; Nicol, David; Mayer, Erik; Dudderidge, Tim; Shah, Nimish C; Gnanapragasam, Vincent; Voet, Thierry; Campbell, Peter; Futreal, Andrew; Easton, Douglas; Warren, Anne Y; Foster, Christopher S; Stratton, Michael R; Whitaker, Hayley C; McDermott, Ultan; Brewer, Daniel S; Neal, David E

2015-04-01

Genome-wide DNA sequencing was used to decrypt the phylogeny of multiple samples from distinct areas of cancer and morphologically normal tissue taken from the prostates of three men. Mutations were present at high levels in morphologically normal tissue distant from the cancer, reflecting clonal expansions, and the underlying mutational processes at work in morphologically normal tissue were also at work in cancer. Our observations demonstrate the existence of ongoing abnormal mutational processes, consistent with field effects, underlying carcinogenesis. This mechanism gives rise to extensive branching evolution and cancer clone mixing, as exemplified by the coexistence of multiple cancer lineages harboring distinct ERG fusions within a single cancer nodule. Subsets of mutations were shared either by morphologically normal and malignant tissues or between different ERG lineages, indicating earlier or separate clonal cell expansions. Our observations inform on the origin of multifocal disease and have implications for prostate cancer therapy in individual cases.

A stable isotope approach to assessing water loss in fruits and vegetables during storage.

PubMed

Greule, Markus; Rossmann, Andreas; Schmidt, Hanns-Ludwig; Mosandl, Armin; Keppler, Frank

2015-02-25

Plant tissue water is the source of oxygen and hydrogen in organic biomatter. Recently, we demonstrated that the stable hydrogen isotope value (δ(2)H) of plant methoxyl groups is a very reliable and easily available archive for the δ(2)H value of this tissue water. Here we show in a model experiment that the δ(2)H values of methoxyl groups remain unchanged after water loss during storage of fruits and vegetables under controlled conditions, while δ(2)H and δ(18)O values of tissue water increase. This enhancement is plant-dependent, and the correlation differs from the meteoric water line. The δ(18)O value is better correlated to the weight decrease of the samples. Therefore, we postulate that the δ(2)H value of methoxyl groups and the δ(18)O value of tissue water are suitable parameters for checking postharvest alterations of tissue water, either addition or loss.

Scanning system for angle-resolved low-coherence interferometry.

PubMed

Steelman, Zachary A; Ho, Derek; Chu, Kengyeh K; Wax, Adam

2017-11-15

Angle-resolved low-coherence interferometry (a/LCI) detects precancer by enabling depth-resolved measurements of nuclear morphology in vivo. A significant limitation of a/LCI is the point-probe nature of the method, sampling <0.5  mm 2 before probe relocation is necessary. In this work, we demonstrate a scanning method capable of assessing an area >100  mm 2 without repositioning. By utilizing a reflection-only three-optic rotator prism and a two-axis scanning mirror, we demonstrate radial scans of a sample with a linear range of 12 mm and a full rotational range of 180°. Use of this design will improve the diagnostic utility of a/LCI for wide-area screening of tissue health.

A scanning system for angle-resolved low-coherence interferometry

PubMed Central

Steelman, Zachary A.; Ho, Derek; Chu, Kengyeh K.; Wax, Adam

2018-01-01

Angle-resolved low-coherence interferometry (a/LCI) detects precancer by enabling depth-resolved measurements of nuclear morphology in vivo. A significant limitation of a/LCI is the point-probe nature of the method, sampling <0.5 mm2 before probe relocation is necessary. In this work, we demonstrate a scanning method capable of assessing an area >100 mm2 without repositioning. By utilizing a reflection-only three-optic rotator (ROTOR) prism and two-axis scanning mirror, we demonstrate radial scans of a sample with a linear range of 12 mm and a full rotational range of 180°. Use of this design will improve the diagnostic utility of a/LCI for wide-area screening of tissue health. PMID:29140317

Lipoaspirate fluid proteome: A preliminary investigation by LC-MS top-down/bottom-up integrated platform of a high potential biofluid in regenerative medicine.

PubMed

Inserra, Ilaria; Martelli, Claudia; Cipollina, Mara; Cicione, Claudia; Iavarone, Federica; Taranto, Giuseppe Di; Barba, Marta; Castagnola, Massimo; Desiderio, Claudia; Lattanzi, Wanda

2016-04-01

The lipoaspirate fluid (LAF) is emerging as a potentially valuable source in regenerative medicine. In particular, our group recently demonstrated that it is able to exert osteoinductive properties in vitro. This original observation stimulated the investigation of the proteomic component of LAF, by means of LC-ESI-LTQ-Orbitrap-MS top-down/bottom-up integrated approach, which represents the object of the present study. Top-down analyses required the optimization of sample pretreatment procedures to enable the correct investigation of the intact proteome. Bottom-up analyses have been directly applied to untreated samples after monodimensional SDS-PAGE separation. The analysis of the acid-soluble fraction of LAF by top-down approach allowed demonstrating the presence of albumin and hemoglobin fragments (i.e. VV- and LVV-hemorphin-7), thymosins β4 and β10 peptides, ubiquitin and acyl-CoA binding protein; adipogenesis regulatory factor, perilipin-1 fragments, and S100A6, along with their PTMs. Part of the bottom-up proteomic profile was reproducibly found in both tested samples. The bottom-up approach allowed demonstrating the presence of proteins, listed among the components of adipose tissue and/or comprised within the ASCs intracellular content and secreted proteome. Our data provide a first glance on the LAF molecular profile, which is consistent with its tissue environment. LAF appeared to contain bioactive proteins, peptides and paracrine factors, suggesting its potential translational exploitation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigation of stress-induced birefringence of tissue determined with polarisation sensitive optical coherence tomography (Conference Presentation)

NASA Astrophysics Data System (ADS)

Karnowski, Karol; Li, Qingyun; Villiger, Martin; Sampson, David D.

2017-02-01

Polarisation sensitive optical coherence tomography (PS-OCT) offers additional intrinsic contrast to probe differences between healthy tissue and cancer that are often barely visible due to limited scattering contrast in an OCT image. PS-OCT reconstructs tissue birefringence from phase-sensitive measurements of orthogonal polarisation components of backscattering. In material science, polarisation has been used to study stress distribution, including the birefringence induced by stress in an otherwise isotropic material. Similar effects in biological tissues have not been well studied yet; however, may have application to tissues subjected to stress, e.g., tendons, muscles, lens, cornea or airway smooth muscle (ASM). The objective of this work is to explore stress-induced birefringence in tissue. We employ an advanced swept source-based PS-OCT system capable of measurement of tissue local polarisation properties. The sample in both cases is illuminated with orthogonal, passively depth-encoded polarisation states. Light returning from the tissue is detected via a polarisation-diversity detection module and a Mueller formalism is used to reconstruct polarisation properties (including retardation, diattenuation, and depolarisation) of the tissue. In this study, we demonstrate the measurement of stress-induced birefringence in phantoms and in soft tissues with polarisation sensitive optical coherence tomography.

Magnetoacoustic tomography with magnetic induction for high-resolution bioimepedance imaging through vector source reconstruction under the static field of MRI magnet.

PubMed

Mariappan, Leo; Hu, Gang; He, Bin

2014-02-01

Magnetoacoustic tomography with magnetic induction (MAT-MI) is an imaging modality to reconstruct the electrical conductivity of biological tissue based on the acoustic measurements of Lorentz force induced tissue vibration. This study presents the feasibility of the authors' new MAT-MI system and vector source imaging algorithm to perform a complete reconstruction of the conductivity distribution of real biological tissues with ultrasound spatial resolution. In the present study, using ultrasound beamformation, imaging point spread functions are designed to reconstruct the induced vector source in the object which is used to estimate the object conductivity distribution. Both numerical studies and phantom experiments are performed to demonstrate the merits of the proposed method. Also, through the numerical simulations, the full width half maximum of the imaging point spread function is calculated to estimate of the spatial resolution. The tissue phantom experiments are performed with a MAT-MI imaging system in the static field of a 9.4 T magnetic resonance imaging magnet. The image reconstruction through vector beamformation in the numerical and experimental studies gives a reliable estimate of the conductivity distribution in the object with a ∼ 1.5 mm spatial resolution corresponding to the imaging system frequency of 500 kHz ultrasound. In addition, the experiment results suggest that MAT-MI under high static magnetic field environment is able to reconstruct images of tissue-mimicking gel phantoms and real tissue samples with reliable conductivity contrast. The results demonstrate that MAT-MI is able to image the electrical conductivity properties of biological tissues with better than 2 mm spatial resolution at 500 kHz, and the imaging with MAT-MI under a high static magnetic field environment is able to provide improved imaging contrast for biological tissue conductivity reconstruction.

Messenger RNA biomarker signatures for forensic body fluid identification revealed by targeted RNA sequencing.

PubMed

Hanson, E; Ingold, S; Haas, C; Ballantyne, J

2018-05-01

The recovery of a DNA profile from the perpetrator or victim in criminal investigations can provide valuable 'source level' information for investigators. However, a DNA profile does not reveal the circumstances by which biological material was transferred. Some contextual information can be obtained by a determination of the tissue or fluid source of origin of the biological material as it is potentially indicative of some behavioral activity on behalf of the individual that resulted in its transfer from the body. Here, we sought to improve upon established RNA based methods for body fluid identification by developing a targeted multiplexed next generation mRNA sequencing assay comprising a panel of approximately equal sized gene amplicons. The multiplexed biomarker panel includes several highly specific gene targets with the necessary specificity to definitively identify most forensically relevant biological fluids and tissues (blood, semen, saliva, vaginal secretions, menstrual blood and skin). In developing the biomarker panel we evaluated 66 gene targets, with a progressive iteration of testing target combinations that exhibited optimal sensitivity and specificity using a training set of forensically relevant body fluid samples. The current assay comprises 33 targets: 6 blood, 6 semen, 6 saliva, 4 vaginal secretions, 5 menstrual blood and 6 skin markers. We demonstrate the sensitivity and specificity of the assay and the ability to identify body fluids in single source and admixed stains. A 16 sample blind test was carried out by one lab with samples provided by the other participating lab. The blinded lab correctly identified the body fluids present in 15 of the samples with the major component identified in the 16th. Various classification methods are being investigated to permit inference of the body fluid/tissue in dried physiological stains. These include the percentage of reads in a sample that are due to each of the 6 tissues/body fluids tested and inter-sample differential gene expression revealed by agglomerative hierarchical clustering. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of Differentially Expressed K-Ras Transcript Variants in Patients With Leiomyoma.

PubMed

Zolfaghari, Nooshin; Shahbazi, Shirin; Torfeh, Mahnaz; Khorasani, Maryam; Hashemi, Mehrdad; Mahdian, Reza

2017-10-01

Molecular studies have demonstrated a wide range of gene expression variations in uterine leiomyoma. The rat sarcoma virus/rapidly accelerated fibrosarcoma/mitogen-activated protein kinase (RAS/RAF/MAPK) is the crucial cellular pathway in transmitting external signals into nucleus. Deregulation of this pathway contributes to excessive cell proliferation and tumorigenesis. The present study aims to investigate the expression profile of the K-Ras transcripts in tissue samples from patients with leiomyoma. The patients were leiomyoma cases who had no mutation in mediator complex subunit 12 ( MED12) gene. A quantitative approach has been applied to determine the difference in the expression of the 2 main K-Ras messenger RNA (mRNA) variants. The comparison between gene expression levels in leiomyoma and normal myometrium group was performed using relative expression software tool. The expression of K-Ras4B gene was upregulated in leiomyoma group ( P = .016), suggesting the involvement of K-Ras4B in the disease pathogenesis. Pairwise comparison of the K-Ras4B expression between each leiomyoma tissue and its matched adjacent normal myometrium revealed gene upregulation in 68% of the cases. The expression of K-Ras4A mRNA was relatively upregulated in leiomyoma group ( P = .030). In addition, the mean expression of K-Ras4A gene in leiomyoma tissues relative to normal samples was 4.475 (95% confidence interval: 0.10-20.42; standard error: 0.53-12.67). In total, 58% of the cases showed more than 2-fold increase in K-Ras4A gene expression. Our results demonstrated increased expression of both K-Ras mRNA splicing variants in leiomyoma tissue. However, the ultimate result of KRAS expression on leiomyoma development depends on the overall KRAS isoform balance and, consequently, on activated signaling pathways.

Exploring Genome-Wide Expression Profiles Using Machine Learning Techniques.

PubMed

Kebschull, Moritz; Papapanou, Panos N

2017-01-01

Although contemporary high-throughput -omics methods produce high-dimensional data, the resulting wealth of information is difficult to assess using traditional statistical procedures. Machine learning methods facilitate the detection of additional patterns, beyond the mere identification of lists of features that differ between groups.Here, we demonstrate the utility of (1) supervised classification algorithms in class validation, and (2) unsupervised clustering in class discovery. We use data from our previous work that described the transcriptional profiles of gingival tissue samples obtained from subjects suffering from chronic or aggressive periodontitis (1) to test whether the two diagnostic entities were also characterized by differences on the molecular level, and (2) to search for a novel, alternative classification of periodontitis based on the tissue transcriptomes.Using machine learning technology, we provide evidence for diagnostic imprecision in the currently accepted classification of periodontitis, and demonstrate that a novel, alternative classification based on differences in gingival tissue transcriptomes is feasible. The outlined procedures allow for the unbiased interrogation of high-dimensional datasets for characteristic underlying classes, and are applicable to a broad range of -omics data.

FACS-based Isolation of Neural and Glioma Stem Cell Populations from Fresh Human Tissues Utilizing EGF Ligand

PubMed Central

Tome-Garcia, Jessica; Doetsch, Fiona; Tsankova, Nadejda M.

2018-01-01

Direct isolation of human neural and glioma stem cells from fresh tissues permits their biological study without prior culture and may capture novel aspects of their molecular phenotype in their native state. Recently, we demonstrated the ability to prospectively isolate stem cell populations from fresh human germinal matrix and glioblastoma samples, exploiting the ability of cells to bind the Epidermal Growth Factor (EGF) ligand in fluorescence-activated cell sorting (FACS). We demonstrated that FACS-isolated EGF-bound neural and glioblastoma populations encompass the sphere-forming colonies in vitro, and are capable of both self-renewal and multilineage differentiation. Here we describe in detail the purification methodology of EGF-bound (i.e., EGFR+) human neural and glioma cells with stem cell properties from fresh postmortem and surgical tissues. The ability to prospectively isolate stem cell populations using native ligand-binding ability opens new doors for understanding both normal and tumor cell biology in uncultured conditions, and is applicable for various downstream molecular sequencing studies at both population and single-cell resolution. PMID:29516026

Assessing reproductive status in elasmobranch fishes using steroid hormones extracted from skeletal muscle tissue

PubMed Central

Prohaska, Bianca K.; Tsang, Paul C. W.; Driggers, William B.; Hoffmayer, Eric R.; Wheeler, Carolyn R.; Brown, A. Christine; Sulikowski, James A.

2013-01-01

Elasmobranch fishes (sharks, skates, and rays) are particularly susceptible to anthropogenic threats, making a thorough understanding of their life history characteristics essential for proper management. Historically, elasmobranch reproductive data have been collected by lethal sampling, an approach that is problematic for threatened and endangered species. However, recent studies have demonstrated that non-lethal approaches can be as effective as lethal ones for assessment of the reproductive status of an animal. For example, plasma has been used to examine concentrations of steroid hormones. Additionally, skeletal muscle tissue, which can be obtained non-lethally and with minimal stress, can also be used to quantify concentrations of steroid hormones. Skeletal muscle progesterone, testosterone, and estradiol concentrations were determined to be statistically significant indicators of reproductive status in the oviparous Leucoraja erinacea, the yolk-dependent viviparous Squalus acanthias, and the yolk-sac placental viviparous Rhizoprionodon terraenovae. The results of the present study demonstrate that steroid hormones present in non-lethally harvested skeletal muscle tissue can be used as reliable indicators of reproductive status in elasmobranchs. PMID:27293612

A Novel Application for the Cavalieri Principle: A Stereological and Methodological Study

PubMed Central

Altunkaynak, Berrin Zuhal; Altunkaynak, Eyup; Unal, Deniz; Unal, Bunyamin

2009-01-01

Objective The Cavalieri principle was applied to consecutive pathology sections that were photographed at the same magnification and used to estimate tissue volumes via superimposing a point counting grid on these images. The goal of this study was to perform the Cavalieri method quickly and practically. Materials and Methods In this study, 10 adult female Sprague Dawley rats were used. Brain tissue was removed and sampled both systematically and randomly. Brain volumes were estimated using two different methods. First, all brain slices were scanned with an HP ScanJet 3400C scanner, and their images were shown on a PC monitor. Brain volume was then calculated based on these images. Second, all brain slices were photographed in 10× magnification with a microscope camera, and brain volumes were estimated based on these micrographs. Results There was no statistically significant difference between the volume measurements of the two techniques (P>0.05; Paired Samples t Test). Conclusion This study demonstrates that personal computer scanning of serial tissue sections allows for easy and reliable volume determination based on the Cavalieri method. PMID:25610077

A novel application for the cavalieri principle: a stereological and methodological study.

