Automatic detection of spermatozoa for laser capture microdissection.

PubMed

Vandewoestyne, Mado; Van Hoofstat, David; Van Nieuwerburgh, Filip; Deforce, Dieter

2009-03-01

In sexual assault crimes, differential extraction of spermatozoa from vaginal swab smears is often ineffective, especially when only a few spermatozoa are present in an overwhelming amount of epithelial cells. Laser capture microdissection (LCM) enables the precise separation of spermatozoa and epithelial cells. However, standard sperm-staining techniques are non-specific and rely on sperm morphology for identification. Moreover, manual screening of the microscope slides is time-consuming and labor-intensive. Here, we describe an automated screening method to detect spermatozoa stained with Sperm HY-LITER. Different ratios of spermatozoa and epithelial cells were used to assess the automatic detection method. In addition, real postcoital samples were also screened. Detected spermatozoa were isolated using LCM and DNA analysis was performed. Robust DNA profiles without allelic dropout could be obtained from as little as 30 spermatozoa recovered from postcoital samples, showing that the staining had no significant influence on DNA recovery.

Gene expression analysis of immunostained endothelial cells isolated from formaldehyde-fixated paraffin embedded tumors using laser capture microdissection--a technical report.

PubMed

Kaneko, Tomoatsu; Okiji, Takashi; Kaneko, Reika; Suda, Hideaki; Nör, Jacques E

2009-12-01

Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by reverse transcription-PCR (RT-PCR) and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4 degrees C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. Glyceraldehyde-3-phosphate dehydrogenase and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM.

Neuronal Type-Specific Gene Expression Profiling and Laser-Capture Microdissection

PubMed Central

Pietersen, Charmaine Y.; Lim, Maribel P.; Macey, Laurel; Woo, Tsung-Ung W.; Sonntag, Kai C.

2014-01-01

The human brain is an exceptionally heterogeneous structure. In order to gain insight into the neurobiological basis of neural circuit disturbances in various neurologic or psychiatric diseases, it is often important to define the molecular cascades that are associated with these disturbances in a neuronal type-specific manner. This can be achieved by the use of laser microdissection, in combination with molecular techniques such as gene expression profiling. To identify neurons in human postmortem brain tissue, one can use the inherent properties of the neuron, such as pigmentation and morphology or its structural composition through immunohistochemistry (IHC). Here, we describe the isolation of homogeneous neuronal cells and high-quality RNA from human postmortem brain material using a combination of rapid IHC, Nissl staining, or simple morphology with Laser-Capture Microdissection (LCM) or Laser Microdissection (LMD). PMID:21761317

Neuronal type-specific gene expression profiling and laser-capture microdissection.

PubMed

Pietersen, Charmaine Y; Lim, Maribel P; Macey, Laurel; Woo, Tsung-Ung W; Sonntag, Kai C

2011-01-01

The human brain is an exceptionally heterogeneous structure. In order to gain insight into the neurobiological basis of neural circuit disturbances in various neurologic or psychiatric diseases, it is often important to define the molecular cascades that are associated with these disturbances in a neuronal type-specific manner. This can be achieved by the use of laser microdissection, in combination with molecular techniques such as gene expression profiling. To identify neurons in human postmortem brain tissue, one can use the inherent properties of the neuron, such as pigmentation and morphology or its structural composition through immunohistochemistry (IHC). Here, we describe the isolation of homogeneous neuronal cells and high-quality RNA from human postmortem brain material using a combination of rapid IHC, Nissl staining, or simple morphology with Laser-Capture Microdissection (LCM) or Laser Microdissection (LMD).

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA.

PubMed

Wang, Hongyang; Owens, James D; Shih, Joanna H; Li, Ming-Chung; Bonner, Robert F; Mushinski, J Frederic

2006-04-27

Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA

PubMed Central

Wang, Hongyang; Owens, James D; Shih, Joanna H; Li, Ming-Chung; Bonner, Robert F; Mushinski, J Frederic

2006-01-01

Background Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. Results The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. Conclusion RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts. PMID:16643667

[The estimation of possibilities for the application of the laser capture microdissection technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA].

PubMed

Ivanov, P L; Leonov, S N; Zemskova, E Iu

2012-01-01

The present study was designed to estimate the possibilities of application of the laser capture microdissection (LCM) technology for the molecular-genetic expert analysis (genotyping) of human chromosomal DNA. The experimental method employed for the purpose was the multiplex multilocus analysis of autosomal DNA polymorphism in the preparations of buccal epitheliocytes obtained by LCM. The key principles of the study were the application of physical methods for contrast enhancement of the micropreparations (such as phase-contrast microscopy and dark-field microscopy) and PCR-compatible cell lysis. Genotyping was carried out with the use of AmpFISTR Minifiler TM PCR Amplification Kits ("Applied Biosynthesis", USA). It was shown that the technique employed in the present study ensures reliable genotyping of human chromosomal DNA in the pooled preparations containing 10-20 dissected diploid cells each. This result fairly well agrees with the calculated sensitivity of the method. A few practical recommendations are offered.

Spatial and molecular resolution of diffuse malignant mesothelioma heterogeneity by integrating label-free FTIR imaging, laser capture microdissection and proteomics

NASA Astrophysics Data System (ADS)

Großerueschkamp, Frederik; Bracht, Thilo; Diehl, Hanna C.; Kuepper, Claus; Ahrens, Maike; Kallenbach-Thieltges, Angela; Mosig, Axel; Eisenacher, Martin; Marcus, Katrin; Behrens, Thomas; Brüning, Thomas; Theegarten, Dirk; Sitek, Barbara; Gerwert, Klaus

2017-03-01

Diffuse malignant mesothelioma (DMM) is a heterogeneous malignant neoplasia manifesting with three subtypes: epithelioid, sarcomatoid and biphasic. DMM exhibit a high degree of spatial heterogeneity that complicates a thorough understanding of the underlying different molecular processes in each subtype. We present a novel approach to spatially resolve the heterogeneity of a tumour in a label-free manner by integrating FTIR imaging and laser capture microdissection (LCM). Subsequent proteome analysis of the dissected homogenous samples provides in addition molecular resolution. FTIR imaging resolves tumour subtypes within tissue thin-sections in an automated and label-free manner with accuracy of about 85% for DMM subtypes. Even in highly heterogeneous tissue structures, our label-free approach can identify small regions of interest, which can be dissected as homogeneous samples using LCM. Subsequent proteome analysis provides a location specific molecular characterization. Applied to DMM subtypes, we identify 142 differentially expressed proteins, including five protein biomarkers commonly used in DMM immunohistochemistry panels. Thus, FTIR imaging resolves not only morphological alteration within tissue but it resolves even alterations at the level of single proteins in tumour subtypes. Our fully automated workflow FTIR-guided LCM opens new avenues collecting homogeneous samples for precise and predictive biomarkers from omics studies.

Spatial and molecular resolution of diffuse malignant mesothelioma heterogeneity by integrating label-free FTIR imaging, laser capture microdissection and proteomics.

PubMed

Großerueschkamp, Frederik; Bracht, Thilo; Diehl, Hanna C; Kuepper, Claus; Ahrens, Maike; Kallenbach-Thieltges, Angela; Mosig, Axel; Eisenacher, Martin; Marcus, Katrin; Behrens, Thomas; Brüning, Thomas; Theegarten, Dirk; Sitek, Barbara; Gerwert, Klaus

2017-03-30

Diffuse malignant mesothelioma (DMM) is a heterogeneous malignant neoplasia manifesting with three subtypes: epithelioid, sarcomatoid and biphasic. DMM exhibit a high degree of spatial heterogeneity that complicates a thorough understanding of the underlying different molecular processes in each subtype. We present a novel approach to spatially resolve the heterogeneity of a tumour in a label-free manner by integrating FTIR imaging and laser capture microdissection (LCM). Subsequent proteome analysis of the dissected homogenous samples provides in addition molecular resolution. FTIR imaging resolves tumour subtypes within tissue thin-sections in an automated and label-free manner with accuracy of about 85% for DMM subtypes. Even in highly heterogeneous tissue structures, our label-free approach can identify small regions of interest, which can be dissected as homogeneous samples using LCM. Subsequent proteome analysis provides a location specific molecular characterization. Applied to DMM subtypes, we identify 142 differentially expressed proteins, including five protein biomarkers commonly used in DMM immunohistochemistry panels. Thus, FTIR imaging resolves not only morphological alteration within tissue but it resolves even alterations at the level of single proteins in tumour subtypes. Our fully automated workflow FTIR-guided LCM opens new avenues collecting homogeneous samples for precise and predictive biomarkers from omics studies.

Application of laser-capture microdissection to analysis of gene expression in the testis.

PubMed

Sluka, Pavel; O'Donnell, Liza; McLachlan, Robert I; Stanton, Peter G

2008-01-01

The isolation and molecular analysis of highly purified cell populations from complex, heterogeneous tissues has been a challenge for many years. Spermatogenesis in the testis is a particularly difficult process to study given the unique multiple cellular associations within the seminiferous epithelium, making the isolation of specific cell types difficult. Laser-capture microdissection (LCM) is a recently developed technique that enables the isolation of individual cell populations from complex tissues. This technology has enhanced our ability to directly examine gene expression in enriched testicular cell populations by routine methods of gene expression analysis, such as real-time RT-PCR, differential display, and gene microarrays. The application of LCM has however introduced methodological hurdles that have not been encountered with more conventional molecular analyses of whole tissue. In particular, tissue handling (i.e. fixation, storage, and staining), consumables (e.g. slide choice), staining reagents (conventional H&E vs. fluorescence), extraction methods, and downstream applications have all required re-optimisation to facilitate differential gene expression analysis using the small amounts of material obtained using LCM. This review will discuss three critical issues that are essential for successful procurement of cells from testicular tissue sections; tissue morphology, capture success, and maintenance of molecular integrity. The importance of these issues will be discussed with specific reference to the two most commonly used LCM systems; the Arcturus PixCell IIe and PALM systems. The rat testis will be used as a model, and emphasis will be placed on issues of tissue handling, processing, and staining methods, including the application of fluorescence techniques to assist in the identification of cells of interest for the purposes of mRNA expression analysis.