PubMed

Altunkaynak, Berrin Zuhal; Altunkaynak, Eyup; Unal, Deniz; Unal, Bunyamin

2009-08-01

The Cavalieri principle was applied to consecutive pathology sections that were photographed at the same magnification and used to estimate tissue volumes via superimposing a point counting grid on these images. The goal of this study was to perform the Cavalieri method quickly and practically. In this study, 10 adult female Sprague Dawley rats were used. Brain tissue was removed and sampled both systematically and randomly. Brain volumes were estimated using two different methods. First, all brain slices were scanned with an HP ScanJet 3400C scanner, and their images were shown on a PC monitor. Brain volume was then calculated based on these images. Second, all brain slices were photographed in 10× magnification with a microscope camera, and brain volumes were estimated based on these micrographs. There was no statistically significant difference between the volume measurements of the two techniques (P>0.05; Paired Samples t Test). This study demonstrates that personal computer scanning of serial tissue sections allows for easy and reliable volume determination based on the Cavalieri method.

Mitochondrial imaging in live or fixed tissues using a luminescent iridium complex.

PubMed

Sorvina, Alexandra; Bader, Christie A; Darby, Jack R T; Lock, Mitchell C; Soo, Jia Yin; Johnson, Ian R D; Caporale, Chiara; Voelcker, Nicolas H; Stagni, Stefano; Massi, Massimiliano; Morrison, Janna L; Plush, Sally E; Brooks, Douglas A

2018-05-29

Mitochondrial morphology is important for the function of this critical organelle and, accordingly, altered mitochondrial structure is exhibited in many pathologies. Imaging of mitochondria can therefore provide important information about disease presence and progression. However, mitochondrial imaging is currently limited by the availability of agents that have the capacity to image mitochondrial morphology in both live and fixed samples. This can be particularly problematic in clinical studies or large, multi-centre cohort studies, where tissue archiving by fixation is often more practical. We previously reported the synthesis of an iridium coordination complex [Ir(ppy) 2 (MeTzPyPhCN)] + ; where ppy is a cyclometalated 2-phenylpyridine and TzPyPhCN is the 5-(5-(4-cyanophen-1-yl)pyrid-2-yl)tetrazolate ligand; and showed that this complex (herein referred to as IraZolve-Mito) has a high specificity for mitochondria in live cells. Here we demonstrate that IraZolve-Mito can also effectively stain mitochondria in both live and fixed tissue samples. The staining protocol proposed is versatile, providing a universal procedure for cell biologists and pathologists to visualise mitochondria.

Polyurethane/fluor-hydroxyapatite nanocomposite scaffolds for bone tissue engineering. Part I: morphological, physical, and mechanical characterization

PubMed Central

Asefnejad, Azadeh; Behnamghader, Aliasghar; Khorasani, Mohammad Taghi; Farsadzadeh, Babak

2011-01-01

In this study, new nano-fluor-hydroxyapatite (nFHA)/polyurethane composite scaffolds were fabricated for potential use in bone tissue engineering. Polyester urethane samples were synthesized from polycaprolactone, hexamethylene diisocyanate, and 1,4-butanediol as chain extender. Nano fluor-hydroxyapatite (nFHA) was successfully synthesized by sol-gel method. The solid–liquid phase separation and solvent sublimation methods were used for preparation of the porous composites. Mechanical properties, chemical structure, and morphological characteristics of the samples were investigated by compressive test, Fourier transform infrared, and scanning electron microscopy (SEM) techniques, respectively. The effect of nFHA powder content on porosity and pore morphology was investigated. SEM images demonstrated that the scaffolds were constituted of interconnected and homogeneously distributed pores. The pore size of the scaffolds was in the range 50–250 μm. The result obtained in this research revealed that the porosity and pore average size decreased and compressive modulus increased with nFHA percentage. Considering morphological, physical, and mechanical properties, the scaffold with a higher ratio of nFHA has suitable potential use in tissue regeneration. PMID:21289986

Quantitative image analysis of cellular heterogeneity in breast tumors complements genomic profiling.

PubMed

Yuan, Yinyin; Failmezger, Henrik; Rueda, Oscar M; Ali, H Raza; Gräf, Stefan; Chin, Suet-Feung; Schwarz, Roland F; Curtis, Christina; Dunning, Mark J; Bardwell, Helen; Johnson, Nicola; Doyle, Sarah; Turashvili, Gulisa; Provenzano, Elena; Aparicio, Sam; Caldas, Carlos; Markowetz, Florian

2012-10-24

Solid tumors are heterogeneous tissues composed of a mixture of cancer and normal cells, which complicates the interpretation of their molecular profiles. Furthermore, tissue architecture is generally not reflected in molecular assays, rendering this rich information underused. To address these challenges, we developed a computational approach based on standard hematoxylin and eosin-stained tissue sections and demonstrated its power in a discovery and validation cohort of 323 and 241 breast tumors, respectively. To deconvolute cellular heterogeneity and detect subtle genomic aberrations, we introduced an algorithm based on tumor cellularity to increase the comparability of copy number profiles between samples. We next devised a predictor for survival in estrogen receptor-negative breast cancer that integrated both image-based and gene expression analyses and significantly outperformed classifiers that use single data types, such as microarray expression signatures. Image processing also allowed us to describe and validate an independent prognostic factor based on quantitative analysis of spatial patterns between stromal cells, which are not detectable by molecular assays. Our quantitative, image-based method could benefit any large-scale cancer study by refining and complementing molecular assays of tumor samples.

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.

A flow-proteometric platform for analyzing protein concentration (FAP): Proof of concept for quantification of PD-L1 protein in cells and tissues.

PubMed

Chou, Chao-Kai; Huang, Po-Jung; Tsou, Pei-Hsiang; Wei, Yongkun; Lee, Heng-Huan; Wang, Ying-Nai; Liu, Yen-Liang; Shi, Colin; Yeh, Hsin-Chih; Kameoka, Jun; Hung, Mien-Chie

2018-05-29

Protein expression level is critically related to the cell physiological function. However, current methodologies such as Western blot (WB) and Immunohistochemistry (IHC) in analyzing the protein level are rather semi-quantitative and without the information of actual protein concentration. We have developed a microfluidic technique termed a "flow-proteometric platform for analyzing protein concentration (FAP)" that can measure the concentration of a target protein in cells or tissues without the requirement of a calibration standard, e.g., the purified target molecules. To validate our method, we tested a number of control samples with known target protein concentrations and showed that the FAP measurement resulted in concentrations that well matched the actual concentrations in the control samples (coefficient of determination [R 2 ], 0.998), demonstrating a dynamic range of concentrations from 0.13 to 130 pM of a detection for 2 min. We successfully determined a biomarker protein (for predicting the treatment response of cancer immune check-point therapy) PD-L1 concentration in cancer cell lines (HeLa PD-L1 and MDA-MB-231) and breast cancer patient tumor tissues without any prior process of sample purification and standard line construction. Therefore, FAP is a simple, faster, and reliable method to measure the protein concentration in cells and tissues, which can support the conventional methods such as WB and IHC to determine the actual protein level. Copyright © 2018 Elsevier B.V. All rights reserved.

Integrated analyses of proteins and their glycans in a magnetic bead-based multiplex assay format.

PubMed

Li, Danni; Chiu, Hanching; Chen, Jing; Zhang, Hui; Chan, Daniel W

2013-01-01

Well-annotated clinical samples are valuable resources for biomarker discovery and validation. Multiplex and integrated methods that simultaneously measure multiple analytes and generate integrated information about these analytes from a single measurement are desirable because these methods help conserve precious samples. We developed a magnetic bead-based system for multiplex and integrated glycoprotein quantification by immunoassays and glycan detection by lectin immunosorbent assays (LISAs). Magnetic beads coupled with antibodies were used for capturing proteins of interest. Biotinylated antibodies in combination with streptavidin-labeled phycoerythrin were used for protein quantification. In the LISAs, biotinylated detection antibodies were replaced by biotinylated lectins for glycan detection. Using tissue inhibitor of metallopeptidase 1 (TIMP-1), tissue plasminogen activator, membrane metallo-endopeptidase, and dipeptidyl peptidase-IV (DPP-4) as models, we found that the multiplex integrated system was comparable to single immunoassays in protein quantification and LISAs in glycan detection. The merits of this system were demonstrated when applied to well-annotated prostate cancer tissues for validation of biomarkers in aggressive prostate cancer. Because of the system's multiplex ability, we used only 300 ng of tissue protein for the integrated detection of glycans in these proteins. Fucosylated TIMP-1 and DPP-4 offered improved performance over the proteins in distinguishing aggressive and nonaggressive prostate cancer. The multiplex and integrated system conserves samples and is a useful tool for validation of glycoproteins and their glycoforms as biomarkers. © 2012 American Association for Clinical Chemistry

Inflammatory cytokine expression following the use of bipolar electrocoagulation, ultracision harmonic scalpel and cold knife biopsy.

PubMed

Litta, Pietro; Saccardi, Carlo; Gizzo, Salvatore; Conte, Lorena; Ambrosi, Giulia; Sissi, Claudia; Palumbo, Manlio

2015-08-01

Electrical surgical devices may determine tissue damage through lateral thermal spread and activation of inflammatory processes. Several tissue effects are associated with the use of different surgical instruments. The aim of the present study was to compare tissue damage following the application of cold knife biopsy, bipolar electrocoagulation and the ultracision harmonic scalpel, through the analysis of inflammatory gene mediator expression. Three fragments of the round ligament (length 0.5 cm) were obtained from 22 females who had undergone total or subtotal laparoscopic hysterectomy using three different modes of resection: Cold knife biopsy, bipolar electrocoagulation and ultracision harmonic scalpel. The tissue fragments were examined by quantitative polymerase chain reaction (qPCR) analysis of selected cytokines. Gene expression analysis demonstrated large standard deviations due to individual variability among patients and indicated variability in the concentrations of cytokines in the three different samples. The quantity of cytokine mRNA in the cold knife biopsy samples was generally greater than those obtained by other techniques. Tumor necrosis factor-α expression was significantly higher in the sample obtained with the ultracision harmonic scalpel and bipolar electrocoagulation (P=0.033) when compared with cold knife biopsy. The inflammatory response was analyzed by the quantification of gene expression through the use of qPCR. The ultracision harmonic scalpel and bipolar electrocoagulation triggered the inflammatory cascade and resulted in an increased production of cytokines compared with cold knife biopsy.

Induction of Macrophage Chemotaxis by Aortic Extracts from Patients with Marfan Syndrome Is Related to Elastin Binding Protein

PubMed Central

Guo, Gao; Gehle, Petra; Doelken, Sandra; Martin-Ventura, José Luis; von Kodolitsch, Yskert; Hetzer, Roland; Robinson, Peter N.

2011-01-01

Marfan syndrome is an autosomal dominantly inherited disorder of connective tissue with prominent skeletal, ocular, and cardiovascular manifestations. Aortic aneurysm and dissection are the major determinants of premature death in untreated patients. In previous work, we showed that extracts of aortic tissues from the mgR mouse model of Marfan syndrome showed increased chemotactic stimulatory activity related to the elastin-binding protein. Aortic samples were collected from 6 patients with Marfan syndrome and 8 with isolated aneurysms of the ascending aorta. Control samples were obtained from 11 organ donors without known vascular or connective tissue diseases. Soluble proteins extracted from the aortic samples of the two patient groups were compared against buffer controls and against the aortic samples from controls with respect to the ability to induce macrophage chemotaxis as measured using a modified Boyden chamber, as well as the reactivity to a monoclonal antibody BA4 against bioactive elastin peptides using ELISA. Samples from Marfan patients displayed a statistically significant increase in chemotactic inductive activity compared to control samples. Additionally, reactivity to BA4 was significantly increased. Similar statistically significant increases were identified for the samples from patients with idiopathic thoracic aortic aneurysm. There was a significant correlation between the chemotactic index and BA4 reactivity, and the increases in chemotactic activity of extracts from Marfan patients could be inhibited by pretreatment with lactose, VGVAPG peptides, or BA4, which indicates the involvement of EBP in mediating the effects. Our results demonstrate that aortic extracts of patients with Marfan syndrome can elicit macrophage chemotaxis, similar to our previous study on aortic extracts of the mgR mouse model of Marfan syndrome (Guo et al., Circulation 2006; 114:1855-62). PMID:21647416

funtooNorm: an R package for normalization of DNA methylation data when there are multiple cell or tissue types.

PubMed

Oros Klein, Kathleen; Grinek, Stepan; Bernatsky, Sasha; Bouchard, Luigi; Ciampi, Antonio; Colmegna, Ines; Fortin, Jean-Philippe; Gao, Long; Hivert, Marie-France; Hudson, Marie; Kobor, Michael S; Labbe, Aurelie; MacIsaac, Julia L; Meaney, Michael J; Morin, Alexander M; O'Donnell, Kieran J; Pastinen, Tomi; Van Ijzendoorn, Marinus H; Voisin, Gregory; Greenwood, Celia M T

2016-02-15

DNA methylation patterns are well known to vary substantially across cell types or tissues. Hence, existing normalization methods may not be optimal if they do not take this into account. We therefore present a new R package for normalization of data from the Illumina Infinium Human Methylation450 BeadChip (Illumina 450 K) built on the concepts in the recently published funNorm method, and introducing cell-type or tissue-type flexibility. funtooNorm is relevant for data sets containing samples from two or more cell or tissue types. A visual display of cross-validated errors informs the choice of the optimal number of components in the normalization. Benefits of cell (tissue)-specific normalization are demonstrated in three data sets. Improvement can be substantial; it is strikingly better on chromosome X, where methylation patterns have unique inter-tissue variability. An R package is available at https://github.com/GreenwoodLab/funtooNorm, and has been submitted to Bioconductor at http://bioconductor.org. © The Author 2015. Published by Oxford University Press.

Ultrasound elastography to determine the layered mechanical properties of articular cartilage and the importance of such structural characteristics under load.

PubMed

McCredie, Alexandra J; Stride, Eleanor; Saffari, Nader

2009-01-01

Articular cartilage is an important load bearing surface in joints. Prone to damage and with limited self-repair ability, it is of interest to tissue engineers. Tissue implant design requires full mechanical characterisation of healthy native tissue. A layered organisation of reinforcing collagen fibrils exists in healthy articular cartilage and is believed to be important for correct tissue function. However, the effect of this on the local depth-dependent elasticity is poorly characterised. In this study, quasi-static ultrasound elastography is used both to compare the depth-dependent elastic properties of cartilage structures with two different fibril arrangements and to monitor changes in the elastic properties of engineered samples during development. Results show global and local elastic properties of the native tissues and highlight the differences caused by fibril architecture. At increasing culture periods, results from the engineered tissue demonstrate an increase in elastic stiffness and the time taken to reach equilibrium under a quasi-static displacement. The study suggests suitability of ultrasound elastography for design and monitoring engineered articular cartilage.

Novel technique for online characterization of cartilaginous tissue properties.

PubMed

Yuan, Tai-Yi; Huang, Chun-Yuh; Yong Gu, Wei

2011-09-01

The goal of tissue engineering is to use substitutes to repair and restore organ function. Bioreactors are an indispensable tool for monitoring and controlling the unique environment for engineered constructs to grow. However, in order to determine the biochemical properties of engineered constructs, samples need to be destroyed. In this study, we developed a novel technique to nondestructively online-characterize the water content and fixed charge density of cartilaginous tissues. A new technique was developed to determine the tissue mechano-electrochemical properties nondestructively. Bovine knee articular cartilage and lumbar annulus fibrosus were used in this study to demonstrate that this technique could be used on different types of tissue. The results show that our newly developed method is capable of precisely predicting the water volume fraction (less than 3% disparity) and fixed charge density (less than 16.7% disparity) within cartilaginous tissues. This novel technique will help to design a new generation of bioreactors which are able to actively determine the essential properties of the engineered constructs, as well as regulate the local environment to achieve the optimal conditions for cultivating constructs.

Nonnegative constraint analysis of key fluorophores within human breast cancer using native fluorescence spectroscopy excited by selective wavelength of 300 nm

NASA Astrophysics Data System (ADS)

Pu, Yang; Sordillo, Laura A.; Alfano, Robert R.

2015-03-01

Native fluorescence spectroscopy offers an important role in cancer discrimination. It is widely acknowledged that the emission spectrum of tissue is a superposition of spectra of various salient fluorophores. In this study, the native fluorescence spectra of human cancerous and normal breast tissues excited by selected wavelength of 300 nm are used to investigate the key building block fluorophores: tryptophan and reduced nicotinamide adenine dinucleotide (NADH). The basis spectra of these key fluorophores' contribution to the tissue emission spectra are obtained by nonnegative constraint analysis. The emission spectra of human cancerous and normal tissue samples are projected onto the fluorophore spectral subspace. Since previous studies indicate that tryptophan and NADH are key fluorophores related with tumor evolution, it is essential to obtain their information from tissue fluorescence but discard the redundancy. To evaluate the efficacy of for cancer detection, linear discriminant analysis (LDA) classifier is used to evaluate the sensitivity, and specificity. This research demonstrates that the native fluorescence spectroscopy measurements are effective to detect changes of fluorophores' compositions in tissues due to the development of cancer.

Maximizing fluorescence collection efficiency in multiphoton microscopy

PubMed Central

Zinter, Joseph P.; Levene, Michael J.

2011-01-01

Understanding fluorescence propagation through a multiphoton microscope is of critical importance in designing high performance systems capable of deep tissue imaging. Optical models of a scattering tissue sample and the Olympus 20X 0.95NA microscope objective were used to simulate fluorescence propagation as a function of imaging depth for physiologically relevant scattering parameters. The spatio-angular distribution of fluorescence at the objective back aperture derived from these simulations was used to design a simple, maximally efficient post-objective fluorescence collection system. Monte Carlo simulations corroborated by data from experimental tissue phantoms demonstrate collection efficiency improvements of 50% – 90% over conventional, non-optimized fluorescence collection geometries at large imaging depths. Imaging performance was verified by imaging layer V neurons in mouse cortex to a depth of 850 μm. PMID:21934897

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.

Three-Dimensional Gene Map of Cancer Cell Types: Structural Entropy Minimisation Principle for Defining Tumour Subtypes

PubMed Central

Li, Angsheng; Yin, Xianchen; Pan, Yicheng

2016-01-01

In this study, we propose a method for constructing cell sample networks from gene expression profiles, and a structural entropy minimisation principle for detecting natural structure of networks and for identifying cancer cell subtypes. Our method establishes a three-dimensional gene map of cancer cell types and subtypes. The identified subtypes are defined by a unique gene expression pattern, and a three-dimensional gene map is established by defining the unique gene expression pattern for each identified subtype for cancers, including acute leukaemia, lymphoma, multi-tissue, lung cancer and healthy tissue. Our three-dimensional gene map demonstrates that a true tumour type may be divided into subtypes, each defined by a unique gene expression pattern. Clinical data analyses demonstrate that most cell samples of an identified subtype share similar survival times, survival indicators and International Prognostic Index (IPI) scores and indicate that distinct subtypes identified by our algorithms exhibit different overall survival times, survival ratios and IPI scores. Our three-dimensional gene map establishes a high-definition, one-to-one map between the biologically and medically meaningful tumour subtypes and the gene expression patterns, and identifies remarkable cells that form singleton submodules. PMID:26842724

Pharmacodynamic and pharmacokinetic studies and prostatic tissue distribution of fosfomycin tromethamine in bacterial prostatitis or normal rats.

PubMed

Fan, L; Shang, X; Zhu, J; Ma, B; Zhang, Q

2018-05-02

In this study, we assessed the therapeutic effects of fosfomycin tromethamine (FT) in a bacterial prostatitis (BP) rat model. The BP model was induced by Escherichia coli and was demonstrated after 7 days microbiologically and histologically. Then, 25 BP rats selected were randomly divided into five treatment groups: model group, positive group, FT-3 day group, FT-7 day group and FT-14 day group. Ventral lobes of prostate from all animals were removed, and the serum samples were collected at the end of the experiments. Microbiological cultures and histological findings of the prostate samples demonstrated reduced bacterial growth and improved inflammatory responses in FT-treatment groups compared with the model group, indicating that FT against prostatic infection induced by E. coli showed good antibacterial effects. Moreover, plasma pharmacokinetics and prostatic distribution of fosfomycin were studied and compared in BP and normal rats. The concentrations of fosfomycin in samples were analysed by liquid chromatography-tandem mass spectrometry. There were no differences in plasma pharmacokinetic parameters between two groups. But significantly higher penetration of fosfomycin into prostatic tissues was found in BP rats. We therefore suggested that FT had a good therapeutic effect on BP and it might be used in curing masculine reproductive system diseases. © 2018 Blackwell Verlag GmbH.

The detection of Giardia muris and Giardia lamblia cysts by immunofluorescence in animal tissues and fecal samples subjected to cycles of freezing and thawing.

PubMed

Erlandsen, S L; Sherlock, L A; Bemrick, W J

1990-04-01

The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). Giardia muris cysts were obtained from either animal carcasses, fecal pellets, or isolated cyst preparations, whereas Giardia lamblia cysts were isolated from fecal samples. These samples were stained using an immunofluorescence technique after 1-3 freezing (-16 C) and thawing (20 C) cycles. Cysts were detected successfully by immunofluorescence in all samples. However, in those samples subjected to freeze-thawing, the cyst walls often became distorted and then were not detectable by bright field microscopy. Low voltage SEM demonstrated that the filaments in the distorted cyst wall underwent rearrangements of interfilament spacing. Quantitation of cyst recovery after freezing and thawing demonstrated that a substantial loss occurred after 1 cycle of alternating temperature when low concentrations of cysts were used, but not with high concentrations of cysts. Cyst recovery, after 3 freezing and thawing cycles, was dramatically lowered irrespective of the initial cyst concentration. These results demonstrated that immunofluorescence was an effective technique for the detection of Giardia spp. cysts in frozen samples and would suggest that freezing and thawing of fecal samples could prevent the detection of cysts when only bright field microscopy was employed.

Photoacoustic bio-quantification of graphene based nanomaterials at a single cell level (Conference Presentation)

NASA Astrophysics Data System (ADS)

Nedosekin, Dmitry A.; Nolan, Jacqueline; Biris, Alexandru S.; Zharov, Vladimir P.

2017-03-01

Arkansas Nanomedicine Center at the University of Arkansas for Medical Sciences in collaboration with other Arkansas Universities and the FDA-based National Center of Toxicological Research in Jefferson, AR is developing novel techniques for rapid quantification of graphene-based nanomaterials (GBNs) in various biological samples. All-carbon GBNs have wide range of potential applications in industry, agriculture, food processing and medicine; however, quantification of GBNs is difficult in carbon reach biological tissues. The accurate quantification of GBNs is essential for research on material toxicity and the development of GBNs-based drug delivery platforms. We have developed microscopy and cytometry platforms for detection and quantification of GBNs in single cells, tissue and blood samples using photoacoustic contrast of GBNs. We demonstrated PA quantification of individual graphene uptake by single cells. High-resolution PA microscopy provided mapping of GBN distribution within live cells to establish correlation with intracellular toxic phenomena using apoptotic and necrotic assays. This new methodology and corresponding technical platform provide the insight on possible toxicological risks of GBNs at singe cells levels. In addition, in vivo PA image flow cytometry demonstrated the capability to monitor of GBNs pharmacokinetics in mouse model and to map the resulting biodistribution of GBNs in mouse tissues. The integrated PA platform provided an unprecedented sensitivity toward GBNs and allowed to enhance conventional toxicology research by providing a direct correlation between uptake of GBNs at a single cell level and cell viability status.

Cytokine stimulation of MUC4 expression in human female reproductive tissue carcinoma cell lines and endometrial cancer.

PubMed

Chapela, Patricia J; Broaddus, Russell R; Hawkins, Shannon M; Lessey, Bruce A; Carson, Daniel D

2015-11-01

MUC4, a transmembrane glycoprotein, interferes with cell adhesion, and promotes EGFR signaling in cancer. Studies in rat models have demonstrated steroid hormonal regulation of endometrial MUC4 expression. In this study, qRT-PCR screening of mouse tissues determined that Muc4 mRNA also was robustly expressed in mouse uteri. Previous studies from our labs have demonstrated MUC4 mRNA was expressed at levels <1% of MUC1 mRNA in human endometrium and endometriotic tissue. Multiple human endometrial adenocarcinoma cell lines were assayed for MUC4 mRNA expression revealing extremely low basal expression in the Ishikawa, RL-95-2, AN3CA, and KLE lines. Moderate to high expression was observed in HEC50 and HEC-1A cells. MUC4 mRNA expression was not affected by progesterone and/or estrogen treatment, but was greatly stimulated at both mRNA and protein levels by proinflammatory cytokines (IFN-γ and TNF-α), particularly when used in combination. In endometrial tissue, MUC4 mRNA levels did not change significantly between normal or cancerous samples; although, a subset of patients with grade 1 and 2 tumors displayed substantially higher expression. Likewise, immunostaining of human endometrial adenocarcinoma tissues revealed little to no staining in many patients (low MUC4), but strong staining in some patients (high MUC4) independent of cancer grade. In cases where staining was observed, it was heterogeneous with some cells displaying robust MUC4 expression and others displaying little or no staining. Collectively, these observations demonstrate that while MUC4 is highly expressed in the mouse uterus, it is not a major mucin in normal human endometrium. Rather, MUC4 is a potential marker of endometrial adenocarcinoma in a subset of patients. © 2015 Wiley Periodicals, Inc.

On-chip clearing of arrays of 3-D cell cultures and micro-tissues.

PubMed

Grist, S M; Nasseri, S S; Poon, T; Roskelley, C; Cheung, K C

2016-07-01

Three-dimensional (3-D) cell cultures are beneficial models for mimicking the complexities of in vivo tissues, especially in tumour studies where transport limitations can complicate response to cancer drugs. 3-D optical microscopy techniques are less involved than traditional embedding and sectioning, but are impeded by optical scattering properties of the tissues. Confocal and even two-photon microscopy limit sample imaging to approximately 100-200 μm depth, which is insufficient to image hypoxic spheroid cores. Optical clearing methods have permitted high-depth imaging of tissues without physical sectioning, but they are difficult to implement for smaller 3-D cultures due to sample loss in solution exchange. In this work, we demonstrate a microfluidic platform for high-throughput on-chip optical clearing of breast cancer spheroids using the SeeDB, Clear(T2), and ScaleSQ clearing methods. Although all three methods are able to effectively clear the spheroids, we find that SeeDB and ScaleSQ more effectively clear the sample than Clear(T2); however, SeeDB induces green autofluorescence while ScaleS causes sample expansion. Our unique on-chip implementation permits clearing arrays of 3-D cultures using perfusion while monitoring the 3-D cultures throughout the process, enabling visualization of the clearing endpoint as well as monitoring of transient changes that could induce image artefacts. Our microfluidic device is compatible with on-chip 3-D cell culture, permitting the use of on-chip clearing at the endpoint after monitoring the same spheroids during their culture. This on-chip method has the potential to improve readout from 3-D cultures, facilitating their use in cell-based assays for high-content drug screening and other applications.

Loss of coxsackie and adenovirus receptor expression in human colorectal cancer: A potential impact on the efficacy of adenovirus-mediated gene therapy in Chinese Han population.

PubMed

Ma, Ying-Yu; Wang, Xiao-Jun; Han, Yong; Li, Gang; Wang, Hui-Ju; Wang, Shi-Bing; Chen, Xiao-Yi; Liu, Fan-Long; He, Xiang-Lei; Tong, Xiang-Min; Mou, Xiao-Zhou

2016-09-01

The coxsackie and adenovirus receptor (CAR) is considered a tumor suppressor and critical factor for the efficacy of therapeutic strategies that employ the adenovirus. However, data on CAR expression levels in colorectal cancer are conflicting and its clinical relevance remains to be elucidated. Immunohistochemistry was performed on tissue microarrays containing 251 pairs of colon cancer and adjacent normal tissue samples from Chinese Han patients to assess the expression levels of CAR. Compared with healthy mucosa, decreased CAR expression (40.6% vs. 95.6%; P<0.001) was observed in colorectal cancer samples. The CAR immunopositivity in tumor tissues was not significantly associated with gender, age, tumor size, differentiation, TNM stage, lymph node metastasis or distant metastasis in patients with colon cancer. However, expression of CAR is present in 83.3% of the tumor tissues from patient with colorectal liver metastasis, which was significantly higher than those without liver metastasis (39.6%; P=0.042). At the plasma membrane, CAR was observed in 29.5% normal mucosa samples, which was significantly higher than in colorectal cancer samples (4.0%; P<0.001). In addition, the survival analysis demonstrated that the expression level of CAR has no association with the prognosis of colorectal cancer. CAR expression was observed to be downregulated in colorectal cancer, and it exerts complex effects during colorectal carcinogenesis, potentially depending on the stage of the cancer development and progression. High CAR expression may promote liver metastasis. With regard to oncolytic therapy, CAR expression analysis should be performed prior to adenoviral oncolytic treatment to stratify Chinese Han patients for treatment.

MicroRNA-300 inhibited glioblastoma progression through ROCK1.

PubMed

Zhou, Fucheng; Li, Yang; Hao, Zhen; Liu, Xuanxi; Chen, Liang; Cao, Yu; Liang, Zuobin; Yuan, Fei; Liu, Jie; Wang, Jianjiao; Zheng, Yongri; Dong, Deli; Bian, Shan; Yang, Baofeng; Jiang, Chuanlu; Li, Qingsong

2016-06-14

Glioblastoma is a common type of brain aggressive tumors and has a poor prognosis. MicroRNAs (miRNAs) are a class of small, endogenous and non-coding RNAs that play crucial roles in cell proliferation, survival and invasion. Deregulated expression of miR-300 has been studied in a lot of cancers. However, the role of miR-300 in glioblastoma is still unknown. In this study, we demonstrated that miR-300 expression was downregulated in glioblastoma tissues compared with the normal tissues. Lower expression level of miR-300 was observed in thirty cases (75 %, 30/40) of glioblastoma samples compared with the normal samples. Moreover, the overall survival of glioblastoma patients with lower miR-300 expression level was shorter than those with higher miR-300 expression level. In addition, miR-300 expression was also downregulated in glioblastoma cell lines. Overexpression of miR-300 inhibited cell proliferation, cell cycle and invasion in glioblastoma cell line U87 and U251. Moreover, we identified ROCK1 as a direct target of miR-300 in U87 and U251 cells. Overexpression of ROCK1 partially rescued the miR-300-mediated cell growth. ROCK1 expression levels in glioblastoma tissues were higher than that in normal tissues. ROCK1 expression levels were higher in thirty-one cases of glioblastoma samples than their normal samples. Furthermore, the expression level ROCK1 was inversely correlated with the expression level of miR-300. Importantly, overexpression of miR-300 suppressed glioblastoma progression in an established xenograft model. In conclusion, we revealed that miR-300 might act as a tumor suppressor gene through inhibiting ROCK1 in glioblastoma.

MicroRNA-300 inhibited glioblastoma progression through ROCK1

PubMed Central

Hao, Zhen; Liu, Xuanxi; Chen, Liang; Cao, Yu; Liang, Zuobin; Yuan, Fei; Liu, Jie; Wang, Jianjiao; Zheng, Yongri; Dong, Deli; Bian, Shan; Yang, Baofeng; Jiang, Chuanlu; Li, Qingsong

2016-01-01

Glioblastoma is a common type of brain aggressive tumors and has a poor prognosis. MicroRNAs (miRNAs) are a class of small, endogenous and non-coding RNAs that play crucial roles in cell proliferation, survival and invasion. Deregulated expression of miR-300 has been studied in a lot of cancers. However, the role of miR-300 in glioblastoma is still unknown. In this study, we demonstrated that miR-300 expression was downregulated in glioblastoma tissues compared with the normal tissues. Lower expression level of miR-300 was observed in thirty cases (75 %, 30/40) of glioblastoma samples compared with the normal samples. Moreover, the overall survival of glioblastoma patients with lower miR-300 expression level was shorter than those with higher miR-300 expression level. In addition, miR-300 expression was also downregulated in glioblastoma cell lines. Overexpression of miR-300 inhibited cell proliferation, cell cycle and invasion in glioblastoma cell line U87 and U251. Moreover, we identified ROCK1 as a direct target of miR-300 in U87 and U251 cells. Overexpression of ROCK1 partially rescued the miR-300-mediated cell growth. ROCK1 expression levels in glioblastoma tissues were higher than that in normal tissues. ROCK1 expression levels were higher in thirty-one cases of glioblastoma samples than their normal samples. Furthermore, the expression level ROCK1 was inversely correlated with the expression level of miR-300. Importantly, overexpression of miR-300 suppressed glioblastoma progression in an established xenograft model. In conclusion, we revealed that miR-300 might act as a tumor suppressor gene through inhibiting ROCK1 in glioblastoma. PMID:27145462

Simultaneous analysis of aminoglycosides with many other classes of drug residues in bovine tissues by ultrahigh-performance liquid chromatography-tandem mass spectrometry using an ion-pairing reagent added to final extracts.