Using laser capture microdissection to study fiber specific signaling in locomotor muscle in COPD: A pilot study.

PubMed

Mohan, Divya; Lewis, Amy; Patel, Mehul S; Curtis, Katrina J; Lee, Jen Y; Hopkinson, Nicholas S; Wilkinson, Ian B; Kemp, Paul R; Polkey, Michael I

2017-06-01

Quadriceps dysfunction is important in chronic obstructive pulmonary disease (COPD), with an associated increased proportion of type II fibers. Investigation of protein synthesis and degradation has yielded conflicting results, possibly due to study of whole biopsy samples, whereas signaling may be fiber-specific. Our objective was to develop a method for fiber-specific gene expression analysis. 12 COPD and 6 healthy subjects underwent quadriceps biopsy. Cryosections were immunostained for type II fibers, which were separated using laser capture microdissection (LCM). Whole muscle and different fiber populations were subject to quantitative polymerase chain reaction. Levels of muscle-RING-finger-protein-1 and Atrogin-1 were lower in type II fibers of COPD versus healthy subjects (P = 0.02 and P = 0.03, respectively), but differences were not apparent in whole muscle or type I fibers. We describe a novel method for studying fiber-specific gene expression in optimum cutting temperature compound-embedded muscle specimens. LCM offers a more sensitive way to identify molecular changes in COPD muscle. Muscle Nerve 55: 902-912, 2017. © 2016 Wiley Periodicals, Inc.

Recovery of high-quality RNA from laser capture microdissected human and rodent pancreas.

PubMed

Butler, Alexandra E; Matveyenko, Aleksey V; Kirakossian, David; Park, Johanna; Gurlo, Tatyana; Butler, Peter C

Laser capture microdissection (LCM) is a powerful method to isolate specific populations of cells for subsequent analysis such as gene expression profiling, for example, microarrays or ribonucleic (RNA)-Seq. This technique has been applied to frozen as well as formalin-fixed, paraffin-embedded (FFPE) specimens with variable outcomes regarding quality and quantity of extracted RNA. The goal of the study was to develop the methods to isolate high-quality RNA from islets of Langerhans and pancreatic duct glands (PDG) isolated by LCM. We report an optimized protocol for frozen sections to minimize RNA degradation and maximize recovery of expected transcripts from the samples using quantitative real-time polymerase chain reaction (RT-PCR) by adding RNase inhibitors at multiple steps during the experiment. This technique reproducibly delivered intact RNA (RIN values 6-7). Using quantitative RT-PCR, the expected profiles of insulin, glucagon, mucin6 (Muc6), and cytokeratin-19 (CK-19) mRNA in PDGs and pancreatic islets were detected. The described experimental protocol for frozen pancreas tissue might also be useful for other tissues with moderate to high levels of intrinsic ribonuclease (RNase) activity.

Production of high quality brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) RNA from isolated populations of rat spinal cord motor neurons obtained by Laser Capture Microdissection (LCM).

PubMed

Mehta, Prachi; Premkumar, Brian; Morris, Renée

2016-08-03

The mammalian central nervous system (CNS) is composed of multiple cellular elements, making it challenging to segregate one particular cell type to study their gene expression profile. For instance, as motor neurons represent only 5-10% of the total cell population of the spinal cord, meaningful transcriptional analysis on these neurons is almost impossible to achieve from homogenized spinal cord tissue. A major challenge faced by scientists is to obtain good quality RNA from small amounts of starting material. In this paper, we used Laser Capture Microdissection (LCM) techniques to identify and isolate spinal cord motor neurons. The present analysis revealed that perfusion with paraformaldehyde (PFA) does not alter RNA quality. RNA integrity numbers (RINs) of tissue samples from rubrospinal tract (RST)-transected, intact spinal cord or from whole spinal cord homogenate were all above 8, which indicates intact, high-quality RNA. Levels of mRNA for brain-derived neurotrophic factor (BDNF) or for its tropomyosin receptor kinase B (TrkB) were not affected by rubrospinal tract (RST) transection, a surgical procedure that deprive motor neurons from one of their main supraspinal input. The isolation of pure populations of neurons with LCM techniques allows for robust transcriptional characterization that cannot be achieved with spinal cord homogenates. Such preparations of pure population of motor neurons will provide valuable tools to advance our understanding of the molecular mechanisms underlying spinal cord injury and neuromuscular diseases. In the near future, LCM techniques might be instrumental to the success of gene therapy for these debilitating conditions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Notochord isolation using laser capture microdissection.

PubMed

Santegoeds, R G C; Yakkioui, Y; Jahanshahi, A; Raven, G; Van Overbeeke, J J; Herrler, A; Temel, Y

2017-03-01

Chordoma are malignant tumors of the axial skeleton, which arise from remnants of the notochord. The Notochord (chorda dorsalis) is an essential embryonic structure involved in the development of the nervous system and axial skeleton. Therefore, the notochord seems to be the most biologically relevant control tissue to study chordoma in molecular biology research. Nevertheless, up to now mainly different tissues but not the notochord have been used as control for chordoma, due to difficulty of isolating notochordal tissue. Here, we describe a fast and precise method of isolating notochordal cells. Examination of human fetuses, with a gestation of 9, 11 and 13 weeks, using (immuno)histochemical methods was performed. To isolate pure notochord cells for further molecular biology investigation five flash frozen fetuses between 9 and 10 weeks of gestation were dissected by microtome slicing. Thereafter pure notochord cells for further molecular biology investigation where harvested by using laser capture microdissection (LCM). RNA was extracted from these samples and used in quantitative PCR. This study illustrates notochord of embryonic spines in three different stages of gestation (9-11-13 weeks). Immunohistochemical staining with brachyury showed strong staining of the notochord, but also weak staining of the intervertebral disc and vertebral body. LCM of notochord slices and subsequent total RNA extraction resulted in a good yield of total RNA. qPCR analysis of two housekeeping genes confirmed the quality of the RNA. LCM is a fast and precise method to isolate notochord and the quality and yield RNA extracted from this tissue is sufficient for qPCR analysis. Therefore early embryo notochord isolated by LCM is suggested to be the gold standard for future research in chordoma development, classification and diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

The Isolation of Pure Populations of Neurons by Laser Capture Microdissection: Methods and Application in Neuroscience.

PubMed

Morris, Renée; Mehta, Prachi

2018-01-01

In mammals, the central nervous system (CNS) is constituted of various cellular elements, posing a challenge to isolating specific cell types to investigate their expression profile. As a result, tissue homogenization is not amenable to analyses of motor neurons profiling as these represent less than 10% of the total spinal cord cell population. One way to tackle the problem of tissue heterogeneity and obtain meaningful genomic, proteomic, and transcriptomic profiling is to use laser capture microdissection technology (LCM). In this chapter, we describe protocols for the capture of isolated populations of motor neurons from spinal cord tissue sections and for downstream transcriptomic analysis of motor neurons with RT-PCR. We have also included a protocol for the immunological confirmation that the captured neurons are indeed motor neurons. Although focused on spinal cord motor neurons, these protocols can be easily optimized for the isolation of any CNS neurons.

Laser capture microdissection: Big data from small samples

PubMed Central

Datta, Soma; Malhotra, Lavina; Dickerson, Ryan; Chaffee, Scott; Sen, Chandan K.; Roy, Sashwati

2015-01-01

Any tissue is made up of a heterogeneous mix of spatially distributed cell types. In response to any (patho) physiological cue, responses of each cell type in any given tissue may be unique and cannot be homogenized across cell-types and spatial co-ordinates. For example, in response to myocardial infarction, on one hand myocytes and fibroblasts of the heart tissue respond differently. On the other hand, myocytes in the infarct core respond differently compared to those in the peri-infarct zone. Therefore, isolation of pure targeted cells is an important and essential step for the molecular analysis of cells involved say in the progression of disease. Laser capture microdissection (LCM) is powerful to obtain a pure targeted cell subgroup, or even a single cell, quickly and precisely under the microscope, successfully tackling the problem of tissue heterogeneity in molecular analysis. This review presents an overview of LCM technology, the principles, advantages and limitations and its down-stream applications in the fields of proteomics, genomics and transcriptomics. With powerful technologies and appropriate applications, this technique provides unprecedented insights into cell biology from cells grown in their natural tissue habitat as opposed to those cultured in artificial petri dish conditions. PMID:25892148

Laser capture microdissection: Big data from small samples.

PubMed

Datta, Soma; Malhotra, Lavina; Dickerson, Ryan; Chaffee, Scott; Sen, Chandan K; Roy, Sashwati

2015-11-01

Any tissue is made up of a heterogeneous mix of spatially distributed cell types. In response to any (patho) physiological cue, responses of each cell type in any given tissue may be unique and cannot be homogenized across cell-types and spatial co-ordinates. For example, in response to myocardial infarction, on one hand myocytes and fibroblasts of the heart tissue respond differently. On the other hand, myocytes in the infarct core respond differently compared to those in the peri-infarct zone. Therefore, isolation of pure targeted cells is an important and essential step for the molecular analysis of cells involved in the progression of disease. Laser capture microdissection (LCM) is powerful to obtain a pure targeted cell subgroup, or even a single cell, quickly and precisely under the microscope, successfully tackling the problem of tissue heterogeneity in molecular analysis. This review presents an overview of LCM technology, the principles, advantages and limitations and its down-stream applications in the fields of proteomics, genomics and transcriptomics. With powerful technologies and appropriate applications, this technique provides unprecedented insights into cell biology from cells grown in their natural tissue habitat as opposed to those cultured in artificial petri dish conditions.

Laser capture microdissection tailored to type 1 diabetes mellitus research.

PubMed

Szulawski, Robert; Nakazawa, Masato; McCall, Kelly D; James, Calvin B L; Schwartz, Frank L

2016-01-01

RNA isolation from pancreatic islets poses unique challenges. Here, we present a reproducible means of obtaining high-quality RNA from juvenile rodent islets in sufficient quantities for use in ex vivo expression studies. Tissue was extracted from female non-obese diabetic (NOD) toll-like receptor 3 (TLR3)(+/+) and (TLR3)(-/-) mice in the pre-diabetic stage. Samples were frozen in liquid nitrogen, sectioned, fixed in a highly alcoholic solution, and stained with an alcoholic cresyl violet (CV) solution. Rehydration of the fixed sections was minimized. Islets were identified visually and isolated with the Leica LMD6000 laser capture microdissection (LCM) system to yield samples highly enriched in islet RNA. Real time qPCR was performed on the islet cDNA using probes for CXC chemokine ligand 10 (CXCL10), an inflammatory marker that plays a critical role in the pathogenesis of type 1 diabetes mellitus (TIDM). This method represents an improvement over currently described LCM techniques for rodent pancreatic islets and makes feasible expression studies using small amounts of starting tissue without the need for RNA pre-amplification. This has immediate implications for ongoing TIDM studies using the NOD mouse.

ACVP-12: Quantitative Assessment of HIV/SIV Viral DNA in Laser Capture Microdissected (LCM) CD4+ T cell and/or Macrophage Populations from Formalin-Fixed Tissue Specimens | Frederick National Laboratory for Cancer Research

Cancer.gov

The Tissue Analysis Core (TAC) within the AIDS and Cancer Virus Program will process, embed, and perform microtomy on fixed tissue samples presented in ethanol. CD4 (DAB) and CD68/CD163 (FastRed) double immunohistochemistry will be performed, allowin

Microarray analysis of laser capture microdissected-anulus cells from the human intervertebral disc.

PubMed

Gruber, Helen E; Mougeot, Jean-Luc; Hoelscher, Gretchen; Ingram, Jane A; Hanley, Edward N

2007-05-15

Five Thompson Grade I/II discs (Group 1), 7 Grade III discs (Group 2), and 3 Grade IV discs (Group IV) were studied here in a project approved by the authors' Human Subjects Institutional Review Board. Our objective was to use laser capture microdissection (LCM) to harvest cells from the human anulus and to derive gene expression profiles using microarray analysis. Appropriate gene expression is essential in the intervertebral disc for maintenance of extracellular matrix (ECM), ECM remodeling, and maintenance of a viable disc cell population. During disc degeneration, cell numbers drop, making gene expression studies challenging. LCM was used to harvest cells from paraffin-embedded sections of human anulus tissue. Gene profiling used Affymetrix GeneChip Human X3P arrays. ANOVA and SAM permutation analysis were applied to dCHIP normalized, filtered, and log-transformed gene expression data ( approximately 33,500 probes), and data analyzed to identify genes that were significantly differentially expressed between the 3 groups. We identified 47 genes that were significantly differentially expressed between the 3 groups (P < 0.001 and lowest q values). Compared with the healthiest discs (Grade I/II), 13 genes were up-regulated and 19 down-regulated in both the Grade III and the Grade IV discs. Genes with biologic significance regulated during degeneration involved cell senescence, low cell division rates, hypoxia-related genes, heat-shock protein 70 interacting protein, neuropilin 2, and interleukin-23p19 (interleukin-12 family). Results expand our understanding of disc aging and degeneration and show that LCM is a valuable technique that can be used to collect mRNA amounts adequate for microarray analysis from the sparse cell population of the human anulus.

Analysis of Temporal-spatial Co-variation within Gene Expression Microarray Data in an Organogenesis Model

NASA Astrophysics Data System (ADS)

Ehler, Martin; Rajapakse, Vinodh; Zeeberg, Barry; Brooks, Brian; Brown, Jacob; Czaja, Wojciech; Bonner, Robert F.

The gene networks underlying closure of the optic fissure during vertebrate eye development are poorly understood. We used a novel clustering method based on Laplacian Eigenmaps, a nonlinear dimension reduction method, to analyze microarray data from laser capture microdissected (LCM) cells at the site and developmental stages (days 10.5 to 12.5) of optic fissure closure. Our new method provided greater biological specificity than classical clustering algorithms in terms of identifying more biological processes and functions related to eye development as defined by Gene Ontology at lower false discovery rates. This new methodology builds on the advantages of LCM to isolate pure phenotypic populations within complex tissues and allows improved ability to identify critical gene products expressed at lower copy number. The combination of LCM of embryonic organs, gene expression microarrays, and extracting spatial and temporal co-variations appear to be a powerful approach to understanding the gene regulatory networks that specify mammalian organogenesis.

Human Papillomavirus (HPV) Genotypes in Condylomas, Intraepithelial Neoplasia, and Invasive Carcinoma of the Penis Using Laser Capture Microdissection (LCM)-PCR: A Study of 191 Lesions in 43 Patients.

PubMed

Fernández-Nestosa, María J; Guimerà, Nuria; Sanchez, Diego F; Cañete-Portillo, Sofía; Velazquez, Elsa F; Jenkins, David; Quint, Wim; Cubilla, Antonio L

2017-06-01

Laser capture microdissection-polymerase chain reaction (LCM-PCR) supported by p16 was used for the first time to demonstrate human papillomavirus (HPV) DNA in histologically specific penile lesions, which were as follows: squamous hyperplasia (12 lesions, 10 patients), flat lesions (12 lesions, 5 patients), condylomas (26 lesions, 7 patients), penile intraepithelial neoplasia (PeIN) (115 lesions, 43 patients), and invasive squamous cell carcinomas (26 lesions, 26 patients). HPV was detected by whole-tissue section and LCM-PCR. LCM proved to be more precise than whole-tissue section in assigning individual genotypes to specific lesions. HPV was negative or very infrequent in squamous hyperplasia, differentiated PeIN, and low-grade keratinizing variants of carcinomas. HPV was strongly associated with condylomas, warty/basaloid PeIN, adjacent flat lesions, and warty/basaloid carcinomas. A single HPV genotype was found in each lesion. Some condylomas and flat lesions, especially those with atypia, were preferentially associated with high-risk HPV. Unlike invasive carcinoma, in which few genotypes of HPV were involved, there were 18 HPV genotypes in PeIN, usually HPV 16 in basaloid PeIN but marked HPV heterogeneity in warty PeIN (11 different genotypes). Variable and multiple HPV genotypes were found in multicentric PeIN, whereas unicentric PeIN was usually related to a single genotype. There was a correspondence among HPV genotypes in invasive and associated PeIN. p16 was positive in the majority of HPV-positive lesions except condylomas containing LR-HPV. p16 was usually negative in squamous hyperplasia, differentiated PeIN, and low-grade keratinizing variants of squamous cell carcinomas. In summary, we demonstrated that LCM-PCR was a superior research technique for investigating HPV genotypes in intraepithelial lesions. A significant finding was the heterogeneity of HPV genotypes in PeIN and the differential association of HPV genotypes with subtypes of PeIN. The presence of atypia and high-risk HPV in condylomas and adjacent flat lesions suggests a precursor role, and the correspondence of HPV genotypes in invasive carcinomas and associated PeIN indicates a causal relation. Data presented support the bimodal hypothesis of penile cancer carcinogenesis in HPV-driven and non-HPV-driven carcinomas and justify the current WHO pathologic classification of PeIN in special subtypes.

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.

Spatial distributions of Kv4 channels and KChip2 isoforms in the murine heart based on laser capture microdissection.

PubMed

Teutsch, Christine; Kondo, Richard P; Dederko, Dorothy A; Chrast, Jacqueline; Chien, Kenneth R; Giles, Wayne R

2007-03-01

Regional differences in repolarizing K(+) current densities and expression levels of their molecular components are important for coordinating the pattern of electrical excitation and repolarization of the heart. The small size of hearts from mice may obscure these interventricular and/or transmural expression differences of K(+) channels. We have examined this possibility in adult mouse ventricle using a technology that provides very high spatial resolution of tissue collection. Conventional manual dissection and laser capture microdissection (LCM) were utilized to dissect tissue from distinct ventricular regions. RNA was isolated from epicardial, mid-myocardial and endocardial layers of both the right and left ventricles. Real-time RT-PCR was used to quantify the transcript expression in these different regions. LCM revealed significant interventricular and transmural gradients for both Kv4.2 and the alpha-subunit of KChIP2. The expression profile of a second K(+) channel transcript, Kir2.1, which is responsible for the inwardly rectifying K(+) current I(k1), showed no interventricular or transmural gradients and therefore served as a negative control. Our findings are in contrast to previous reports of a relatively uniform left ventricular transmural pattern of expression of Kv4.2, Kv4.3 and KChIP2 in adult mouse heart, which appear to be different than that in larger mammals. Specifically, our results demonstrate significant epi- to endocardial differences in the patterns of expression of both Kv4.2 and KChIP2.

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.

Determination of EGFR mutations in single cells microdissected from enriched lung tumor cells in peripheral blood.