PubMed

Lehotay, Steven J; Lightfield, Alan R

2018-01-01

The way to maximize scope of analysis, sample throughput, and laboratory efficiency in the monitoring of veterinary drug residues in food animals is to determine as many analytes as possible as fast as possible in as few methods as possible. Capital and overhead expenses are also reduced by using fewer instruments in the overall monitoring scheme. Traditionally, the highly polar aminoglycoside antibiotics require different chromatographic conditions from other classes of drugs, but in this work, we demonstrate that an ion-pairing reagent (sodium 1-heptanesulfonate) added to the combined final extracts from two sample preparation methods attains good separation of 174 targeted drugs, including 9 aminoglycosides, in the same 10.5-min ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The full method was validated in bovine kidney, liver, and muscle tissues according to US regulatory protocols, and 137-146 (79-84%) of the drugs gave between 70 and 120% average recoveries with ≤ 25% RSDs in the different types of tissues spiked at 0.5, 1, and 2 times the regulatory levels of interest (10-1000 ng/g depending on the drug). This method increases sample throughput and the possible number of drugs monitored in the US National Residue Program, and requires only one UHPLC-MS/MS method and instrument for analysis rather than two by the previous scheme. Graphical abstract Outline of the streamlined approach to monitor 174 veterinary drugs, including aminoglycosides, in bovine tissues by combining two extracts of the same sample with an ion-pairing reagent for analysis by UHPLC-MS/MS.

Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA

PubMed Central

Zill, Oliver A.; Sebisanovic, Dragan; Lopez, Rene; Blau, Sibel; Collisson, Eric A.; Divers, Stephen G.; Hoon, Dave S. B.; Kopetz, E. Scott; Lee, Jeeyun; Nikolinakos, Petros G.; Baca, Arthur M.; Kermani, Bahram G.; Eltoukhy, Helmy; Talasaz, AmirAli

2015-01-01

Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital SequencingTM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing. PMID:26474073

The effect of electronically steering a phased array ultrasound transducer on near-field tissue heating.

PubMed

Payne, Allison; Vyas, Urvi; Todd, Nick; de Bever, Joshua; Christensen, Douglas A; Parker, Dennis L

2011-09-01

This study presents the results obtained from both simulation and experimental techniques that show the effect of mechanically or electronically steering a phased array transducer on proximal tissue heating. The thermal response of a nine-position raster and a 16-mm diameter circle scanning trajectory executed through both electronic and mechanical scanning was evaluated in computer simulations and experimentally in a homogeneous tissue-mimicking phantom. Simulations were performed using power deposition maps obtained from the hybrid angular spectrum (HAS) method and applying a finite-difference approximation of the Pennes' bioheat transfer equation for the experimentally used transducer and also for a fully sampled transducer to demonstrate the effect of acoustic window, ultrasound beam overlap and grating lobe clutter on near-field heating. Both simulation and experimental results show that electronically steering the ultrasound beam for the two trajectories using the 256-element phased array significantly increases the thermal dose deposited in the near-field tissues when compared with the same treatment executed through mechanical steering only. In addition, the individual contributions of both beam overlap and grating lobe clutter to the near-field thermal effects were determined through comparing the simulated ultrasound beam patterns and resulting temperature fields from mechanically and electronically steered trajectories using the 256-randomized element phased array transducer to an electronically steered trajectory using a fully sampled transducer with 40 401 phase-adjusted sample points. Three distinctly different three distinctly different transducers were simulated to analyze the tradeoffs of selected transducer design parameters on near-field heating. Careful consideration of design tradeoffs and accurate patient treatment planning combined with thorough monitoring of the near-field tissue temperature will help to ensure patient safety during an MRgHIFU treatment.

Pathological consequences of systemic measles virus infection.

PubMed

Ludlow, Martin; McQuaid, Stephen; Milner, Dan; de Swart, Rik L; Duprex, W Paul

2015-01-01

The identification of poliovirus receptor-like 4 (PVRL4) as the second natural receptor for measles virus (MV) has closed a major gap in our understanding of measles pathogenesis, and explains how this predominantly lymphotropic virus breaks through epithelial barriers to transmit to a susceptible host. Advances in the development of wild-type, recombinant MVs which express fluorescent proteins making infected cells readily detectable in living tissues and animals, has also increased our understanding of this important and highly transmissible human disease. Thus, it is timely to review how these advances have provided new insights into MV infection of immune, epithelial and neural cells. This demands access to primate samples that help us understand the early and acute stages of the disease, which are challenging to dissect due to the mild/self-limiting nature of the infection. It also requires well-characterized and rather rare human tissue samples from patients who succumb to neurological sequelae to help study the consequences of the long-term persistence of this RNA virus in vivo. Collectively, these studies have provided unique insights into how the use of two cellular receptors, CD150 and PVRL4, governs the in vivo tissue-specific temporal patterns of virus spread and resulting pathological lesions. Analysis of tissue samples has also demonstrated the importance of differing mechanisms of virus cell-to-cell spread within lymphoid, epithelial and neural tissues in the dissemination of MV during acute and long-term persistent infections. Given the incentive to eradicate MV globally, and the inevitable question as to whether or not vaccination should cease in light of the existence of closely related morbilliviruses, a thorough understanding of measles pathological lesions is essential. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Multi-distance diffuse optical spectroscopy with a single optode via hypotrochoidal scanning.

PubMed

Applegate, Matthew B; Roblyer, Darren

2018-02-15

Frequency-domain diffuse optical spectroscopy (FD-DOS) is an established technique capable of determining optical properties and chromophore concentrations in biological tissue. Most FD-DOS systems use either manually positioned, handheld probes or complex arrays of source and detector fibers to acquire data from many tissue locations, allowing for the generation of 2D or 3D maps of tissue. Here, we present a new method to rapidly acquire a wide range of source-detector (SD) separations by mechanically scanning a single SD pair. The source and detector fibers are mounted on a scan head that traces a hypotrochoidal pattern over the sample that, when coupled with a high-speed FD-DOS system, enables the rapid collection of dozens of SD separations for depth-resolved imaging. We demonstrate that this system has an average error of 4±2.6% in absorption and 2±1.8% in scattering across all SD separations. Additionally, by linearly translating the device, the size and location of an absorbing inhomogeneity can be determined through the generation of B-scan images in a manner conceptually analogous to ultrasound imaging. This work demonstrates the potential of single optode diffuse optical scanning for depth resolved visualization of heterogeneous biological tissues at near real-time rates.

Strategies to overcome photobleaching in algorithm-based adaptive optics for nonlinear in-vivo imaging.

PubMed

Caroline Müllenbroich, M; McGhee, Ewan J; Wright, Amanda J; Anderson, Kurt I; Mathieson, Keith

2014-01-01

We have developed a nonlinear adaptive optics microscope utilizing a deformable membrane mirror (DMM) and demonstrated its use in compensating for system- and sample-induced aberrations. The optimum shape of the DMM was determined with a random search algorithm optimizing on either two photon fluorescence or second harmonic signals as merit factors. We present here several strategies to overcome photobleaching issues associated with lengthy optimization routines by adapting the search algorithm and the experimental methodology. Optimizations were performed on extrinsic fluorescent dyes, fluorescent beads loaded into organotypic tissue cultures and the intrinsic second harmonic signal of these cultures. We validate the approach of using these preoptimized mirror shapes to compile a robust look-up table that can be applied for imaging over several days and through a variety of tissues. In this way, the photon exposure to the fluorescent cells under investigation is limited to imaging. Using our look-up table approach, we show signal intensity improvement factors ranging from 1.7 to 4.1 in organotypic tissue cultures and freshly excised mouse tissue. Imaging zebrafish in vivo, we demonstrate signal improvement by a factor of 2. This methodology is easily reproducible and could be applied to many photon starved experiments, for example fluorescent life time imaging, or when photobleaching is a concern.

Downregulated SASH1 expression indicates poor clinical prognosis in gastric cancer.

PubMed

Zhou, Nan; Liu, Can; Wang, Xudong; Mao, Qinsheng; Jin, Qin; Li, Peng

2018-04-01

SASH1 (SAM- and SH3-domain containing 1), a novel candidate tumor suppressor, has attracted attention due to its role in intracellular signal transduction and its tumor prognostic value in diverse cancers. Reports have demonstrated that reduced SASH1 expression correlates with tumor proliferation, invasion, and metastasis. However, the expression and prognostic significance of SASH1 in gastric cancer (GC) remain unclear. In this study, 8 paired fresh-frozen GC tissues and corresponding gastric mucosal tissues were examined by Western blot to analyze the protein expression of SASH1. Seven hundred twenty-six formalin-fixed, paraffin-embedded (FFPE) gastric tissue samples were evaluated by immunohistochemical (IHC) to determine the correlations of SASH1 expression with clinicopathological factors and prognosis. Compared with adjacent noncancerous tissues, SASH1 was significantly downregulated in GC specimens. Analysis using the χ 2 test revealed that low SASH1 expression was significantly associated with advanced TNM stage (P < .001) in GC. Cox regression multivariable analyses demonstrated that SASH1 expression (P < .001), TNM stage (P < .001), preoperative CEA level (P = .003) and preoperative CA19-9 level (P = .002) were independent prognostic factors. Our clinical findings suggest that downregulated SASH1 expression could be used as an independent biomarker for poor prognosis in GC. Copyright © 2018. Published by Elsevier Inc.

Experimental characterization of intrapulse tissue conductivity changes for electroporation.

PubMed

Neal, Robert E; Garcia, Paulo A; Robertson, John L; Davalos, Rafael V

2011-01-01

Cells exposed to short electric pulses experience a change in their transmembrane potential, which can lead to increased membrane permeability of the cell. When the energy of the pulses surpasses a threshold, the cell dies in a non-thermal manner known as irreversible electroporation (IRE). IRE has shown promise in the focal ablation of pathologic tissues. Its non-thermal mechanism spares sensitive structures and facilitates rapid lesion resolution. IRE effects depend on the electric field distribution, which can be predicted with numerical modeling. When the cells become permeabilized, the bulk tissue properties change, affecting this distribution. For IRE to become a reliable and successful treatment of diseased tissues, robust predictive treatment planning methods must be developed. It is vital to understand the changes in tissue properties undergoing the electric pulses to improve numerical models and predict treatment volumes. We report on the experimental characterization of these changes for kidney tissue. Tissue samples were pulsed between plate electrodes while intrapulse voltage and current data were measured to determine the conductivity of the tissue during the pulse. Conductivity was then established as a function of the electric field to which the tissue is exposed. This conductivity curve was used in a numerical model to demonstrate the impact of accounting for these changes when modeling electric field distributions to develop treatment plans.

The bereavement process of tissue donors' family members: responses of grief, posttraumatic stress, personal growth, and ongoing attachment.

PubMed

Hogan, Nancy; Schmidt, Lee; Coolican, Maggie

2014-09-01

Donated tissues can save lives of critically burned patients and those needing a heart valve replacement. Tissues enhance the lives of a million recipients annually through transplants of corneas, bones, tendons, and vein grafts. Unfortunately, the need for some tissues exceeds their availability. The goal of the quantitative component of this mixed methods study was to identify the grief, posttraumatic stress, personal growth, and ongoing attachment response of tissue donors' family members during a 2-year period. Simultaneous mixed methods design. The sample for this study consisted of 52 tissue donors' family members, mostly widows (83%). Data were collected for 2 years to test changes in grief, posttraumatic stress, panic behavior, personal growth, and ongoing attachment. The bereaved participants experienced significantly fewer grief reactions, less posttraumatic stress, and greater personal growth. There was no significant difference in the ongoing attachment to their deceased loved ones. The results of this study may reinforce the positive meaning that tissue donors' family members can find in tissue donation. Findings also demonstrate that the bereavement process corroborates contemporary bereavement and attachment theories. Health professionals are encouraged to seek donations with less worry that tissue donors' family members will experience adverse outcomes during bereavement.

Controlling for anthropogenically induced atmospheric variation in stable carbon isotope studies

USGS Publications Warehouse

Long, E.S.; Sweitzer, R.A.; Diefenbach, D.R.; Ben-David, M.

2005-01-01

Increased use of stable isotope analysis to examine food-web dynamics, migration, transfer of nutrients, and behavior will likely result in expansion of stable isotope studies investigating human-induced global changes. Recent elevation of atmospheric CO2 concentration, related primarily to fossil fuel combustion, has reduced atmospheric CO2 ??13C (13C/12C), and this change in isotopic baseline has, in turn, reduced plant and animal tissue ??13C of terrestrial and aquatic organisms. Such depletion in CO2 ??13C and its effects on tissue ??13C may introduce bias into ??13C investigations, and if this variation is not controlled, may confound interpretation of results obtained from tissue samples collected over a temporal span. To control for this source of variation, we used a high-precision record of atmospheric CO2 ??13C from ice cores and direct atmospheric measurements to model modern change in CO2 ??13C. From this model, we estimated a correction factor that controls for atmospheric change; this correction reduces bias associated with changes in atmospheric isotopic baseline and facilitates comparison of tissue ??13C collected over multiple years. To exemplify the importance of accounting for atmospheric CO2 ??13C depletion, we applied the correction to a dataset of collagen ??13C obtained from mountain lion (Puma concolor) bone samples collected in California between 1893 and 1995. Before correction, in three of four ecoregions collagen ??13C decreased significantly concurrent with depletion of atmospheric CO2 ??13C (n ??? 32, P ??? 0.01). Application of the correction to collagen ??13C data removed trends from regions demonstrating significant declines, and measurement error associated with the correction did not add substantial variation to adjusted estimates. Controlling for long-term atmospheric variation and correcting tissue samples for changes in isotopic baseline facilitate analysis of samples that span a large temporal range. ?? Springer-Verlag 2005.

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

Polarization properties of amyloid-beta plaques in Alzheimer's disease (Conference Presentation)

NASA Astrophysics Data System (ADS)

Baumann, Bernhard; Wöhrer, Adelheid; Ricken, Gerda; Pircher, Michael; Kovacs, Gabor G.; Hitzenberger, Christoph K.

2016-03-01

In histopathological practice, birefringence is used for the identification of amyloidosis in numerous tissues. Amyloid birefringence is caused by the parallel arrangement of fibrous protein aggregates. Since neurodegenerative processes in Alzheimer's disease (AD) are also linked to the formation of amyloid-beta (Aβ) plaques, optical methods sensitive to birefringence may act as non-invasive tools for Aβ identification. At last year's Photonics West, we demonstrated polarization-sensitive optical coherence tomography (PS-OCT) imaging of ex vivo cerebral tissue of advanced stage AD patients. PS-OCT provides volumetric, structural imaging based on both backscatter contrast and tissue polarization properties. In this presentation, we report on polarization-sensitive neuroimaging along with numerical simulations of three-dimensional Aβ plaques. High speed PS-OCT imaging was performed using a spectral domain approach based on polarization maintaining fiber optics. The sample beam was interfaced to a confocal scanning microscope arrangement. Formalin-fixed tissue samples as well as thin histological sections were imaged. For comparison to the PS-OCT results, ray propagation through plaques was modeled using Jones analysis and various illumination geometries and plaque sizes. Characteristic polarization patterns were found. The results of this study may not only help to understand PS-OCT imaging of neuritic Aβ plaques but may also have implications for polarization-sensitive imaging of other fibrillary structures.

The potential of optical coherence tomography for diagnosing meniscal pathology

NASA Astrophysics Data System (ADS)

Hang-Yin Ling, Carrie; Pozzi, Antonio; Thieman, Kelley M.; Tonks, Catherine A.; Guo, Shuguang; Xie, Huikai; Horodyski, MaryBeth

2010-04-01

Meniscal tears are often associated with anterior cruciate ligament (ACL) injury and may lead to pain and discomfort in humans. Maximal preservation of meniscal tissue is highly desirable to mitigate the progression of osteoarthritis. Guidelines of which meniscal tears are amenable to repair and what part of damaged tissues should be removed are elusive and lacking consensus. Images of microstructural changes in meniscus would potentially guide the surgeons to manage the meniscal tears better, but the resolution of current diagnostic techniques is limited for this application. In this study, we demonstrated the feasibility of using optical coherence tomography (OCT) for the diagnosis of meniscal pathology. Torn medial menisci were collected from dogs with ACL insufficiency. The torn meniscus was divided into three tissue samples and scanned by OCT and scanning electron microscopy (SEM). OCT and SEM images of torn menisci were compared. Each sample was evaluated for gross and microstructural abnormalities and reduction or loss of birefringence from the OCT images. The abnormalities detected with OCT were described for each type of tear. OCT holds promise in non-destructive and fast assessment of microstructural changes and tissue birefringence of meniscal tears. Future development of intraoperative OCT may help surgeons in the decision making of meniscal treatment.

Tissue Banking, Bioinformatics, and Electronic Medical Records: The Front-End Requirements for Personalized Medicine

PubMed Central

Suh, K. Stephen; Sarojini, Sreeja; Youssif, Maher; Nalley, Kip; Milinovikj, Natasha; Elloumi, Fathi; Russell, Steven; Pecora, Andrew; Schecter, Elyssa; Goy, Andre

2013-01-01

Personalized medicine promises patient-tailored treatments that enhance patient care and decrease overall treatment costs by focusing on genetics and “-omics” data obtained from patient biospecimens and records to guide therapy choices that generate good clinical outcomes. The approach relies on diagnostic and prognostic use of novel biomarkers discovered through combinations of tissue banking, bioinformatics, and electronic medical records (EMRs). The analytical power of bioinformatic platforms combined with patient clinical data from EMRs can reveal potential biomarkers and clinical phenotypes that allow researchers to develop experimental strategies using selected patient biospecimens stored in tissue banks. For cancer, high-quality biospecimens collected at diagnosis, first relapse, and various treatment stages provide crucial resources for study designs. To enlarge biospecimen collections, patient education regarding the value of specimen donation is vital. One approach for increasing consent is to offer publically available illustrations and game-like engagements demonstrating how wider sample availability facilitates development of novel therapies. The critical value of tissue bank samples, bioinformatics, and EMR in the early stages of the biomarker discovery process for personalized medicine is often overlooked. The data obtained also require cross-disciplinary collaborations to translate experimental results into clinical practice and diagnostic and prognostic use in personalized medicine. PMID:23818899

Cardiac effects of magnesium sulfate pretreatment on acute dichlorvos-induced organophosphate poisoning: an experimental study in rats.