PubMed

Ran, Ran; Li, Longyun; Wang, Mengzhao; Wang, Shulan; Zheng, Zhi; Lin, Peter Ping

2013-09-01

A minimally invasive and repeatable approach for real-time epidermal growth factor receptor (EGFR) mutation surveillance would be highly beneficial for individualized therapy of late stage lung cancer patients whose surgical specimens are often not available. We aim to develop a viable method to detect EGFR mutations in single circulating tumor cells (CTCs). Using a model CTC system of spiked tumor cells in whole blood, we evaluated EGFR mutation determination in single tumor cells enriched from blood. We used magnetic beads labeled with antibody against leukocyte surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level. We then used laser cell microdissection (LCM) to isolate individual CTCs, followed by whole-genome amplification of the DNA for exon 19 microdeletion, L858R and T790M mutation detection by PCR sequencing. EGFR mutations were successfully measured in individual spiked tumor cells enriched from 7.5 ml whole blood. Whole-genome amplification provided sufficient DNA for mutation determination at multiple sites. Ninety-five percent of the single CTCs microdissected by LCM (19/20) yielded PCR amplicons for at least one of the three mutation sites. The amplification success rates were 55 % (11/20) for exon 19 deletion, 45 % (9/20) for T790M, and 85 % (17/20) for L858R. Sequencing of the amplicons showed allele dropout in the amplification reactions, but mutations were correctly identified in 80 % of the amplicons. EGFR mutation determination from single captured tumor cells from blood is feasible with the approach described here. However, to overcome allele dropout and to obtain reliable information about the tumor's EGFR status, multiple individual tumor cells should be assayed.

A novel whole tooth-in-jaw-bone culture of rat molars: morphological, immunohistochemical, and laser capture microdissection analysis.

PubMed

Chokechanachaisakul, Uraiwan; Kaneko, Tomoatsu; Yamanaka, Yusuke; Okiji, Takashi; Suda, Hideaki

2012-10-01

In conventional whole-tooth culture systems, limitation exists regarding maintenance of the vitality of the dental pulp, because this tissue is encased in rigid dentin walls that hinder nutrition supply. We here report a whole tooth-in-jaw-bone culture system of rat mandibular first molars, where transcardiac perfusion with culture medium was carried out before placement of the jaw bone into culture medium, aiming to facilitate longer time preservation of the dental pulp tissue. Following 7 days of culture, the pulp tissues were analyzed by histology and immunohistochemistry to ED2 (antiresident macrophage). ED2-positive macrophages were also analyzed for their Class II MHC, interleukin-6 (IL-6), and p53 mRNA expression levels by means of immune-laser capture microdissection (immune-LCM). Dentin sialophosphoprotein (DSPP) mRNA expression in odontobalstic layer was also examined by LCM. Teeth cultured following saline-perfusion and nonperfusion served as cultured controls. Normal teeth also served as noncultured controls. Histological examination demonstrated that the structure of the pulp tissue was well preserved in the medium-perfused explants in contrast to the cultured control groups. The Class II MHC, IL-6, and p53 mRNA expression levels of ED2-positive cells and DSPP expression levels of odontoblastic layer tissues in the pulp of medium-perfused explants were not significantly different from those in the noncultured normal teeth. In conclusion, the structural integrity and mRNA expression in the pulp were maintained at the in vivo level in the ex vivo whole tooth-in-jaw-bone culture system. The system may lay the foundation for studies aiming at defining further histological and molecular mechanism of the pulp. Copyright © 2012 Wiley Periodicals, Inc.

Single Cell Multiplex Protein Measurements through Rare Earth Element Immunolabeling, Laser Capture Microdissection and Inductively Coupled Mass Spectrometry.

PubMed

Liba, Amir; Wanagat, Jonathan

2014-11-01

Complex diseases such as heart disease, stroke, cancer, and aging are the primary causes of death in the US. These diseases cause heterogeneous conditions among cells, conditions that cannot be measured in tissue homogenates and require single cell approaches. Understanding protein levels within tissues is currently assayed using various molecular biology techniques (e.g., Western blots) that rely on milligram to gram quantities of tissue homogenates or immunofluorescent (IF) techniques that are limited by spectral overlap. Tissue homogenate studies lack references to tissue structure and mask signals from individual or rare cellular events. Novel techniques are required to bring protein measurement sensitivity to the single cell level and offer spatiotemporal resolution and scalability. We are developing a novel approach to protein quantification by exploiting the inherently low concentration of rare earth elements (REE) in biological systems. By coupling REE-antibody immunolabeling of cells with laser capture microdissection (LCM) and ICP-QQQ, we are achieving multiplexed protein measurement in histological sections of single cells. This approach will add to evolving single cell techniques and our ability to understand cellular heterogeneity in complex biological systems and diseases.

Spatial transcriptomic analysis of cryosectioned tissue samples with Geo-seq.

PubMed

Chen, Jun; Suo, Shengbao; Tam, Patrick Pl; Han, Jing-Dong J; Peng, Guangdun; Jing, Naihe

2017-03-01

Conventional gene expression studies analyze multiple cells simultaneously or single cells, for which the exact in vivo or in situ position is unknown. Although cellular heterogeneity can be discerned when analyzing single cells, any spatially defined attributes that underpin the heterogeneous nature of the cells cannot be identified. Here, we describe how to use Geo-seq, a method that combines laser capture microdissection (LCM) and single-cell RNA-seq technology. The combination of these two methods enables the elucidation of cellular heterogeneity and spatial variance simultaneously. The Geo-seq protocol allows the profiling of transcriptome information from only a small number cells and retains their native spatial information. This protocol has wide potential applications to address biological and pathological questions of cellular properties such as prospective cell fates, biological function and the gene regulatory network. Geo-seq has been applied to investigate the spatial transcriptome of mouse early embryo, mouse brain, and pathological liver and sperm tissues. The entire protocol from tissue collection and microdissection to sequencing requires ∼5 d, Data analysis takes another 1 or 2 weeks, depending on the amount of data and the speed of the processor.

Identification of the Neuromuscular Junction Transcriptome of Extraocular Muscle by Laser Capture Microdissection

PubMed Central

Ketterer, Caroline; Zeiger, Ulrike; Budak, Murat T.; Rubinstein, Neal A.; Khurana, Tejvir S.

2010-01-01

Purpose. To examine and characterize the profile of genes expressed at the synapses or neuromuscular junctions (NMJs) of extraocular muscles (EOMs) compared with those expressed at the tibialis anterior (TA). Methods. Adult rat eyeballs with rectus EOMs attached and TAs were dissected, snap frozen, serially sectioned, and stained for acetylcholinesterase (AChE) to identify the NMJs. Approximately 6000 NMJs for rectus EOM (EOMsyn), 6000 NMJs for TA (TAsyn), equal amounts of NMJ-free fiber regions (EOMfib, TAfib), and underlying myonuclei and RNAs were captured by laser capture microdissection (LCM). RNA was processed for microarray-based expression profiling. Expression profiles and interaction lists were generated for genes differentially expressed at synaptic and nonsynaptic regions of EOM (EOMsyn versus EOMfib) and TA (TAsyn versus TAfib). Profiles were validated by using real-time quantitative polymerase chain reaction (qPCR). Results. The regional transcriptomes associated with NMJs of EOMs and TAs were identified. Two hundred seventy-five genes were preferentially expressed in EOMsyn (compared with EOMfib), 230 in TAsyn (compared with TAfib), and 288 additional transcripts expressed in both synapses. Identified genes included novel genes as well as well-known, evolutionarily conserved synaptic markers (e.g., nicotinic acetylcholine receptor (AChR) alpha (Chrna) and epsilon (Chrne) subunits and nestin (Nes). Conclusions. Transcriptome level differences exist between EOM synaptic regions and TA synaptic regions. The definition of the synaptic transcriptome provides insight into the mechanism of formation and functioning of the unique synapses of EOM and their differential involvement in diseases noted in the EOM allotype. PMID:20393109

Expression profiling during ocular development identifies 2 Nlz genes with a critical role in optic fissure closure.

PubMed

Brown, Jacob D; Dutta, Sunit; Bharti, Kapil; Bonner, Robert F; Munson, Peter J; Dawid, Igor B; Akhtar, Amana L; Onojafe, Ighovie F; Alur, Ramakrishna P; Gross, Jeffrey M; Hejtmancik, J Fielding; Jiao, Xiaodong; Chan, Wai-Yee; Brooks, Brian P

2009-02-03

The gene networks underlying closure of the optic fissure during vertebrate eye development are poorly understood. Here, we profile global gene expression during optic fissure closure using laser capture microdissected (LCM) tissue from the margins of the fissure. From these data, we identify a unique role for the C(2)H(2) zinc finger proteins Nlz1 and Nlz2 in normal fissure closure. Gene knockdown of nlz1 and/or nlz2 in zebrafish leads to a failure of the optic fissure to close, a phenotype which closely resembles that seen in human uveal coloboma. We also identify misregulation of pax2 in the developing eye of morphant fish, suggesting that Nlz1 and Nlz2 act upstream of the Pax2 pathway in directing proper closure of the optic fissure.

Bioinformatic investigation of the role of ubiquitins in cucumber flower morphogenesis

NASA Astrophysics Data System (ADS)

Pawełkowicz, Magdalena; Osipowski, Paweł; Wojcieszek, Michał; Kowalczuk, Cezary; PlÄ der, Wojciech; Przybecki, Zbigniew

2016-09-01

Three cDNA clones were used to screen cucumber genome in order to find genes and proteins. Functional annotation reveals that they are correlated with ubiquitination pathways. Various bioinformatics tools were used to screen and check protein sequences features such as: the presence of specific domains, transmembrane regions, cleavage site and cellular placement. The computational analysis for promotor region shows many binding sites for transcription factors, which could regulate the expression of genes. In order to check gene expression levels in developing flower buds of monoecious (B10) and gynoecious (2gg) cucumber lines, the real - time PCR technique was applied. The expression was checked for the whole buds and only for the 3rd and 4th whorls of bud when generative organ are form which were obtained by Laser Capture Microdissection (LCM) technique.

Quantitative proteomic analysis of microdissected oral epithelium for cancer biomarker discovery.

PubMed

Xiao, Hua; Langerman, Alexander; Zhang, Yan; Khalid, Omar; Hu, Shen; Cao, Cheng-Xi; Lingen, Mark W; Wong, David T W

2015-11-01

Specific biomarkers are urgently needed for the detection and progression of oral cancer. The objective of this study was to discover cancer biomarkers from oral epithelium through utilizing high throughput quantitative proteomics approaches. Morphologically malignant, epithelial dysplasia, and adjacent normal epithelial tissues were laser capture microdissected (LCM) from 19 patients and used for proteomics analysis. Total proteins from each group were extracted, digested and then labelled with corresponding isobaric tags for relative and absolute quantitation (iTRAQ). Labelled peptides from each sample were combined and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) for protein identification and quantification. In total, 500 proteins were identified and 425 of them were quantified. When compared with adjacent normal oral epithelium, 17 and 15 proteins were consistently up-regulated or down-regulated in malignant and epithelial dysplasia, respectively. Half of these candidate biomarkers were discovered for oral cancer for the first time. Cornulin was initially confirmed in tissue protein extracts and was further validated in tissue microarray. Its presence in the saliva of oral cancer patients was also explored. Myoglobin and S100A8 were pre-validated by tissue microarray. These data demonstrated that the proteomic biomarkers discovered through this strategy are potential targets for oral cancer detection and salivary diagnostics. Copyright © 2015 Elsevier Ltd. All rights reserved.

Global proteome profiling of dental cementum under experimentally-induced apposition.

PubMed

Salmon, Cristiane R; Giorgetti, Ana Paula O; Paes Leme, Adriana Franco; Domingues, Romênia R; Sallum, Enilson Antonio; Alves, Marcelo C; Kolli, Tamara N; Foster, Brian L; Nociti, Francisco H

2016-06-01

Dental cementum (DC) covers the tooth root and has important functions in tooth attachment and position. DC can be lost to disease, and regeneration is currently unpredictable due to limited understanding of DC formation. This study used a model of experimentally-induced apposition (EIA) in mice to identify proteins associated with new DC formation. Mandibular first molars were induced to super-erupt for 6 and 21days after extracting opposing maxillary molars. Decalcified and formalin-fixed paraffin-embedded mandible sections were prepared for laser capture microdissection. Microdissected protein extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and the data submitted to repeated measure ANOVA test (RM-ANOVA, alpha=5%). A total of 519 proteins were identified, with 97 (18.6%) proteins found exclusively in EIA sites and 50 (9.6%) proteins exclusively expressed in control sites. Fifty six (10.7%) proteins were differentially regulated by RM-ANOVA (p<0.05), with 24 regulated by the exclusive effect of EIA (12 proteins) or the interaction between EIA and time (12 proteins), including serpin 1a, procollagen C-endopeptidase enhancer, tenascin X (TNX), and asporin (ASPN). In conclusion, proteomic analysis demonstrated significantly altered protein profile in DC under EIA, providing new insights on DC biology and potential candidates for tissue engineering applications. Dental cementum (DC) is a mineralized tissue that covers the tooth root surface and has important functions in tooth attachment and position. DC and other periodontal tissues can be lost to disease, and regeneration is currently unpredictable due to lack of understanding of DC formation. This study used a model of experimentally-induced apposition (EIA) in mice to promote new cementum formation, followed by laser capture microdissection (LCM) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) proteomic analysis. This approach identified proteins associated with new cementum formation that may be targets for promoting cementum regeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

Transcript analysis of laser capture microdissected white matter astrocytes and higher phenol sulfotransferase 1A1 expression during autoimmune neuroinflammation.

PubMed

Guillot, Flora; Garcia, Alexandra; Salou, Marion; Brouard, Sophie; Laplaud, David A; Nicot, Arnaud B

2015-07-04

Astrocytes, the most abundant cell population in mammal central nervous system (CNS), contribute to a variety of functions including homeostasis, metabolism, synapse formation, and myelin maintenance. White matter (WM) reactive astrocytes are important players in amplifying autoimmune demyelination and may exhibit different changes in transcriptome profiles and cell function in a disease-context dependent manner. However, their transcriptomic profile has not yet been defined because they are difficult to purify, compared to gray matter astrocytes. Here, we isolated WM astrocytes by laser capture microdissection (LCM) in a murine model of multiple sclerosis to better define their molecular profile focusing on selected genes related to inflammation. Based on previous data indicating anti-inflammatory effects of estrogen only at high nanomolar doses, we also examined mRNA expression for enzymes involved in steroid inactivation. Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL6 mice with MOG35-55 immunization. Fluorescence activated cell sorting (FACS) analysis of a portion of individual spinal cords at peak disease was used to assess the composition of immune cell infiltrates. Using custom Taqman low-density-array (TLDA), we analyzed mRNA expression of 40 selected genes from immuno-labeled laser-microdissected WM astrocytes from lumbar spinal cord sections of EAE and control mice. Immunohistochemistry and double immunofluorescence on control and EAE mouse spinal cord sections were used to confirm protein expression in astrocytes. The spinal cords of EAE mice were infiltrated mostly by effector/memory T CD4+ cells and macrophages. TLDA-based profiling of LCM-astrocytes identified EAE-induced gene expression of cytokines and chemokines as well as inflammatory mediators recently described in gray matter reactive astrocytes in other murine CNS disease models. Strikingly, SULT1A1, but not other members of the sulfotransferase family, was expressed in WM spinal cord astrocytes. Moreover, its expression was further increased in EAE. Immunohistochemistry on spinal cord tissues confirmed preferential expression of this enzyme in WM astrocytic processes but not in gray matter astrocytes. We described here for the first time the mRNA expression of several genes in WM astrocytes in a mouse model of multiple sclerosis. Besides expected pro-inflammatory chemokines and specific inflammatory mediators increased during EAE, we evidenced relative high astrocytic expression of the cytoplasmic enzyme SULT1A1. As the sulfonation activity of SULT1A1 inactivates estradiol among other phenolic substrates, its high astrocytic expression may account for the relative resistance of this cell population to the anti-neuroinflammatory effects of estradiol. Blocking the activity of this enzyme during neuroinflammation may thus help the injured CNS to maintain the anti-inflammatory activity of endogenous estrogens or limit the dose of estrogen co-regimens for therapeutical purposes.

Maturation of the developing human fetal prostate in a rodent xenograft model

PubMed Central

Saffarini, Camelia M.; McDonnell, Elizabeth V.; Amin, Ali; Spade, Daniel J.; Huse, Susan M.; Kostadinov, Stefan; Hall, Susan J.; Boekelheide, Kim

2015-01-01

Background Prostate cancer is the most commonly diagnosed non-skin cancer in men. The etiology of prostate cancer is unknown, although both animal and epidemiologic data suggest that early life exposures to various toxicants, may impact DNA methylation status during development, playing an important role. Methods We have developed a xenograft model to characterize the growth and differentiation of human fetal prostate implants (gestational age 12-24 weeks) that can provide new data on the potential role of early life stressors on prostate cancer. The expression of key immunohistochemical markers responsible for prostate maturation was evaluated, including p63, cytokeratin 18, α-smooth muscle actin, vimentin, caldesmon, Ki-67, prostate specific antigen, estrogen receptor-α, and androgen receptor. Xenografts were separated into epithelial and stromal compartments using laser capture microdissection (LCM), and the DNA methylation status was assessed in >480,000 CpG sites throughout the genome. Results Xenografts demonstrated growth and maturation throughout the 200 days of post-implantation evaluation. DNA methylation profiles of laser capture micro-dissected tissue demonstrated tissue-specific markers clustered by their location in either the epithelium or stroma of human prostate tissue. Differential methylated promoter region CpG-associated gene analysis revealed significantly more stromal than epithelial DNA methylation in the 30 and 90-day xenografts. Functional classification analysis identified CpG-related gene clusters in methylated epithelial and stromal human xenografts. Conclusion This study of human fetal prostate tissue establishes a xenograft model that demonstrates dynamic growth and maturation, allowing for future mechanistic studies of the developmental origins of later life proliferative prostate disease. PMID:24038131

Distinct Transcriptional Changes and Epithelial-stromal Interactions are Altered in Early Stage Colon Cancer Development

PubMed Central

Mo, Allen; Jackson, Stephen; Varma, Kamini; Carpino, Alan; Giardina, Charles; Devers, Thomas J.; Rosenberg, Daniel W.

2016-01-01

While the progression of mutated colonic cells is dependent upon interactions between the initiated epithelium and surrounding stroma, the nature of these interactions is poorly understood. Here the development of an ultra-sensitive laser-capture microdissection (LCM)/RNA-seq approach for studying the epithelial and stromal compartments of aberrant crypt foci (ACF) is described. ACF are the earliest identifiable pre-neoplastic lesion found within the human colon and are detected using high-definition endoscopy with contrast dye-spray. The current analysis focused on the epithelium of ACF with somatic mutations to either KRAS, BRAF, or APC, with expression patterns compared to normal mucosa from each patient. By comparing gene expression patterns between groups, an increase in a number of pro-inflammatory NF-κB target genes were identified that were specific to ACF epithelium, including TIMP1, RELA and RELB. Distinct transcriptional changes associated with each somatic mutation were observed and a subset display a BRAFV600E-mediated senescence-associated transcriptome characterized by increased expression of CDKN2A. Finally, LCM-captured ACF-associated stroma was found to be transcriptionally distinct from normal stroma, with an up-regulation of genes related to immune cell infiltration and fibroblast activation. Immunofluorescence confirmed increased CD3+ T cells within the stromal microenvironment of ACF and an abundance of activated fibroblasts. Collectively, these results provide new insight into the cellular interplay that occurs at the earliest stages of colonic neoplasia, highlighting the important role of NF-kB, activated stromal fibroblasts and lymphocyte infiltration. Implications Fibroblasts and immune cells in the stromal microenvironment play an important role during the earliest stages of colon carcinogenesis. PMID:27353028

Identification of a putative protein profile associating with tamoxifen therapy resistance in breast cancer

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

Umar, Arzu; Kang, Hyuk; Timmermans, A. M.