PubMed

Gunay, Nurullah; Kekec, Zeynep; Demiryurek, Seniz; Kose, Ataman; Namiduru, Emine S; Gunay, Nahide E; Sari, Ibrahim; Demiryurek, Abdullah T

2010-02-01

Although atropine and oximes are traditionally used in the management of organophosphate poisoning, investigations have been directed to finding additional therapeutic approaches. Thus, the aim of this study was to evaluate the cardiac effects of magnesium sulfate pretreatment on dichlorvos intoxication in rats. Rats were randomly divided into three groups as control, dichlorvos, and magnesium sulfate groups. After 6 h of dichlorvos or corn oil (as a vehicle) injection, venous blood samples were collected, and cardiac tissue samples were obtained. Biochemical analyses were performed to measure some parameters on serum and cardiac tissue. Immunohistochemical analyses of apoptosis and inducible nitric oxide (NO) synthase showed no change in cardiac tissue. Serum cholinesterase levels were markedly depressed with dichlorvos, and further suppressed markedly with magnesium sulfate pretreatment. Although we have demonstrated that serum NO levels in dichlorvos and magnesium sulfate groups were lower than the control group, cardiac tissue NO levels in magnesium sulfate group were higher than the other two groups. Mortality was not significantly affected with magnesium sulfate pretreatment. Uncertainty still persists on the right strategies for the treatment of organophosphate acute poisoning; however, it was concluded that our results do not suggest that magnesium sulfate therapy is beneficial in the management of acute dichlorvos-induced organophosphate poisoning, and also further studies are required.

High axial resolution imaging system for large volume tissues using combination of inclined selective plane illumination and mechanical sectioning

PubMed Central

Zhang, Qi; Yang, Xiong; Hu, Qinglei; Bai, Ke; Yin, Fangfang; Li, Ning; Gang, Yadong; Wang, Xiaojun; Zeng, Shaoqun

2017-01-01

To resolve fine structures of biological systems like neurons, it is required to realize microscopic imaging with sufficient spatial resolution in three dimensional systems. With regular optical imaging systems, high lateral resolution is accessible while high axial resolution is hard to achieve in a large volume. We introduce an imaging system for high 3D resolution fluorescence imaging of large volume tissues. Selective plane illumination was adopted to provide high axial resolution. A scientific CMOS working in sub-array mode kept the imaging area in the sample surface, which restrained the adverse effect of aberrations caused by inclined illumination. Plastic embedding and precise mechanical sectioning extended the axial range and eliminated distortion during the whole imaging process. The combination of these techniques enabled 3D high resolution imaging of large tissues. Fluorescent bead imaging showed resolutions of 0.59 μm, 0.47μm, and 0.59 μm in the x, y, and z directions, respectively. Data acquired from the volume sample of brain tissue demonstrated the applicability of this imaging system. Imaging of different depths showed uniform performance where details could be recognized in either the near-soma area or terminal area, and fine structures of neurons could be seen in both the xy and xz sections. PMID:29296503

Validation of microbiological testing in cardiovascular tissue banks: results of a quality round trial.

PubMed

de By, Theo M M H; McDonald, Carl; Süßner, Susanne; Davies, Jill; Heng, Wee Ling; Jashari, Ramadan; Bogers, Ad J J C; Petit, Pieter

2017-11-01

Surgeons needing human cardiovascular tissue for implantation in their patients are confronted with cardiovascular tissue banks that use different methods to identify and decontaminate micro-organisms. To elucidate these differences, we compared the quality of processing methods in 20 tissue banks and 1 reference laboratory. We did this to validate the results for accepting or rejecting tissue. We included the decontamination methods used and the influence of antibiotic cocktails and residues with results and controls. The minor details of the processes were not included. To compare the outcomes of microbiological testing and decontamination methods of heart valve allografts in cardiovascular tissue banks, an international quality round was organized. Twenty cardiovascular tissue banks participated in this quality round. The quality round method was validated first and consisted of sending purposely contaminated human heart valve tissue samples with known micro-organisms to the participants. The participants identified the micro-organisms using their local decontamination methods. Seventeen of the 20 participants correctly identified the micro-organisms; if these samples were heart valves to be released for implantation, 3 of the 20 participants would have decided to accept their result for release. Decontamination was shown not to be effective in 13 tissue banks because of growth of the organisms after decontamination. Articles in the literature revealed that antibiotics are effective at 36°C and not, or less so, at 2-8°C. The decontamination procedure, if it is validated, will ensure that the tissue contains no known micro-organisms. This study demonstrates that the quality round method of sending contaminated tissues and assessing the results of the microbiological cultures is an effective way of validating the processes of tissue banks. Only when harmonization, based on validated methods, has been achieved, will surgeons be able to fully rely on the methods used and have confidence in the consistent sterility of the tissue grafts. Tissue banks should validate their methods so that all stakeholders can trust the outcomes. © The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

Combined Falling Drop/Open Port Sampling Interface System for Automated Flow Injection Mass Spectrometry

DOE PAGES

Van Berkel, Gary J.; Kertesz, Vilmos; Orcutt, Matt; ...

2017-11-07

The aim of this work was to demonstrate and to evaluate the analytical performance of a combined falling drop/open port sampling interface (OPSI) system as a simple noncontact, no-carryover, automated system for flow injection analysis with mass spectrometry. The falling sample drops were introduced into the OPSI using a widely available autosampler platform utilizing low cost disposable pipet tips and conventional disposable microtiter well plates. The volume of the drops that fell onto the OPSI was in the 7–15 μL range with an injected sample volume of several hundred nanoliters. Sample drop height, positioning of the internal capillary on themore » sampling end of the probe, and carrier solvent flow rate were optimized for maximum signal. Sample throughput, signal reproducibility, matrix effects, and quantitative analysis capability of the system were established using the drug molecule propranolol and its isotope labeled internal standard in water, unprocessed river water and two commercially available buffer matrices. A sample-to-sample throughput of ~45 s with a ~4.5 s base-to-base flow injection peak profile was obtained in these experiments. In addition, quantitation with minimally processed rat plasma samples was demonstrated with three different statin drugs (atorvastatin, rosuvastatin, and fluvastatin). Direct characterization capability of unprocessed samples was demonstrated by the analysis of neat vegetable oils. Employing the autosampler system for spatially resolved liquid extraction surface sampling exemplified by the analysis of propranolol and its hydroxypropranolol glucuronide phase II metabolites from a rat thin tissue section was also illustrated.« less

Combined Falling Drop/Open Port Sampling Interface System for Automated Flow Injection Mass Spectrometry

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

Van Berkel, Gary J.; Kertesz, Vilmos; Orcutt, Matt

The aim of this work was to demonstrate and to evaluate the analytical performance of a combined falling drop/open port sampling interface (OPSI) system as a simple noncontact, no-carryover, automated system for flow injection analysis with mass spectrometry. The falling sample drops were introduced into the OPSI using a widely available autosampler platform utilizing low cost disposable pipet tips and conventional disposable microtiter well plates. The volume of the drops that fell onto the OPSI was in the 7–15 μL range with an injected sample volume of several hundred nanoliters. Sample drop height, positioning of the internal capillary on themore » sampling end of the probe, and carrier solvent flow rate were optimized for maximum signal. Sample throughput, signal reproducibility, matrix effects, and quantitative analysis capability of the system were established using the drug molecule propranolol and its isotope labeled internal standard in water, unprocessed river water and two commercially available buffer matrices. A sample-to-sample throughput of ~45 s with a ~4.5 s base-to-base flow injection peak profile was obtained in these experiments. In addition, quantitation with minimally processed rat plasma samples was demonstrated with three different statin drugs (atorvastatin, rosuvastatin, and fluvastatin). Direct characterization capability of unprocessed samples was demonstrated by the analysis of neat vegetable oils. Employing the autosampler system for spatially resolved liquid extraction surface sampling exemplified by the analysis of propranolol and its hydroxypropranolol glucuronide phase II metabolites from a rat thin tissue section was also illustrated.« less

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.

Quantitative Analysis of Microbicide Concentrations in Fluids, Gels and Tissues Using Confocal Raman Spectroscopy

PubMed Central

Chuchuen, Oranat; Henderson, Marcus H.; Sykes, Craig; Kim, Min Sung; Kashuba, Angela D. M.; Katz, David F.

2013-01-01

Topical vaginal anti-HIV microbicides are an important focus in female-based strategies to prevent the sexual transmission of HIV. Understanding microbicide pharmacokinetics is essential to development, characterization and implementation of efficacious microbicide drug delivery formulations. Current methods to measure drug concentrations in tissue (e.g., LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry) are highly sensitive, but destructive and complex. This project explored the use of confocal Raman spectroscopy to detect microbicide drugs and to measure their local concentrations in fluids, drug delivery gels, and tissues. We evaluated three candidate microbicide drugs: tenofovir, Dapivirine and IQP-0528. Measurements were performed in freshly excised porcine buccal tissue specimens, gel vehicles and fluids using two Horiba Raman microscopes, one of which is confocal. Characteristic spectral peak calibrations for each drug were obtained using serial dilutions in the three matrices. These specific Raman bands demonstrated strong linear concentration dependences in the matrices and were characterized with respect to their unique vibrational signatures. At least one specific Raman feature was identified for each drug as a marker band for detection in tissue. Sensitivity of detection was evaluated in the three matrices. A specific peak was also identified for tenofovir diphosphate, the anti-HIV bioactive product of tenofovir after phosphorylation in host cells. Z-scans of drug concentrations vs. depth in excised tissue specimens, incubated under layers of tenofovir solution in a Transwell assay, showed decreasing concentration with depth from the surface into the tissue. Time-dependent concentration profiles were obtained from tissue samples incubated in the Transwell assay, for times ranging 30 minutes - 6 hours. Calibrations and measurements from tissue permeation studies for tenofovir showed good correlation with gold standard LC-MS/MS data. These results demonstrate that confocal Raman spectroscopy holds promise as a tool for practical, minimally invasive, label-free measurement of microbicide drug concentrations in fluids, gels and tissues. PMID:24386455

Synthesis of electroactive tetraaniline grafted polyethylenimine for tissue engineering

NASA Astrophysics Data System (ADS)

Dong, Shilei; Han, Lu; Cai, Muhang; Li, Luhai; Wei, Yan

2015-07-01

Tetraaniline grafted polyethylenimine (AT-PEI) was successfully synthesized in this study. Proton Nuclear Magnetic Resonance (1H NMR) Spectroscopy was used to determine the structure of carboxyl-capped aniline tetramer (AT-COOH) and AT-PEI. UV-Vis spectroscopy and Fourier transform infrared (FT-IR) spectroscopy were employed to characterize the absorption spectrum of the obtained AT-PEI samples. The morphology of AT-PEI copolymers in aqueous solution was determined by Scanning electron microscope (SEM). Moreover, AT-PEI copolymers demonstrated excellent solubility in aqueous solution and possessed electroactivity by cyclic voltammogram (CV) curves, which showed its potential application in the field of tissue engineering.

Multiparameter thermo-mechanical OCT-based characterization of laser-induced cornea reshaping

NASA Astrophysics Data System (ADS)

Zaitsev, Vladimir Yu.; Matveyev, Alexandr L.; Matveev, Lev A.; Gelikonov, Grigory V.; Vitkin, Alex; Omelchenko, Alexander I.; Baum, Olga I.; Shabanov, Dmitry V.; Sovetsky, Alexander A.; Sobol, Emil N.

2017-02-01

Phase-sensitive optical coherence tomography (OCT) is used for visualizing dynamic and cumulative strains and corneashape changes during laser-produced tissue heating. Such non-destructive (non-ablative) cornea reshaping can be used as a basis of emerging technologies of laser vision correction. In experiments with cartilaginous samples, polyacrilamide phantoms and excised rabbit eyes we demonstrate ability of the developed OCT system to simultaneously characterize transient and cumulated strain distributions, surface displacements, scattering tissue properties and possibility of temperature estimation via thermal-expansion measurements. The proposed approach can be implemented in perspective real-time OCT systems for ensuring safety of new methods of laser reshaping of cornea.

Report of the clinical donor case workshop of the European Association of Tissue Banks annual meeting 2012.

PubMed

Beele, Hilde; van Wijk, Marja J; Parker, Robert; Sánchez-Ibáňez, Jacinto; Brubaker, Scott A; Wulff, Birgit; Richters, Cornelia D; Cox, Mike; Warwick, Ruth M; Eastlund, Ted

2013-12-01

The European Association of Tissue Banks (EATB) donor case workshop is a forum held within the program of the EATB annual congress. The workshop offers an opportunity to discuss and evaluate approaches taken to challenging situations regarding donor selection, it promotes consensus development in deciding tissue donor acceptability when donor health issues are not addressed in standards and regulations, and serves to strengthen the professional tissue banking networks across Europe and beyond. This report reflects some of the discussion at the workshop during the annual congress in Vienna in 2012. The cases presented dealt with problems encountered by tissue bank facilities concerning idiopathic thrombocytopenia and auto-immune disorders, hemodilution and blood sample identification, premalignant and malignant lesions, and Huntington's disease. The discussions during the workshop demonstrate that the implications on the safety of tissue transplantation of various tissue donor illnesses, physical findings and behaviours, and the preventive measures taken by tissue facilities, may not always be agreed by tissue facility medical directors and other professionals. Moreover, they reveal that operating procedures, regulations and standards cannot comprehensively cover all tissue donor findings, medical histories and circumstances surrounding the cause of death. For many of the issues raised, there is a need for scientific research to provide a better evidence base for future deliberations about the suitability and eligibility of tissue allograft donors.

Essential tactics of tissue preparation and matrix nano-spotting for successful compound imaging mass spectrometry.

PubMed

Végvári, Akos; Fehniger, Thomas E; Gustavsson, Lena; Nilsson, Anna; Andrén, Per E; Kenne, Kerstin; Nilsson, Johan; Laurell, Thomas; Marko-Varga, György

2010-04-18

The ultimate goal of MALDI-Imaging Mass Spectrometry (MALDI-IMS) is to achieve spatial localization of analytes in tissue sections down to individual tissue compartments or even at the level of a few cells. With compound tissue imaging, it is possible to track the transportation of an unlabelled, inhaled reference compound within lung tissue, through the application of MALDI-IMS. The procedure for isolation and preparation of lung tissues is found to be crucial in order to preserve the anatomy and structure of the pulmonary compartments. To avoid delocalization of analytes within lung tissue compartments we have applied an in-house designed nano-spotter, based on a microdispenser mounted on an XY table, of which movement and spotting functionality were fully computer controlled. We demonstrate the usefulness of this platform in lung tissue sections isolated from rodent in vivo model, applied to compound tissue imaging as exemplified with the determination of the spatial distribution of (1alpha,2beta,4beta,7beta)-7-[(hydroxidi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatricyclo[3.3.1.0(2,4)]nonane, also known as tiotropium. We provide details on tissue preparation protocols and sample spotting technology for successful identification of drug in mouse lung tissue by using MALDI-Orbitrap instrumentation. Copyright 2010 Elsevier B.V. All rights reserved.

rpoB gene mutations among Mycobacterium tuberculosis isolates from extrapulmonary sites.

PubMed

Khosravi, Azar Dokht; Meghdadi, Hossein; Ghadiri, Ata A; Alami, Ameneh; Sina, Amir Hossein; Mirsaeidi, Mehdi

2018-03-01

The aim of this study was to analyze mutations occurring in the rpoB gene of Mycobacterium tuberculosis (MTB) isolates from clinical samples of extrapulmonary tuberculosis (EPTB). Seventy formalin-fixed, paraffin-embedded samples and fresh tissue samples from confirmed EPTB cases were analyzed. Nested PCR based on the rpoB gene was performed on the extracted DNAs, combined with cloning and subsequent sequencing. Sixty-seven (95.7%) samples were positive for nester PCR. Sequence analysis of the 81 bp region of the rpoB gene demonstrated mutations in 41 (61.2%) of 67 sequenced samples. Several point mutations including deletion mutations at codons 510, 512, 513 and 515, with 45% and 51% of the mutations in codons 512 and 513 respectively were seen, along with 26% replacement mutations at codons 509, 513, 514, 518, 520, 524 and 531. The most common alteration was Gln → His, at codon 513, presented in 30 (75.6%) isolates. This study demonstrated sequence alterations in codon 513 of the 81 bp region of the rpoB gene as the most common mutation occurred in 75.6% of molecularly confirmed rifampin-resistant strains. In addition, simultaneous mutation at codons 512 and 513 was demonstrated in 34.3% of the isolates. © 2018 APMIS. Published by John Wiley & Sons Ltd.