2009-06-01

Tamoxifen-resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that associate with tamoxifen-resistance is a first step towards better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy-resistance in breast cancer, using nanoLC coupled with FTICR MS. Comparative proteome analysis was performed on ~5,500 pooled tumor cells (corresponding to ~550 ng protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data setsmore » (n=24 and n=27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag (AMT) reference databases.« less

Identification of a putative protein profile associated with tamoxifen therapy resistance in breast cancer.

PubMed

Umar, Arzu; Kang, Hyuk; Timmermans, Annemieke M; Look, Maxime P; Meijer-van Gelder, Marion E; den Bakker, Michael A; Jaitly, Navdeep; Martens, John W M; Luider, Theo M; Foekens, John A; Pasa-Tolić, Ljiljana

2009-06-01

Tamoxifen resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy resistance in breast cancer using nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on approximately 5,500 pooled tumor cells (corresponding to approximately 550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n = 24 and n = 27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with > or = 2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n = 156). Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant tumors and significantly associated with an earlier tumor progression following first line tamoxifen treatment (hazard ratio, 1.87; 95% confidence interval, 1.25-2.80; p = 0.002). In summary, comparative proteomics performed on laser capture microdissection-derived breast tumor cells using nano-LC-FTICR MS technology revealed a set of putative biomarkers associated with tamoxifen therapy resistance in recurrent breast cancer.

Laser Capture Microdissection Assisted Identification of Epithelial MicroRNA Expression Signatures for Prognosis of Stage I NSCLC

DTIC Science & Technology

2014-12-01

NAME(S) AND ADDRESS(ES) Health Research, Inc. 8. PERFORMING ORGANIZATION REPORT NUMBER Roswell Park Cancer Institute Division Elm and Carlton...Network Core at Roswell Park. Laser capture micro-dissection of these specimens will be performed to separate the epithelial from the stromal components...Buffalo Grove, IL) LMD6000 laser capture microdissection system in the Department of Pathology at Roswell Park Cancer Institute. The dissection was

Somatic mosaicism of androgen receptor CAG repeats in colorectal carcinoma epithelial cells from men.

PubMed

Di Fabio, Francesco; Alvarado, Carlos; Gologan, Adrian; Youssef, Emad; Voda, Linda; Mitmaker, Elliot; Beitel, Lenore K; Gordon, Philip H; Trifiro, Mark

2009-06-01

The X-linked human androgen receptor gene (AR) contains an exonic polymorphic trinucleotide CAG. The length of this encoded CAG tract inversely affects AR transcriptional activity. Colorectal carcinoma is known to express the androgen receptor, but data on somatic CAG repeat lengths variations in malignant and normal epithelial cells are still sporadic. Using laser capture microdissection (LCM), epithelial cells from colorectal carcinoma and normal-appearing mucosa were collected from the fresh tissue of eight consecutive male patients undergoing surgery (mean age, 70 y; range, 54-82). DNA isolated from each LCM sample underwent subsequent PCR and DNA sequencing to precisely determine AR CAG repeat lengths and the presence of microsatellite instability (MSI). Different AR CAG repeat lengths were observed in colorectal carcinoma (ranging from 0 to 36 CAG repeats), mainly in the form of multiple shorter repeat lengths. This genetic heterogeneity (somatic mosaicism) was also found in normal-appearing colorectal mucosa. Half of the carcinoma cases examined tended to have a higher number of AR CAG repeat lengths with a wider range of repeat size variation compared to normal mucosa. MSI carcinomas tended to have longer median AR CAG repeat lengths (n = 17) compared to microsatellite stable carcinomas (n = 14), although the difference was not significant (P = 0.31, Mann-Whitney test). Multiple unique somatic mutations of the AR CAG repeats occur in colorectal mucosa and in carcinoma, predominantly resulting in shorter alleles. Colorectal epithelial cells carrying AR alleles with shorter CAG repeat lengths may be more androgen-sensitive and therefore have a growth advantage.

BCL-2 as a Therapeutic Target in Human Tubulointerstitial Inflammation

PubMed Central

Ko, Kichul; Wang, Jianing; Perper, Stuart; Jiang, Yulei; Yanez, Denisse; Kaverina, Natalya; Ai, Junting; Liarski, Vladimir M.; Chang, Anthony; Peng, Yahui; Lan, Li; Westmoreland, Susan; Olson, Lisa; Giger, Maryellen L.; Wang, Li Chun; Clark, Marcus R.

2016-01-01

Objective In lupus nephritis (LuN), tubulointerstitial inflammation (TII) is associated with in situ adaptive immune cell networks that amplify local tissue damage. As patients with severe TII often fail conventional therapy and develop renal failure, understanding these in situ mechanisms might reveal new therapeutic targets. We hypothesized that in TII, dysregulated apoptotic regulators maintain local adaptive immunity and drive inflammation. Methods We developed novel computational approaches that, when applied to multicolor confocal images, quantified apoptotic regulator protein expression in selected lymphocyte subsets. This approach was validated using laser capture microdissection (LCM) coupled to qPCR. Furthermore, we explored the consequences of dysregulated apoptotic mediator expression in a murine model of LuN. Results Analyses of renal biopsies from LuN and mixed cellular allograft rejection patients revealed that BCL-2 was frequently expressed in infiltrating lymphocytes while expression of MCL-1 was low. In contrast, the reciprocal pattern of expression was observed in tonsil germinal centers. These results were consistent with RNA expression data obtained using LCM and qPCR. BCL-2 was also highly expressed in tubulointerstitial infiltrates of NZB/W F1 mice. Furthermore, treatment of NZB/W F1 mice with ABT-199, a selective oral inhibitor of BCL-2, prolonged survival and prevented proteinuria and development of TII in a prevention model. Interestingly, glomerular immune complexes were partially ameliorated by ABT-199 and serum anti-dsDNA antibody titers were unaffected. Conclusion These data demonstrate BCL-2 as an attractive therapeutic target in LuN manifesting TII. PMID:27159593

Deletions at SLC18A1 increased the risk of CRC and lower SLC18A1 expression associated with poor CRC outcome.

PubMed

Zhang, Dandan; Li, Zhenli; Xu, Xiaohong; Zhou, Dan; Tang, Shunli; Yin, Xiaoyang; Xu, Fangying; Li, Hui; Zhou, Yuan; Zhu, Tao; Deng, Hong; Zhang, Shuai; Huang, Qiong; Wang, Jing; Yin, Wei; Zhu, Yimin; Lai, Maode

2017-10-26

Copy number variations (CNVs) contribute to the development of colorectal cancer (CRC). We conducted a two-stage association study to identify CNV risk loci for CRC. We performed a gene-based rare CNV study on 694 sporadic CRC and 1641 controls using Illumina Human-OmniExpress-12v1.0 BeadChips, and further replicated in 934 CRC cases and 2680 controls for risk CNVs by using TaqMan Copy Number Assay. Tumor buddings, cancer cells in the center of primary tumor and normal intestinal epithelial cells were captured using laser capture microdissection (LCM) and were assayed using AffymetrixGeneChip® Human Genome U133 Plus 2.0 Array. In addition, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus data were assessed for the effects of risk CNVs. We found that germline deletions affecting the last six exons of SLC18A1 significantly associated with CRC with a combined P value of 6.4 × 10-5 by a two-stage analysis. Both in TCGA CRC RNA seq dataset and GDS4382, SLC18A1 was significantly down regulated in CRC tissues than in paired normal tissues (N = 32 and 17 pairs, P = 0.004 and 0.009, respectively). In LCM samples, similar observations were obtained that the expression levels of SLC18A1 in the tumor buddings, cancer cells in the center of primary tumor, and stroma of both tumor budding and cancer cells were lower than normal intestinal epithelial and stromal cells (fold change = 0.17-0.62, 0.12-0.57 and 0.37-0.68, respectively). In summary, the germline deletions at SLC18A1 contributed to the development of CRC. The role of SLC18A1 required further exploration. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Differences in shotgun protein expression profile between superficial bladder transitional cell carcinoma and normal urothelium.

PubMed

Niu, Hai Tao; Zhang, Yi Bing; Jiang, Hai Ping; Cheng, Bo; Sun, Guang; Wang, Yi; E, Ya Jun; Pang, De Quan; Chang, Ji Wu

2009-01-01

This study was undertaken to identify differences in protein expression profiles between superficial bladder transitional cell carcinoma (BTCC) and normal urothelial cells. We used laser capture microdissection (LCM) to harvest purified cells, and used two-dimensional liquid chromatography (2D-LC) followed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to separate and identify the peptide mixture. A total of 440/438 proteins commonly appeared in 4 paired specimens. Multi-step bioinformatic procedures were used for the analysis of identified proteins; 175/179 of the 293/287 proteins that were specific expressed in tumor/normal cells own gene ontology (GO) biological process annotation. Compared with the entire list of the international protein index (IPI), there are 52/46 GO terms exhibited as enriched and 6/10 exhibited as depleted, respectively. Significantly altered pathways between tumor and normal cells mainly include oxidative phosphorylation, focal adhesion, etc. Finally, descriptive statistics show that the shotgun proteomics strategy has practice directive significance for biomarker discovery by two-dimensional electrophoresis (2-DE) technology.