Dendritic cells provide a potential link between smoking and inflammation in rheumatoid arthritis

PubMed Central

2012-01-01

Introduction Smoking increases the risk of developing rheumatoid arthritis (RA) and affects the severity of established RA. Smoking can impact on Th17 lymphocyte differentiation and function through activation of the aryl hydrocarbon receptor (AHR), a process with implications for the pathogenic mechanisms in RA that involve the cytokine, interleukin (IL)-17A. The objective of this study was to establish any effect of smoking on the inflammatory tissue lesions of rheumatoid arthritis via the AHR and IL-17A. Methods Twenty synovial and eighteen subcutaneous nodule tissue samples from 31 patients with RA were studied. Patient smoking status at the time of tissue collection was established. Expression of AHR, CYP1A1, AHRR, IL6, IL17A, IL17F, IL22, IL23, IL23R, IFNG, TBX21, IDO1 and FOXP3 genes were assessed in tissues and cultured cells using real-time PCR. Two-colour immunofluorescence was used to co-localise AHR and CYP1A1 protein in synovial tissues. The response of monocytes and monocyte-derived dendritic cells (mo-DCs) to the AHR agonist, benzo(a)pyrene (BaP) was compared in vitro. Results AHR gene expression was demonstrated in rheumatoid synovial tissues and nodules with significantly greater expression in synovia. Expression was not influenced by smoking in either tissue. Evidence of AHR activation, indicated by CYP1A1 and AHRR gene expression, was found only in synovia from patients who smoked. However, IL17A gene expression was lower in synovia from smokers. TBX21 and FOXP3 expression was not affected by smoking. Within the synovial tissues of smokers the principal cell type with evidence of AHR activation was a subset of synovial DCs. This observation was consistent with the sensitivity of human mo-DCs to BaP stimulation demonstrated in vitro. Exposure to BaP affected mo-DC function as demonstrated by decreased IL6 expression induced by PolyI:C, without affecting indoleamine 2,3 dioxygenase (IDO)1 expression. Conclusion Our findings show that one effect of smoking on inflamed rheumatoid synovial tissue involves activation of the AHR pathway. A subset of synovial DCs is important in the response to cigarette smoke. The potential for smoking to affect DC behaviour in joint tissues has relevance to both early and late phases of RA pathogenesis and warrants further investigation. PMID:23036591

Dehomogenized Elastic Properties of Heterogeneous Layered Materials in AFM Indentation Experiments.

PubMed

Lee, Jia-Jye; Rao, Satish; Kaushik, Gaurav; Azeloglu, Evren U; Costa, Kevin D

2018-06-05

Atomic force microscopy (AFM) is used to study mechanical properties of biological materials at submicron length scales. However, such samples are often structurally heterogeneous even at the local level, with different regions having distinct mechanical properties. Physical or chemical disruption can isolate individual structural elements but may alter the properties being measured. Therefore, to determine the micromechanical properties of intact heterogeneous multilayered samples indented by AFM, we propose the Hybrid Eshelby Decomposition (HED) analysis, which combines a modified homogenization theory and finite element modeling to extract layer-specific elastic moduli of composite structures from single indentations, utilizing knowledge of the component distribution to achieve solution uniqueness. Using finite element model-simulated indentation of layered samples with micron-scale thickness dimensions, biologically relevant elastic properties for incompressible soft tissues, and layer-specific heterogeneity of an order of magnitude or less, HED analysis recovered the prescribed modulus values typically within 10% error. Experimental validation using bilayer spin-coated polydimethylsiloxane samples also yielded self-consistent layer-specific modulus values whether arranged as stiff layer on soft substrate or soft layer on stiff substrate. We further examined a biophysical application by characterizing layer-specific microelastic properties of full-thickness mouse aortic wall tissue, demonstrating that the HED-extracted modulus of the tunica media was more than fivefold stiffer than the intima and not significantly different from direct indentation of exposed media tissue. Our results show that the elastic properties of surface and subsurface layers of microscale synthetic and biological samples can be simultaneously extracted from the composite material response to AFM indentation. HED analysis offers a robust approach to studying regional micromechanics of heterogeneous multilayered samples without destructively separating individual components before testing. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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.

Monitoring Dopamine ex Vivo during Electrical Stimulation Using Liquid-Microjunction Surface Sampling.

PubMed

Gill, Emily L; Marks, Megan; Yost, Richard A; Vedam-Mai, Vinata; Garrett, Timothy J

2017-12-19

Liquid-microjunction surface sampling (LMJ-SS) is an ambient ionization technique based on the continuous flow of solvent using an in situ microextraction device in which solvent moves through the probe, drawing in the analytes in preparation for ionization using an electrospray ionization source. However, unlike traditional mass spectrometry (MS) techniques, it operates under ambient pressure and requires no sample preparation, thereby making it ideal for rapid sampling of thicker tissue sections for electrophysiological and other neuroscientific research studies. Studies interrogating neural synapses, or a specific neural circuit, typically employ thick, ex vivo tissue sections maintained under near-physiological conditions to preserve tissue viability and maintain the neural networks. Deep brain stimulation (DBS) is a surgical procedure used to treat the neurological symptoms that are associated with certain neurodegenerative and neuropsychiatric diseases. Parkinson's disease (PD) is a neurological disorder which is commonly treated with DBS therapy. PD is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta portion of the brain. Here, we demonstrate that the LMJ-SS methodology can provide a platform for ex vivo analysis of the brain during electrical stimulation, such as DBS. We employ LMJ-SS in the ex vivo analysis of mouse brain tissue for monitoring dopamine during electrical stimulation of the striatum region. The mouse brain tissue was sectioned fresh post sacrifice and maintained in artificial cerebrospinal fluid to create near-physiological conditions before direct sampling using LMJ-SS. A selection of metabolites, including time-sensitive metabolites involved in energy regulation in the brain, were identified using standards, and the mass spectral database mzCloud was used to assess the feasibility of the methodology. Thereafter, the intensity of m/z 154 corresponding to protonated dopamine was monitored before and after electrical stimulation of the striatum region, showing an increase in signal directly following a stimulation event. Dopamine is the key neurotransmitter implicated in PD, and although electrochemical detectors have shown such increases in dopamine post-DBS, this is the first study to do so using MS methodologies.

Detection of Chronic Wasting Disease Prions in Salivary, Urinary, and Intestinal Tissues of Deer: Potential Mechanisms of Prion Shedding and Transmission▿

PubMed Central

Haley, Nicholas J.; Mathiason, Candace K.; Carver, Scott; Zabel, Mark; Telling, Glenn C.; Hoover, Edward A.

2011-01-01

Efficient horizontal transmission is a signature trait of chronic wasting disease (CWD) in cervids. Infectious prions shed into excreta appear to play a key role in this facile transmission, as has been demonstrated by bioassays of cervid and transgenic species and serial protein misfolding cyclic amplification (sPMCA). However, the source(s) of infectious prions in these body fluids has yet to be identified. In the present study, we analyzed tissues proximate to saliva, urine, and fecal production by sPMCA in an attempt to elucidate this unique aspect of CWD pathogenesis. Oropharyngeal, urogenital, and gastrointestinal tissues along with blood and obex from CWD-exposed cervids (comprising 27 animals and >350 individual samples) were analyzed and scored based on the apparent relative CWD burden. PrPCWD-generating activity was detected in a range of tissues and was highest in the salivary gland, urinary bladder, and distal intestinal tract. In the same assays, blood from the same animals and unseeded normal brain homogenate controls (n = 116 of 117) remained negative. The PrP-converting activity in peripheral tissues varied from 10−11- to 100-fold of that found in brain of the same animal. Deer with highest levels of PrPCWD amplification in the brain had higher and more widely disseminated prion amplification in excretory tissues. Interestingly, PrPCWD was not demonstrable in these excretory tissues by conventional Western blotting, suggesting a low prion burden or the presence of protease-sensitive infectious prions destroyed by harsh proteolytic treatments. These findings offer unique insights into the transmission of CWD in particular and prion infection and trafficking overall. PMID:21525361

Marsh wrens as bioindicators of mercury in wetlands of Great Salt Lake: do blood and feathers reflect site-specific exposure risk to bird reproduction?

USGS Publications Warehouse

Hartman, C. Alex; Ackerman, Joshua T.; Herring, Garth; Isanhart, John; Herzog, Mark P.

2013-01-01

Nonlethal sampling of bird blood and feathers are among the more common ways of estimating the risk of mercury exposure to songbird reproduction. The implicit assumption is that mercury concentrations in blood or feathers of individuals captured in a given area are correlated with mercury concentrations in eggs from the same area. Yet, this assumption is rarely tested. We evaluated mercury concentrations in blood, feathers, and eggs of marsh wrens in wetlands of Great Salt Lake, Utah, and, at two spatial scales, specifically tested the assumption that mercury concentrations in blood and feather samples from birds captured in a defined area were predictive of mercury concentrations in eggs collected in the same area. Mercury concentrations in blood were not correlated with mercury concentrations in eggs collected within the same wetland unit, and were poorly correlated with mercury concentrations in eggs collected at the smaller home range spatial scale of analysis. Moreover, mercury exposure risk, as estimated via tissue concentrations, differed among wetland units depending upon whether blood or egg mercury concentrations were sampled. Mercury concentrations in feathers also were uncorrelated with mercury concentrations in eggs, and were poorly correlated with mercury concentrations in blood. These results demonstrate the potential for contrasting management actions that may be implemented based solely on the specific avian tissue that is sampled, and highlight the importance of developing avian tissues as biomonitoring tools for assessing local risk of mercury exposure to bird reproduction.

Coordinated release of acylation stimulating protein (ASP) and triacylglycerol clearance by human adipose tissue in vivo in the postprandial period.

PubMed

Saleh, J; Summers, L K; Cianflone, K; Fielding, B A; Sniderman, A D; Frayn, K N

1998-04-01

The objective of this study was to determine whether Acylation Stimulating Protein (ASP) is generated in vivo by human adipose tissue during the postprandial period. After a fat meal, samples from 12 subjects were obtained (up to 6 h) from an arterialized hand vein and an anterior abdominal wall vein that drains adipose tissue. Veno-arterial (V-A) gradients across the subcutaneous adipose tissue bed were calculated. The data demonstrate that ASP is produced in vivo (positive V-A gradient) With maximal production at 3-5 h postprandially. The plasma triacylglycerol (TAG) clearance was evidenced by a negative V-A gradient. It increased substantially after 3 h and remained prominent until the final time point. There was, therefore, a close temporal coordination between ASP generation and TAG clearance. In contrast, plasma insulin and non-esterified fatty acid (NEFA) had an early (1-2 h) postprandial change. Fatty acid incorporation into adipose tissue (FIAT) was calculated from V-A glycerol and non-esterified fatty acid (NEFA) differences postprandially. FIAT was negative during the first hour, implying net fat mobilization. FIAT then became increasingly positive, implying net fat deposition, and overall followed the same time course as ASP and TAG clearance. There was a direct positive correlation between total ASP production and total FIAT (r = 0.566, P < 0.05). These data demonstrate that ASP is generated in vivo by human adipocytes and that this process is accentuated postprandially, supporting the concept that ASP plays an important role in clearance of TAG from plasma and fatty acid storage in adipose tissue.

Non-destructive optical clearing technique enhances optical coherence tomography (OCT) for real-time, 3D histomorphometry of brain tissue (Conference Presentation)

NASA Astrophysics Data System (ADS)

Paul, Akshay; Chang, Theodore H.; Chou, Li-Dek; Ramalingam, Tirunelveli S.

2016-03-01

Evaluation of neurodegenerative disease often requires examination of brain morphology. Volumetric analysis of brain regions and structures can be used to track developmental changes, progression of disease, and the presence of transgenic phenotypes. Current standards for microscopic investigation of brain morphology are limited to detection of superficial structures at a maximum depth of 300μm. While histological techniques can provide detailed cross-sections of brain structures, they require complicated tissue preparation and the ultimate destruction of the sample. A non-invasive, label-free imaging modality known as Optical Coherence Tomography (OCT) can produce 3-dimensional reconstructions through high-speed, cross-sectional scans of biological tissue. Although OCT allows for the preservation of intact samples, the highly scattering and absorbing properties of biological tissue limit imaging depth to 1-2mm. Optical clearing agents have been utilized to increase imaging depth by index matching and lipid digestion, however, these contemporary techniques are expensive and harsh on tissues, often irreversibly denaturing proteins. Here we present an ideal optical clearing agent that offers ease-of-use and reversibility. Similar to how SeeDB has been effective for microscopy, our fructose-based, reversible optical clearing technique provides improved OCT imaging and functional immunohistochemical mapping of disease. Fructose is a natural, non-toxic sugar with excellent water solubility, capable of increasing tissue transparency and reducing light scattering. We will demonstrate the improved depth-resolving performance of OCT for enhanced whole-brain imaging of normal and diseased murine brains following a fructose clearing treatment. This technique potentially enables rapid, 3-dimensional evaluation of biological tissues at axial and lateral resolutions comparable to histopathology.

Existence of life-time stable proteins in mature rats—Dating of proteins’ age by repeated short-term exposure to labeled amino acids throughout age

PubMed Central

Schjerling, Peter; Bornø, Andreas; Holm, Lars

2017-01-01

In vivo turnover rates of proteins covering the processes of protein synthesis and breakdown rates have been measured in many tissues and protein pools using various techniques. Connective tissue and collagen protein turnover is of specific interest since existing results are rather diverging. The aim of this study is to investigate whether we can verify the presence of protein pools within the same tissue with very distinct turnover rates over the life-span of rats with special focus on connective tissue. Male and female Lewis rats (n = 35) were injected with five different isotopically labeled amino acids tracers. The tracers were injected during fetal development (Day -10 to -2), after birth (Day 5–9), at weaning (Day 25–32) at puberty (Day 54–58) and at adulthood (Day 447–445). Subgroups of rats were euthanized three days after every injection period, at different time point between injection periods and lastly at day 472. Tissue (liver, muscle, eye lens and patellar tendon) and blood samples were collected after euthanization. The enrichment of the labeled amino acids in the tissue or blood samples was measured using GC-MS-MS. In muscle and liver we demonstrated a rapid decrease of tracer enrichments throughout the rat’s life, indicating that myofibrillar and cytoskeleton proteins have a high turnover. In contrast, the connective tissue protein in the eye lens and patellar tendon of the mature rat showed detainment of tracer enrichment injected during fetal development and first living days, indicating very slow turnover. The data support the hypothesis that some proteins synthesized during the early development and growth still exist much later in life of animals and hence has a very slow turnover rate. PMID:28957442

Normal breast tissue DNA methylation differences at regulatory elements are associated with the cancer risk factor age.

PubMed

Johnson, Kevin C; Houseman, E Andres; King, Jessica E; Christensen, Brock C

2017-07-10

The underlying biological mechanisms through which epidemiologically defined breast cancer risk factors contribute to disease risk remain poorly understood. Identification of the molecular changes associated with cancer risk factors in normal tissues may aid in determining the earliest events of carcinogenesis and informing cancer prevention strategies. Here we investigated the impact cancer risk factors have on the normal breast epigenome by analyzing DNA methylation genome-wide (Infinium 450 K array) in cancer-free women from the Susan G. Komen Tissue Bank (n = 100). We tested the relation of established breast cancer risk factors, age, body mass index, parity, and family history of disease, with DNA methylation adjusting for potential variation in cell-type proportions. We identified 787 cytosine-guanine dinucleotide (CpG) sites that demonstrated significant associations (Q value <0.01) with subject age. Notably, DNA methylation was not strongly associated with the other evaluated breast cancer risk factors. Age-related DNA methylation changes are primarily increases in methylation enriched at breast epithelial cell enhancer regions (P = 7.1E-20), and binding sites of chromatin remodelers (MYC and CTCF). We validated the age-related associations in two independent populations, using normal breast tissue samples (n = 18) and samples of normal tissue adjacent to tumor tissue (n = 97). The genomic regions classified as age-related were more likely to be regions altered in both pre-invasive (n = 40, P = 3.0E-03) and invasive breast tumors (n = 731, P = 1.1E-13). DNA methylation changes with age occur at regulatory regions, and are further exacerbated in cancer, suggesting that age influences breast cancer risk in part through its contribution to epigenetic dysregulation in normal breast tissue.

Rapid In Situ Profiling of Lipid C═C Location Isomers in Tissue Using Ambient Mass Spectrometry with Photochemical Reactions.