Comparative transcriptional survey between laser-microdissected cells from laminar abscission zone and petiolar cortical tissue during ethylene-promoted abscission in citrus leaves

PubMed Central

Agustí, Javier; Merelo, Paz; Cercós, Manuel; Tadeo, Francisco R; Talón, Manuel

2009-01-01

Background Abscission is the cell separation process by which plants are able to shed organs. It has a great impact on the yield of most crop plants. At the same time, the process itself also constitutes an excellent model to study cell separation processes, since it occurs in concrete areas known as abscission zones (AZs) which are composed of a specific cell type. However, molecular approaches are generally hampered by the limited area and cell number constituting the AZ. Therefore, detailed studies at the resolution of cell type are of great relevance in order to accurately describe the process and to identify potential candidate genes for biotechnological applications. Results Efficient protocols for the isolation of specific citrus cell types, namely laminar abscission zone (LAZ) and petiolar cortical (Pet) cells based on laser capture microdissection (LCM) and for RNA microextraction and amplification have been developed. A comparative transcriptome analysis between LAZ and Pet from citrus leaf explants subjected to an in-vitro 24 h ethylene treatment was performed utilising microarray hybridization and analysis. Our analyses of gene functional classes differentially represented in ethylene-treated LAZ revealed an activation program dominated by the expression of genes associated with protein synthesis, protein fate, cell type differentiation, development and transcription. The extensive repertoire of genes associated with cell wall biosynthesis and metabolism strongly suggests that LAZ layers activate both catabolic and anabolic wall modification pathways during the abscission program. In addition, over-representation of particular members of different transcription factor families suggests important roles for these genes in the differentiation of the effective cell separation layer within the many layers contained in the citrus LAZ. Preferential expression of stress-related and defensive genes in Pet reveals that this tissue is likely to be reprogrammed to prevent pathogen attacks and general abiotic stresses after organ shedding. Conclusion The LCM-based data generated in this survey represent the most accurate description of the main biological processes and genes involved in organ abscission in citrus. This study provides novel molecular insight into ethylene-promoted leaf abscission and identifies new putative target genes for characterization and manipulation of organ abscission in citrus. PMID:19852773

Gene Expression in Accumbens GABA Neurons from Inbred Rats with Different Drug-Taking Behavior

PubMed Central

Sharp, B.M.; Chen, H.; Gong, S.; Wu, X.; Liu, Z.; Hiler, K.; Taylor, W.L.; Matta, S.G.

2011-01-01

Inbred Lewis and Fisher 344 rat strains differ greatly in drug self-administration; Lewis rats operantly self-administer drugs of abuse including nicotine, whereas Fisher self-administer poorly. As shown herein, operant food self-administration is similar. Based on their pivotal role in drug reward, we hypothesized that differences in basal gene expression in GABAergic neurons projecting from nucleus accumbens (NAcc) to ventral pallidum (VP) play a role in vulnerability to drug taking behavior. The transcriptomes of NAcc shell-VP GABAergic neurons from these two strains were analyzed in adolescents, using a multidisciplinary approach that combined stereotaxic ionotophoretic brain microinjections, laser-capture microdissection (LCM) and microarray measurement of transcripts. LCM enriched the gene transcripts detected in GABA neurons compared to the residual NAcc tissue: a ratio of neuron/residual > 1 and false discovery rate (FDR) <5% yielded 6,623 transcripts, whereas a ratio of >3 yielded 3,514. Strain-dependent differences in gene expression within GABA neurons were identified; 322 vs. 60 transcripts showed 1.5-fold vs. 2-fold differences in expression (FDR<5%). Classification by gene ontology showed these 322 transcripts were widely distributed, without categorical enrichment. This is most consistent with a global change in GABA neuron function. Literature-mining by Chilibot found 38 genes related to synaptic plasticity, signaling and gene transcription, all of which determine drug-abuse; 33 genes have no known association with addiction or nicotine. In Lewis rats, upregulation of Mint-1, Cask, CamkIIδ, Ncam1, Vsnl1, Hpcal1 and Car8 indicates these transcripts likely contribute to altered signaling and synaptic function in NAcc GABA projection neurons to VP. PMID:21745336

Suppression of Radiation-Induced Testicular Germ Cell Apoptosis by 2,5-Hexanedione Pretreatment. III. Candidate Gene Analysis Identifies a Role for Fas in the Attenuation of X-ray–Induced Apoptosis

PubMed Central

Campion, Sarah N.; Sandrof, Moses A.; Yamasaki, Hideki; Boekelheide, Kim

2010-01-01

Germ cell apoptosis directly induced by x-radiation (x-ray) exposure is stage specific, with a higher incidence in stage II/III seminiferous tubules. A priming exposure to the Sertoli cell toxicant 2,5-hexanedione (HD) results in a marked reduction in x-ray–induced germ cell apoptosis in these affected stages. Because of the stage specificity of these responses, examination of associated gene expression in whole testis tissue has clear limitations. Laser capture microdissection (LCM) of specific cell populations in the testis is a valuable technique for investigating the responses of different cell types following toxicant exposure. LCM coupled with quantitative real-time PCR was performed to examine the expression of apoptosis-related genes at both early (3 h) and later (12 h) time points after x-ray exposure, with or without the priming exposure to HD. The mRNAs examined include Fas, FasL, caspase 3, bcl-2, p53, PUMA, and AEN, which were identified either by literature searches or microarray analysis. Group 1 seminiferous tubules (stages I–VI) exhibited the greatest changes in gene expression. Further analysis of this stage group (SG) revealed that Fas induction by x-ray is significantly attenuated by HD co-exposure. Selecting only for germ cells from seminiferous tubules of the most sensitive SG has provided further insight into the mechanisms involved in the co-exposure response. It is hypothesized that following co-exposure, germ cells adapt to the lack of Sertoli cell support by reducing the Fas response to normal FasL signals. These findings provide a better understanding and appreciation of the tissue complexity and technical difficulties associated with examining gene expression in the testis. PMID:20616204

Antimicrobial peptides and pro-inflammatory cytokines are differentially regulated across epidermal layers following bacterial stimuli.

PubMed

Percoco, Giuseppe; Merle, Chloé; Jaouen, Thomas; Ramdani, Yasmina; Bénard, Magalie; Hillion, Mélanie; Mijouin, Lily; Lati, Elian; Feuilloley, Marc; Lefeuvre, Luc; Driouich, Azeddine; Follet-Gueye, Marie-Laure

2013-12-01

The skin is a natural barrier between the body and the environment and is colonised by a large number of microorganisms. Here, we report a complete analysis of the response of human skin explants to microbial stimuli. Using this ex vivo model, we analysed at both the gene and protein level the response of epidermal cells to Staphylococcus epidermidis (S. epidermidis) and Pseudomonas fluorescens (P. fluorescens), which are present in the cutaneous microbiota. We showed that both bacterial species affect the structure of skin explants without penetrating the living epidermis. We showed by real-time quantitative polymerase chain reaction (qPCR) that S. epidermidis and P. fluorescens increased the levels of transcripts that encode antimicrobial peptides (AMPs), including human β defensin (hBD)2 and hBD3, and the pro-inflammatory cytokines interleukin (IL)-1α and (IL)-1-β, as well as IL-6. In addition, we analysed the effects of bacterial stimuli on the expression profiles of genes related to innate immunity and the inflammatory response across the epidermal layers, using laser capture microdissection (LCM) coupled to qPCR. We showed that AMP transcripts were principally upregulated in suprabasal keratinocytes. Conversely, the expression of pro-inflammatory cytokines was upregulated in the lower epidermis. These findings were confirmed by protein localisation using specific antibodies coupled to optical or electron microscopy. This work underscores the potential value of further studies that use LCM on human skin explants model to study the roles and effects of the epidermal microbiota on human skin physiology. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Molecular profiling of synchronous and metachronous cancers of the pancreas reveal molecular mimicry between samples from the same patient.

PubMed

Talbott, Vanessa A; Yeo, Charles J; Brody, Jonathan R; Witkiewicz, Agnieszka K

2012-07-01

Pancreatic ductal adenocarcinoma (PDA) is rarely a survivable disease. In rare cases, separate synchronous tumors are discovered at the time of resection, while in others, patients present with a metachronous cancer after prior surgical resection. Studying molecular markers of synchronous and metachronous lesions may aid to clarify the biology of this often deadly disease. Two patients presented with synchronous tumors (each one with a tumor in the pancreatic head/neck and the other in the tail, designated patients A and B). An additional patient (patient C) underwent an R0 resection for PDA of the head and recurred 1.5 y later with PDA in the tail. Genomic DNA was laser capture microdissected (LCM) from the tumor and molecular analysis was performed. K-ras status and loss of heterozygosity (LOH) were determined from multiple specimens for each case. All samples from each patient harbored identical K-ras mutations. In patient A, the tumor at the head of the pancreas had more clonal genetic instability as reflected by LOH analysis over multiple LCM samples. Patient B had more genetic instability in the tail lesion compared with the neck. Patient C had virtually the identical molecular profile in both tumors, supporting the notion that both tumors were related. We conclude that the synchronous and metachronous tumors likely are initiated from identical precursor lesions and/or events (i.e., K-ras mutations). Future studies will need to investigate if these tumors will respond similarly to adjuvant therapies targeted against the clonal molecular events in the tumor. Copyright © 2012 Elsevier Inc. All rights reserved.