PubMed

Tang, Fei; Guo, Chengan; Ma, Xiaoxiao; Zhang, Jian; Su, Yuan; Tian, Ran; Shi, Riyi; Xia, Yu; Wang, Xiaohao; Ouyang, Zheng

2018-05-01

Rapid and in situ profiling of lipids using ambient mass spectrometry (AMS) techniques has great potential for clinical diagnosis, biological studies, and biomarker discovery. In this study, the online photochemical reaction involving carbon-carbon double bonds was coupled with a surface sampling technique to develop a direct tissue-analysis method with specificity to lipid C═C isomers. This method enabled the in situ analysis of lipids from the surface of various tissues or tissue sections, which allowed the structural characterization of lipid isomers within 2 min. Under optimized reaction conditions, we have established a method for the relative quantitation of lipid C═C location isomers by comparing the abundances of the diagnostic ions arising from each isomer, which has been proven effective through the established linear relationship ( R 2 = 0.999) between molar ratio and diagnostic ion ratio of the FA 18:1 C═C location isomers. This method was then used for the rapid profiling of unsaturated lipid C═C isomers in the sections of rat brain, lung, liver, spleen, and kidney, as well as in normal and diseased rat tissues. Quantitative information on FA 18:1 and PC 16:0-18:1 C═C isomers was obtained, and significant differences were observed between different samples. To the best of our knowledge, this is the first study to report the direct analysis of lipid C═C isomers in tissues using AMS. Our results demonstrated that this method can serve as a rapid analytical approach for the profiling of unsaturated lipid C═C isomers in biological tissues and should contribute to functional lipidomics and clinical diagnosis.

Transcriptional Profiling of Synovial Macrophages Using Minimally Invasive Ultrasound-Guided Synovial Biopsies in Rheumatoid Arthritis.

PubMed

Mandelin, Arthur M; Homan, Philip J; Shaffer, Alexander M; Cuda, Carla M; Dominguez, Salina T; Bacalao, Emily; Carns, Mary; Hinchcliff, Monique; Lee, Jungwha; Aren, Kathleen; Thakrar, Anjali; Montgomery, Anna B; Bridges, S Louis; Bathon, Joan M; Atkinson, John P; Fox, David A; Matteson, Eric L; Buckley, Christopher D; Pitzalis, Costantino; Parks, Deborah; Hughes, Laura B; Geraldino-Pardilla, Laura; Ike, Robert; Phillips, Kristine; Wright, Kerry; Filer, Andrew; Kelly, Stephen; Ruderman, Eric M; Morgan, Vince; Abdala-Valencia, Hiam; Misharin, Alexander V; Budinger, G Scott; Bartom, Elizabeth T; Pope, Richard M; Perlman, Harris; Winter, Deborah R

2018-06-01

Currently, there are no reliable biomarkers for predicting therapeutic response in patients with rheumatoid arthritis (RA). The synovium may unlock critical information for determining efficacy, since a reduction in the numbers of sublining synovial macrophages remains the most reproducible biomarker. Thus, a clinically actionable method for the collection of synovial tissue, which can be analyzed using high-throughput strategies, must become a reality. This study was undertaken to assess the feasibility of utilizing synovial biopsies as a precision medicine-based approach for patients with RA. Rheumatologists at 6 US academic sites were trained in minimally invasive ultrasound-guided synovial tissue biopsy. Biopsy specimens obtained from patients with RA and synovial tissue from patients with osteoarthritis (OA) were subjected to histologic analysis, fluorescence-activated cell sorting, and RNA sequencing (RNA-seq). An optimized protocol for digesting synovial tissue was developed to generate high-quality RNA-seq libraries from isolated macrophage populations. Associations were determined between macrophage transcriptional profiles and clinical parameters in RA patients. Patients with RA reported minimal adverse effects in response to synovial biopsy. Comparable RNA quality was observed from synovial tissue and isolated macrophages between patients with RA and patients with OA. Whole tissue samples from patients with RA demonstrated a high degree of transcriptional heterogeneity. In contrast, the transcriptional profile of isolated RA synovial macrophages highlighted different subpopulations of patients and identified 6 novel transcriptional modules that were associated with disease activity and therapy. Performance of synovial tissue biopsies by rheumatologists in the US is feasible and generates high-quality samples for research. Through the use of cutting-edge technologies to analyze synovial biopsy specimens in conjunction with corresponding clinical information, a precision medicine-based approach for patients with RA is attainable. © 2018, American College of Rheumatology.

Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

PubMed Central

Aboushousha, Tarek; Mamdouh, Samah; Hamdy, Hussam; Helal, Noha; Khorshed, Fatma; Safwat, Gehan; Seleem, Mohamed

2018-01-01

Objective: To investigate the expression of TTF-1, RAGE, GLUT1 and SOX2 in HCV-associated HCCs and in surrounding non-tumorous liver tissue. Material and Methods: Tissue material from partial hepatectomy cases for HCC along with corresponding serum samples and 30 control serum samples from healthy volunteers were studied. Biopsies were classified into: non-tumor hepatic tissue (36 sections); HCC (33 sections) and liver cell dysplasia (LCD) (15 sections). All cases were positive for HCV. Immunohistochemistry (IHC), gene extraction and quantitative real-time reverse-transcription assays (qRT-PCR) were applied. Results: By IHC, LCD and HCC showed significantly high percentages of positive cases with all markers. SOX2 showed significant increase with higher HCC grades, while RAGE demonstrated an inverse relation and GLUT-1 and TTF-1 lacked any correlation. In nontumorous-HCV tissue, we found significantly high TTF-1, low RAGE and negative SOX2 expression. RAGE, GLUT-1 and SOX2 show non-significant elevation positivity in high grade HCV compared to low grade lesions. TTF-1, RAGE and SOX2 exhibited low expression in cirrhosis compared to fibrosis. Biochemical studies on serum and tissue extracts revealed significant down-regulation of RAGE, GLUT-1 and SOX2 genes, as well as significant up-regulation of the TTF-1 gene in HCC cases compared to controls. All studied genes show significant correlation with HCC grade. In non-tumor tissue, only TTF-1 gene expression had a significant correlation with the fibrosis score. Conclusion: Higher expression of TTF-1, RAGE, GLUT-1 and SOX2 in HCC and dysplasia compared to non-tumor tissues indicates up-regulation of these markers as early events during the development of HCV-associated HCC. PMID:29373917

Immunohistochemical and Biochemical Expression Patterns of TTF-1, RAGE, GLUT-1 and SOX2 in HCV-Associated Hepatocellular Carcinomas

PubMed

Aboushousha, Tarek; Mamdouh, Samah; Hamdy, Hussam; Helal, Noha; Khorshed, Fatma; Safwat, Gehan; Seleem, Mohamed

2018-01-27

Objective: To investigate the expression of TTF-1, RAGE, GLUT1 and SOX2 in HCV-associated HCCs and in surrounding non-tumorous liver tissue. Material and Methods: Tissue material from partial hepatectomy cases for HCC along with corresponding serum samples and 30 control serum samples from healthy volunteers were studied. Biopsies were classified into: non-tumor hepatic tissue (36 sections); HCC (33 sections) and liver cell dysplasia (LCD) (15 sections). All cases were positive for HCV. Immunohistochemistry (IHC), gene extraction and quantitative real-time reverse-transcription assays (qRT-PCR) were applied. Results: By IHC, LCD and HCC showed significantly high percentages of positive cases with all markers. SOX2 showed significant increase with higher HCC grades, while RAGE demonstrated an inverse relation and GLUT-1 and TTF-1 lacked any correlation. In nontumorous-HCV tissue, we found significantly high TTF-1, low RAGE and negative SOX2 expression. RAGE, GLUT-1 and SOX2 show non-significant elevation positivity in high grade HCV compared to low grade lesions. TTF-1, RAGE and SOX2 exhibited low expression in cirrhosis compared to fibrosis. Biochemical studies on serum and tissue extracts revealed significant down-regulation of RAGE, GLUT-1 and SOX2 genes, as well as significant up-regulation of the TTF-1 gene in HCC cases compared to controls. All studied genes show significant correlation with HCC grade. In non-tumor tissue, only TTF-1 gene expression had a significant correlation with the fibrosis score. Conclusion: Higher expression of TTF-1, RAGE, GLUT-1 and SOX2 in HCC and dysplasia compared to non-tumor tissues indicates up-regulation of these markers as early events during the development of HCV-associated HCC. Creative Commons Attribution License

Design and characterization of dexamethasone-loaded poly (glycerol sebacate)-poly caprolactone/gelatin scaffold by coaxial electro spinning for soft tissue engineering.

PubMed

Nadim, Afsaneh; Khorasani, Saied Nouri; Kharaziha, Mahshid; Davoodi, Seyyed Mohammadreza

2017-09-01

The aim of this research was to fabricate dexamethasone (Dex)-loaded poly (glycerol sebacate) (PGS)-poly (caprolactone) (PCL)/gelatin (Gt) (PGS-PCL/Gt-Dex) fibrous scaffolds in the form of core/shell structure which have potential application in soft tissues. In this regard, after synthesize and characterizations of PGS, PGS-PCL and gelatin fibrous scaffolds were separately developed in order to optimize the electrospinning parameters. In the next step, coaxial electrospun fibrous scaffold of PGS-PCL/Gt fibrous scaffold with PGS-PCL as core and Gt as shell was developed and its mechanical, physical and chemical properties were characterized. Moreover, degradability, hydrophilicity and biocompatibility of PGS-PCL/Gt fibrous scaffold were evaluated. In addition, Dex was encapsulated in PGS-PCL/Gt fibrous scaffold and drug release was assessed for tissue engineering application. Results demonstrated the formation of coaxial fibrous scaffold with average porosity of 79% and average fiber size of 294nm. Moreover, PGS-PCL/Gt fibrous scaffold revealed lower elastic modulus, ultimate tensile and ultimate elongation than those of PGS-PCL scaffold and more close to mechanical properties of natural tissue. Furthermore, lower contact angle of PGS-PCL/Gt than that of PGS-PCL demonstrated improved surface hydrophilicity of scaffold. DEX release was sustained over a period time of 30days from the scaffolds via three steps consisting of an initial burst release, secondary linear phase release pattern with slower rate over 20days followed by an apparent zero-order release phase. MTT observations demonstrated that there was no evidence of toxicity in the samples with and without Dex. Our findings indicated that core/shell PGS-PCL/Gt-Dex fibrous could be used as a carrier for the sustained release of drugs relevant for tissue engineering which makes it appropriate for soft tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

Trace-Element Concentrations in Tissues of Aquatic Organisms from Rivers and Streams of the United States, 1992-1999

USGS Publications Warehouse

DeWeese, Lawrence R.; Stephens, Verlin C.; Short, Terry M.; Dubrovsky, Neil M.

2007-01-01

The U.S. Geological Survey National Water-Quality Assessment Program collected tissue samples from a variety of aquatic organisms during 1992-1999 within 47 study units across the United States. These tissue samples were collected to determine the occurrence and distribution of 20 major and minor trace elements in aquatic organisms. This report presents the tissue trace-element concentration data, sample summaries, and concentration statistics for 1,457 tissue samples representing 76 species or groups of fish, aquatic invertebrates, and plants were collected at 824 sampling sites.

Quantitatively characterizing microstructural variations of skin tissues during ultraviolet radiation damaging process based on Mueller matrix polarimetry

NASA Astrophysics Data System (ADS)

Sheng, Wei; He, Honghui; Dong, Yang; Ma, Hui

2018-02-01

As one of the most fundamental features of light, polarization can be used to develop imaging techniques which can provide insight into the optical and structural properties of tissues. Especially, the Mueller matrix polarimetry is suitable to detect the changes in collagen and elastic fibres, which are the main compositions of skin tissue. Here we demonstrate a novel quantitative, non-contact and in situ technique to monitor the microstructural variations of skin tissue during ultraviolet radiation (UVR) induced photoaging based on Mueller matrix polarimetry. Specifically, we measure the twodimensional (2D) backscattering Mueller matrices of nude mouse skin samples, then calculate and analyze the Mueller matrix derived parameters during the skin photoaging and self-repairing processes. To induce three-day skin photoaging, the back skin of each mouse is irradiated with UVR (0.05J/cm2) for five minutes per day. After UVR, the microstructures of the nude mouse skin are damaged. During the process of UV damage, we measure the backscattering Mueller matrices of the mouse skin samples and examine the relationship between the Mueller matrix parameters and the microstructural variations of skin tissue quantitatively. The comparisons between the UVR damaged groups with and without sunscreens show that the Mueller matrix derived parameters are potential indicators for fibrous microstructure variation in skin tissue. The pathological examinations and Monte Carlo simulations confirm the relationship between the values of Mueller matrix parameters and the changes of fibrous structures. Combined with smart phones or wearable devices, this technique may have a good application prospect in the fields of cosmetics and dermatological health.

Aberrant expression of stress-induced phosphoprotein 1 in colorectal cancer and its clinicopathologic significance.

PubMed

Zhang, Zhimei; Ren, Hui; Yang, Liang; Zhang, Xinhua; Liang, Wei; Wu, Hui; Huang, Leilei; Kang, Jihui; Xu, Jianbo; Zhai, Ertao; Cai, Shirong; He, Yulong

2018-06-04

Stress-Inducible Phosphoprotein1 (STIP1) is an adaptor protein that bridges HSP70 and HSP90 folding and a secretory protein that regulates malignant tumor progression. The aim of the present study was to demonstrate the clinicopathological significance and prognostic role of STIP1 in colorectal cancer (CRC). We used data from The Cancer Genome Atlas (TCGA) to analyze STIP1 expression in CRC and utilized 8 pairs of fresh-frozen tissue samples to investigate STIP1 expression in CRC tissues and adjacent normal tissues using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assays. We also used immunohistochemical staining to detect STIP1 expression in 144 formalin-fixed, paraffin-embedded (FFPE) CRC tissue samples and determine the clinical significance of STIP1 expression in CRC. The results of bioinformatics analysis, qRT-PCR, and western blot showed that STIP1 expression was higher in CRC tissues than in adjacent normal tissues. High STIP1 expression was significantly correlated with advanced T stage (P=.01), N stage (P=.001), M stage (P﹤0.001), and TNM stage (P﹤0.001). Moreover, Kaplan-Meier analyses indicated that higher STIP1 expression predicted a worse prognosis in patients with CRC, and Cox regression analysis revealed that STIP1 was an independent prognostic factor for overall survival and disease-free survival in patients with CRC. In conclusion, our results suggest that STIP1 acts as an oncogene in CRC and can therefore serve as a biomarker for the prognosis of patients with CRC. Copyright © 2018. Published by Elsevier Inc.

Adipose Tissue Responses to Breaking Sitting in Men and Women with Central Adiposity.

PubMed

Chen, Yung-Chih; Betts, James A; Walhin, Jean-Philippe; Thompson, Dylan

2018-04-27

Breaking prolonged sitting reduces postprandial glucose and insulin concentrations and influences skeletal muscle molecular signalling pathways but it is unknown whether breaking sitting also affects adipose tissue. Eleven central overweight participants (7 men and 4 post-menopausal women) aged 50 ± 5 years (means ± SD) completed two mixed-meal feeding trials (PROLONGED SITTING versus BREAKING SITTING) in a randomised, counterbalanced design. The BREAKING SITTING intervention comprised walking for 2 min every 20 min over 5.5 h. Blood samples were taken at regular intervals to examine metabolic biomarkers and adipokine concentrations. Adipose tissue samples were taken at baseline and at 5.5 h to examine changes in mRNA expression and secretion of selected adipokines ex-vivo. Postprandial glycaemia and insulinaemia were attenuated by approximately 50% and 40% in BREAKING SITTING compared to PROLONGED SITTING (iAUC: 359 ± 117 versus 697 ± 218 mmol·330 min·L, p = 0.001 and 202 ± 71 versus 346 ± 150 nmol·330 min·L, p = 0.001, respectively). Despite these pronounced and sustained differences in postprandial glucose and insulin concentrations, adipose tissue mRNA expression for various genes (IL-6, leptin, adiponectin, PDK4, IRS1/2, PI3K and Akt1, etc.) and ex-vivo adipose tissue secretion of IL-6, leptin and adiponectin were not different between trials. This study demonstrates that breaking sitting with short bouts of physical activity has very pronounced effects on systemic postprandial glucose and insulin concentrations but this does not translate into corresponding effects within adipose tissue.

An Array-Based Analysis of MicroRNA Expression Comparing Matched Frozen and Formalin-Fixed Paraffin-Embedded Human Tissue Samples

PubMed Central

Zhang, Xiao; Chen, Jiamin; Radcliffe, Tom; LeBrun, Dave P.; Tron, Victor A.; Feilotter, Harriet

2008-01-01

MicroRNAs (miRNAs) are small, noncoding RNAs that suppress gene expression at the posttranscriptional level via an antisense RNA-RNA interaction. miRNAs used for array-based profiling are generally purified from either snap-frozen or fresh samples. Because tissues found in most pathology departments are available only in formalin-fixed and paraffin-embedded (FFPE) states, we sought to evaluate miRNA derived from FFPE samples for microarray analysis. In this study, miRNAs extracted from matched snap-frozen and FFPE samples were profiled using the Agilent miRNA array platform (Agilent, Santa Clara, CA). Each miRNA sample was hybridized to arrays containing probes interrogating 470 human miRNAs. Seven cases were compared in either duplicate or triplicate. Intrachip and interchip analyses demonstrated that the processes of miRNA extraction, labeling, and hybridization from both frozen and FFPE samples are highly reproducible and add little variation to the results; technical replicates showed high correlations with one another (Kendall tau, 0.722 to 0.853; Spearman rank correlation coefficient, 0.891 to 0.954). Our results showed consistent high correlations between matched frozen and FFPE samples (Kendall tau, 0.669 to 0.815; Spearman rank correlation coefficient, 0.847 to 0.948), supporting the use of FFPE-derived miRNAs for array-based, gene expression profiling. PMID:18832457

Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients.

PubMed

Shin, Saeam; Kim, Juwon; Kim, Yoonjung; Cho, Sun-Mi; Lee, Kyung-A

2017-10-26

EGFR mutation is an emerging biomarker for treatment selection in non-small-cell lung cancer (NSCLC) patients. However, optimal mutation detection is hindered by complications associated with the biopsy procedure, tumor heterogeneity and limited sensitivity of test methodology. In this study, we evaluated the diagnostic utility of real-time PCR using malignant pleural effusion samples. A total of 77 pleural fluid samples from 77 NSCLC patients were tested using the cobas EGFR mutation test (Roche Molecular Systems). Pleural fluid was centrifuged, and separated cell pellets and supernatants were tested in parallel. Results were compared with Sanger sequencing and/or peptide nucleic acid (PNA)-mediated PCR clamping of matched tumor tissue or pleural fluid samples. All samples showed valid real-time PCR results in one or more DNA samples extracted from cell pellets and supernatants. Compared with other molecular methods, the sensitivity of real-time PCR method was 100%. Concordance rate of real-time PCR and Sanger sequencing plus PNA-mediated PCR clamping was 98.7%. We have confirmed that real-time PCR using pleural fluid had a high concordance rate compared to conventional methods, with no failed samples. Our data demonstrated that the parallel real-time PCR testing using supernatant and cell pellet could offer reliable and robust surrogate strategy when tissue is not available.

Breast Cancer Diagnosis Using a Microfluidic Multiplexed Immunohistochemistry Platform

PubMed Central

Kim, Minseok S.; Kim, Taemin; Kong, Sun-Young; Kwon, Soim; Bae, Chae Yun; Choi, Jaekyu; Kim, Chul Hwan; Lee, Eun Sook; Park, Je-Kyun

2010-01-01

Background Biomarkers play a key role in risk assessment, assessing treatment response, and detecting recurrence and the investigation of multiple biomarkers may also prove useful in accurate prediction and prognosis of cancers. Immunohistochemistry (IHC) has been a major diagnostic tool to identify therapeutic biomarkers and to subclassify breast cancer patients. However, there is no suitable IHC platform for multiplex assay toward personalized cancer therapy. Here, we report a microfluidics-based multiplexed IHC (MMIHC) platform that significantly improves IHC performance in reduction of time and tissue consumption, quantification, consistency, sensitivity, specificity and cost-effectiveness. Methodology/Principal Findings By creating a simple and robust interface between the device and human breast tissue samples, we not only applied conventional thin-section tissues into on-chip without any additional modification process, but also attained perfect fluid control for various solutions, without any leakage, bubble formation, or cross-contamination. Four biomarkers, estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR) and Ki-67, were examined simultaneously on breast cancer cells and human breast cancer tissues. The MMIHC method improved immunoreaction, reducing time and reagent consumption. Moreover, it showed the availability of semi-quantitative analysis by comparing Western blot. Concordance study proved strong consensus between conventional whole-section analysis and MMIHC (n = 105, lowest Kendall's coefficient of concordance, 0.90). To demonstrate the suitability of MMIHC for scarce samples, it was also applied successfully to tissues from needle biopsies. Conclusions/Significance The microfluidic system, for the first time, was successfully applied to human clinical tissue samples and histopathological diagnosis was realized for breast cancers. Our results showing substantial agreement indicate that several cancer-related proteins can be simultaneously investigated on a single tumor section, giving clear advantages and technical advances over standard immunohistochemical method. This novel concept will enable histopathological diagnosis using numerous specific biomarkers at a time even for small-sized specimens, thus facilitating the individualization of cancer therapy. PMID:20454672

The national DBS brain tissue network pilot study: need for more tissue and more standardization.

PubMed

Vedam-Mai, V; Krock, N; Ullman, M; Foote, K D; Shain, W; Smith, K; Yachnis, A T; Steindler, D; Reynolds, B; Merritt, S; Pagan, F; Marjama-Lyons, J; Hogarth, P; Resnick, A S; Zeilman, P; Okun, M S

2011-08-01

Over 70,000 DBS devices have been implanted worldwide; however, there remains a paucity of well-characterized post-mortem DBS brains available to researchers. We propose that the overall understanding of DBS can be improved through the establishment of a Deep Brain Stimulation-Brain Tissue Network (DBS-BTN), which will further our understanding of DBS and brain function. The objectives of the tissue bank are twofold: (a) to provide a complete (clinical, imaging and pathological) database for DBS brain tissue samples, and (b) to make available DBS tissue samples to researchers, which will help our understanding of disease and underlying brain circuitry. Standard operating procedures for processing DBS brains were developed as part of the pilot project. Complete data files were created for individual patients and included demographic information, clinical information, imaging data, pathology, and DBS lead locations/settings. 19 DBS brains were collected from 11 geographically dispersed centers from across the U.S. The average age at the time of death was 69.3 years (51-92, with a standard deviation or SD of 10.13). The male:female ratio was almost 3:1. Average post-mortem interval from death to brain collection was 10.6 h (SD of 7.17). The DBS targets included: subthalamic nucleus, globus pallidus interna, and ventralis intermedius nucleus of the thalamus. In 16.7% of cases the clinical diagnosis failed to match the pathological diagnosis. We provide neuropathological findings from the cohort, and perilead responses to DBS. One of the most important observations made in this pilot study was the missing data, which was approximately 25% of all available data fields. Preliminary results demonstrated the feasibility and utility of creating a National DBS-BTN resource for the scientific community. We plan to improve our techniques to remedy omitted clinical/research data, and expand the Network to include a larger donor pool. We will enhance sample preparation to facilitate advanced molecular studies and progenitor cell retrieval.

Implementation of Amplicon Parallel Sequencing Leads to Improvement of Diagnosis and Therapy of Lung Cancer Patients.

PubMed

König, Katharina; Peifer, Martin; Fassunke, Jana; Ihle, Michaela A; Künstlinger, Helen; Heydt, Carina; Stamm, Katrin; Ueckeroth, Frank; Vollbrecht, Claudia; Bos, Marc; Gardizi, Masyar; Scheffler, Matthias; Nogova, Lucia; Leenders, Frauke; Albus, Kerstin; Meder, Lydia; Becker, Kerstin; Florin, Alexandra; Rommerscheidt-Fuss, Ursula; Altmüller, Janine; Kloth, Michael; Nürnberg, Peter; Henkel, Thomas; Bikár, Sven-Ernö; Sos, Martin L; Geese, William J; Strauss, Lewis; Ko, Yon-Dschun; Gerigk, Ulrich; Odenthal, Margarete; Zander, Thomas; Wolf, Jürgen; Merkelbach-Bruse, Sabine; Buettner, Reinhard; Heukamp, Lukas C

2015-07-01

The Network Genomic Medicine Lung Cancer was set up to rapidly translate scientific advances into early clinical trials of targeted therapies in lung cancer performing molecular analyses of more than 3500 patients annually. Because sequential analysis of the relevant driver mutations on fixated samples is challenging in terms of workload, tissue availability, and cost, we established multiplex parallel sequencing in routine diagnostics. The aim was to analyze all therapeutically relevant mutations in lung cancer samples in a high-throughput fashion while significantly reducing turnaround time and amount of input DNA compared with conventional dideoxy sequencing of single polymerase chain reaction amplicons. In this study, we demonstrate the feasibility of a 102 amplicon multiplex polymerase chain reaction followed by sequencing on an Illumina sequencer on formalin-fixed paraffin-embedded tissue in routine diagnostics. Analysis of a validation cohort of 180 samples showed this approach to require significantly less input material and to be more reliable, robust, and cost-effective than conventional dideoxy sequencing. Subsequently, 2657 lung cancer patients were analyzed. We observed that comprehensive biomarker testing provided novel information in addition to histological diagnosis and clinical staging. In 2657 consecutively analyzed lung cancer samples, we identified driver mutations at the expected prevalence. Furthermore we found potentially targetable DDR2 mutations at a frequency of 3% in both adenocarcinomas and squamous cell carcinomas. Overall, our data demonstrate the utility of systematic sequencing analysis in a clinical routine setting and highlight the dramatic impact of such an approach on the availability of therapeutic strategies for the targeted treatment of individual cancer patients.

Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection.

PubMed

Verma, Sandeep; Singh, Ruchi; Sharma, Vanila; Bumb, Ram Avtar; Negi, Narendra Singh; Ramesh, V; Salotra, Poonam

2017-03-23

Leishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application. We have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL). The assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse. The study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid molecular diagnosis of VL and PKDL with potential for application in assessment of cure.

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.

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.

Proteomic analysis of human dental cementum and alveolar bone

PubMed Central

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

2013-01-01

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. PMID:24007660

Light sheet theta microscopy for rapid high-resolution imaging of large biological samples.

PubMed

Migliori, Bianca; Datta, Malika S; Dupre, Christophe; Apak, Mehmet C; Asano, Shoh; Gao, Ruixuan; Boyden, Edward S; Hermanson, Ola; Yuste, Rafael; Tomer, Raju

2018-05-29

Advances in tissue clearing and molecular labeling methods are enabling unprecedented optical access to large intact biological systems. These developments fuel the need for high-speed microscopy approaches to image large samples quantitatively and at high resolution. While light sheet microscopy (LSM), with its high planar imaging speed and low photo-bleaching, can be effective, scaling up to larger imaging volumes has been hindered by the use of orthogonal light sheet illumination. To address this fundamental limitation, we have developed light sheet theta microscopy (LSTM), which uniformly illuminates samples from the same side as the detection objective, thereby eliminating limits on lateral dimensions without sacrificing the imaging resolution, depth, and speed. We present a detailed characterization of LSTM, and demonstrate its complementary advantages over LSM for rapid high-resolution quantitative imaging of large intact samples with high uniform quality. The reported LSTM approach is a significant step for the rapid high-resolution quantitative mapping of the structure and function of very large biological systems, such as a clarified thick coronal slab of human brain and uniformly expanded tissues, and also for rapid volumetric calcium imaging of highly motile animals, such as Hydra, undergoing non-isomorphic body shape changes.

Application of RT-PCR in formalin-fixed and paraffin-embedded lung cancer tissues.

PubMed

Zhang, Fan; Wang, Zhuo-min; Liu, Hong-yu; Bai, Yun; Wei, Sen; Li, Ying; Wang, Min; Chen, Jun; Zhou, Qing-hua

2010-01-01

To analyze gene expression in formalin-fixed, paraffin-embedded lung cancer tissues using modified method. Total RNA from frozen tissues was extracted using TRIZOL reagent. RNA was extracted from formalin-fixed, paraffin-embedded tissues by digestion with proteinase K before the acid-phenol:chloroform extraction and carrier precipitation. We modified this method by using a higher concentration of proteinase K and a longer digestion time, optimized to 16 hours. RT-PCR and real-time RT-PCR were used to check reproducibility and the concordance between frozen and paraffin-embedded samples. The results showed that the RNA extracted from the paraffin-embedded lung tissues had high quality with the most fragment length between 28S and 18S bands (about 1000 to 2000 bases). The housekeeping gene GUSB exhibited low variation of expression in frozen and paraffin-embedded lung tissues, whereas PGK1 had the lowest variation in lymphoma tissues. Furthermore, real-time PCR analysis of the expression of known prognostic genes in non-small cell lung carcinoma (NSCLC) demonstrated an extremely high correlation (r>0.880) between the paired frozen and formalin-fixed, paraffin-embedded specimens. This improved method of RNA extraction is suitable for real-time quantitative RT-PCR, and may be used for global gene expression profiling of paraffin-embedded tissues.

Sensitive and specific detection of classical scrapie prions in the brains of goats by real-time quaking-induced conversion.

PubMed

Dassanayake, Rohana P; Orrú, Christina D; Hughson, Andrew G; Caughey, Byron; Graça, Telmo; Zhuang, Dongyue; Madsen-Bouterse, Sally A; Knowles, Donald P; Schneider, David A

2016-03-01

Real-time quaking-induced conversion (RT-QuIC) is a rapid, specific and highly sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect subinfectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully used to detect PrPSc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie have not yet been tested. Therefore, the aims of the present study were to (1) evaluate whether prion seeding activity could be detected in the brain tissues of goats with scrapie using RT-QuIC, (2) optimize reaction conditions to improve scrapie detection in goats, and (3) compare the performance of RT-QuIC for the detection of PrPSc with the more commonly used ELISA and Western blot assays. We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used with 200  mM sodium chloride, both full-length sheep rPrPSen substrates (PrP genotypes A136R154Q171 and V136R154Q171) provided good discrimination between scrapie-infected and normal goat brain samples at 10(- )3 dilution within 15  h. Our findings indicate that RT-QuIC was at least 10,000-fold more sensitive than ELISA and Western blot assays for the detection of scrapie seeding activity in goat brain samples. In addition to PRNP WT samples, positive RT-QuIC reactions were also observed with three PRNP polymorphic goat brain samples (G/S127, I/M142 and H/R143) tested. Taken together, these findings demonstrate that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples.

Sensitive and specific detection of classical scrapie prions in the brains of goats by real-time quaking-induced conversion

PubMed Central

Dassanayake, Rohana P.; Orrú, Christina D.; Hughson, Andrew G.; Caughey, Byron; Graça, Telmo; Zhuang, Dongyue; Madsen-Bouterse, Sally A.; Knowles, Donald P.; Schneider, David A.

2016-01-01

Real-time quaking-induced conversion (RT-QuIC) is a rapid, specific and highly sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect subinfectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully used to detect PrPSc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie have not yet been tested. Therefore, the aims of the present study were to (1) evaluate whether prion seeding activity could be detected in the brain tissues of goats with scrapie using RT-QuIC, (2) optimize reaction conditions to improve scrapie detection in goats, and (3) compare the performance of RT-QuIC for the detection of PrPSc with the more commonly used ELISA and Western blot assays. We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used with 200 mM sodium chloride, both full-length sheep rPrPSen substrates (PrP genotypes A136R154Q171 and V136R154Q171) provided good discrimination between scrapie-infected and normal goat brain samples at 10− 3 dilution within 15 h. Our findings indicate that RT-QuIC was at least 10 000-fold more sensitive than ELISA and Western blot assays for the detection of scrapie seeding activity in goat brain samples. In addition to PRNP WT samples, positive RT-QuIC reactions were also observed with three PRNP polymorphic goat brain samples (G/S127, I/M142 and H/R143) tested. Taken together, these findings demonstrate that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples. PMID:26653410

Raman-spectroscopy-based biosensing for applications in ophthalmology

NASA Astrophysics Data System (ADS)

Rusciano, Giulia; Capriglione, Paola; Pesce, Giuseppe; Zito, Gianluigi; Del Prete, Antonio; Cennamo, Giovanni; Sasso, Antonio

2013-05-01

Cell-based biosensors rely on the detection and identification of single cells as well as monitoring of changes induced by interaction with drugs and/or toxic agents. Raman spectroscopy is a powerful tool to reach this goal, being non-destructive analytical technique, allowing also measurements of samples in aqueous environment. In addition, micro-Raman measurements do not require preliminary sample preparation (as in fluorescence spectroscopy), show a finger-print spectral response, allow a spatial resolution below typical cell sizes, and are relatively fast (few s or even less). All these properties make micro-Raman technique particularly promising for high-throughput on-line analysis integrated in lab-on-a-chip devices. Herein, we demonstrate some applications of Raman analysis in ophthalmology. In particular, we demonstrate that Raman analysis can provide useful information for the therapeutic treatment of keratitis caused by Acanthamoeba Castellanii (A.), an opportunistic protozoan that is widely distributed in the environment and is known to produce blinding keratitis and fatal encephalitis. In particular, by combining Raman analysis with Principal Component Analysis (PCA), we have demonstrated that is possible to distinguish between live and dead cells, enabling, therefore to establish the effectiveness of therapeutic strategies to vanquish the protozoa. As final step, we have analyzed the presence of biochemical differences in the conjunctival epithelial tissues of patients affected by keratitis with respect to healthy people. As a matter of facts, it is possible to speculate some biochemical alterations of the epithelial tissues, rendering more favorable the binding of the protozoan. The epithelial cells were obtained by impression cytology from eyes of both healthy and keratitis-affected individuals. All the samples were analyzed by Raman spectroscopy within a few hours from cells removal from eyes. The results of this analysis are discussed.

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