Retinal cell responses to elevated intraocular pressure: a gene array comparison between the whole retina and retinal ganglion cell layer.

PubMed

Guo, Ying; Cepurna, William O; Dyck, Jennifer A; Doser, Tom A; Johnson, Elaine C; Morrison, John C

2010-06-01

To determine and compare gene expression patterns in the whole retina and retinal ganglion cell layer (RGCL) in a rodent glaucoma model. IOP was unilaterally elevated in Brown Norway rats (N = 26) by injection of hypertonic saline and monitored for 5 weeks. A cDNA microarray was used on whole retinas from one group of eyes with extensive optic nerve injury and on RGCL isolated by laser capture microdissection (LCM) from another group with comparable injury, to determine the significantly up- or downregulated genes and gene categories in both groups. Expression changes of selected genes were examined by quantitative reverse transcription-PCR (qPCR) to verify microarray results. Microarray analysis of the whole retina identified 632 genes with significantly changed expression (335 up, 297 down), associated with 9 upregulated and 3 downregulated biological processes. In contrast, the RGCL microarray yielded 3726 genes with significantly changed expression (2003 up, 1723 down), including 60% of those found in whole retina. Thirteen distinct upregulated biological processes were identified in the RGCL, dominated by protein synthesis. Among 11 downregulated processes, axon extension and dendrite morphogenesis and generation of precursor metabolism and energy were uniquely identified in the RGCL. qPCR confirmed significant changes in 6 selected messages in whole retina and 11 in RGCL. Increased Atf3, the most upregulated gene in the RGCL, was confirmed by immunohistochemistry of RGCs. Isolation of RGCL by LCM allows a more refined detection of gene response to elevated pressure and improves the potential of determining cellular mechanisms in RGCs and their supporting cells that could be targets for enhancing RGC survival.

TβRIII Expression in Human Breast Cancer Stroma and the Role of Soluble TβRIII in Breast Cancer Associated Fibroblasts.

PubMed

Jovanović, Bojana; Pickup, Michael W; Chytil, Anna; Gorska, Agnieszka E; Johnson, Kimberly C; Moses, Harold L; Owens, Philip

2016-11-04

The TGF-β pathway plays a major role in tumor progression through regulation of epithelial and stromal cell signaling. Dysfunction of the pathway can lead to carcinoma progression and metastasis. To gain insight into the stromal role of the TGF-β pathway in breast cancer, we performed laser capture microdissection (LCM) from breast cancer patients and reduction mammoplasty patients. Microdissected tumor stroma and normal breast stroma were examined for gene expression. Expression of the TGF-β type III receptor ( TGFBR3 ) was greatly decreased in the tumor stroma compared to control healthy breast tissue. These results demonstrated a 44-fold decrease in TGFBR3 mRNA in tumor stroma in comparison to control tissue. We investigated publicly available databases, and have identified that TGFBR3 mRNA levels are decreased in tumor stroma. We next investigated fibroblast cell lines derived from cancerous and normal breast tissue and found that in addition to mRNA levels, TβRIII protein levels were significantly reduced. Having previously identified that cancer-associated fibroblasts secrete greater levels of tumor promoting cytokines, we investigated the consequences of soluble-TβRIII (sTβRIII) on fibroblasts. Fibroblast conditioned medium was analyzed for 102 human secreted cytokines and distinct changes in response to sTβRIII were observed. Next, we used the fibroblast-conditioned medium to stimulate human monocyte cell line THP-1. These results indicate a distinct transcriptional response depending on sTβRIII treatment and whether it was derived from normal or cancerous breast tissue. We conclude that the effect of TβRIII has distinct roles not only in cancer-associated fibroblasts but that sTβRIII has distinct paracrine functions in the tumor microenvironment.

Microarray Detection Call Methodology as a Means to Identify and Compare Transcripts Expressed within Syncytial Cells from Soybean (Glycine max) Roots Undergoing Resistant and Susceptible Reactions to the Soybean Cyst Nematode (Heterodera glycines)

PubMed Central

Klink, Vincent P.; Overall, Christopher C.; Alkharouf, Nadim W.; MacDonald, Margaret H.; Matthews, Benjamin F.

2010-01-01

Background. A comparative microarray investigation was done using detection call methodology (DCM) and differential expression analyses. The goal was to identify genes found in specific cell populations that were eliminated by differential expression analysis due to the nature of differential expression methods. Laser capture microdissection (LCM) was used to isolate nearly homogeneous populations of plant root cells. Results. The analyses identified the presence of 13,291 transcripts between the 4 different sample types. The transcripts filtered down into a total of 6,267 that were detected as being present in one or more sample types. A comparative analysis of DCM and differential expression methods showed a group of genes that were not differentially expressed, but were expressed at detectable amounts within specific cell types. Conclusion. The DCM has identified patterns of gene expression not shown by differential expression analyses. DCM has identified genes that are possibly cell-type specific and/or involved in important aspects of plant nematode interactions during the resistance response, revealing the uniqueness of a particular cell population at a particular point during its differentiation process. PMID:20508855

Live cell isolation by laser microdissection with gravity transfer

NASA Astrophysics Data System (ADS)

Podgorny, Oleg V.

2013-05-01

Laser microdissection by pulsing ultraviolet laser allows the isolation and recultivation of live cells based on morphological features or/and fluorescent labelling from adherent cell cultures. Previous investigations described only the use of the laser microdissection and pressure catapulting (LMPC) for live cell isolation. But LMPC requires complex manipulations and some skill. Furthermore, single-cell cloning using laser microdissection has not yet been demonstrated. The first evidence of successful application of laser microdissection with gravity transfer (LMDGT) for capturing and recultivation of live cells is presented. A new strategy for LMDGT is presented because of the failure to reproduce the manufacturer's protocol. Using the new strategy, successful capturing and recultivation of circle-shaped samples from confluent monolayer of HeLa cells was demonstrated. It was found that LMDGT is easier than LMPC because it doesn't require personal participation of investigator in transferring of isolated samples to final culture dishes. Moreover, for the first time, the generation of clonal colonies from single live cells isolated by laser microdissection was demonstrated. Data obtained in this study confirm that LMDGT is a reliable and high-yield method allowing isolation and expansion of both cell clusters and single cells from adherent cell cultures.

DETECTION OF K-RAS AND P53 MUTATIONS IN SPUTUM SAMPLES OF LUNG CANCER PATIENTS USING LASER CAPTURE MICRODISSECTION MICROSCOPE AND MUTATION ANALYSIS

EPA Science Inventory

Detection of K-ras and p53 Mutations in Sputum Samples of Lung Cancer Patients Using Laser Capture Microdissection Microscope and Mutation Analysis

Phouthone Keohavong a,*, Wei-Min Gao a, Kui-Cheng Zheng a, Hussam Mady b, Qing Lan c, Mona Melhem b, and Judy Mumford d.
<...

Phrenic motoneuron expression of serotonergic and glutamatergic receptors following upper cervical spinal cord injury

PubMed Central

Mantilla, Carlos B.; Bailey, Jeffrey P.; Zhan, Wen-Zhi; Sieck, Gary C.

2012-01-01

Following cervical spinal cord injury at C2 (SH hemisection model) there is progressive recovery of phrenic activity. Neuroplasticity in the postsynaptic expression of neurotransmitter receptors may contribute to functional recovery. Phrenic motoneurons express multiple serotonergic (5-HTR) and glutamatergic (GluR) receptors, but the timing and possible role of these different neurotransmitter receptor subtypes in the neuroplasticity following SH are not clear. The current study was designed to test the hypothesis that there is an increased expression of serotonergic and glutamatergic neurotransmitter receptors within phrenic motoneurons after SH. In adult male rats, phrenic motoneurons were labeled retrogradely by intrapleural injection of Alexa 488-conjugated cholera toxin B. In thin (10 μm) frozen sections of the spinal cord, fluorescently-labeled phrenic motoneurons were visualized for laser capture microdissection (LCM). Using quantitative real-time RT-PCR in LCM samples, the time course of changes in 5-HTR and GluR mRNA expression was determined in phrenic motoneurons up to 21 days post-SH. Expression of 5-HTR subtypes 1b, 2a and 2c and GluR subtypes AMPA, NMDA, mGluR1 and mGluR5 was evident in phrenic motoneurons from control and SH rats. Phrenic motoneuron expression of 5-HTR2a increased ~8-fold (relative to control) at 14 days post-SH, whereas NMDA expression increased ~16-fold by 21-days post-SH. There were no other significant changes in receptor expression at any time post-SH. This is the first study to systematically document changes in motoneuron expression of multiple neurotransmitter receptors involved in regulation of motoneuron excitability. By providing information on the neuroplasticity of receptors expressed in a motoneuron pool that is inactivated by a higher-level spinal cord injury, appropriate pharmacological targets can be identified to alter motoneuron excitability. PMID:22227062

4-protein signature predicting tamoxifen treatment outcome in recurrent breast cancer.

PubMed

De Marchi, Tommaso; Liu, Ning Qing; Stingl, Cristoph; Timmermans, Mieke A; Smid, Marcel; Look, Maxime P; Tjoa, Mila; Braakman, Rene B H; Opdam, Mark; Linn, Sabine C; Sweep, Fred C G J; Span, Paul N; Kliffen, Mike; Luider, Theo M; Foekens, John A; Martens, John W M; Umar, Arzu

2016-01-01

Estrogen receptor (ER) positive tumors represent the majority of breast malignancies, and are effectively treated with hormonal therapies, such as tamoxifen. However, in the recurrent disease resistance to tamoxifen therapy is common and a major cause of death. In recent years, in-depth proteome analyses have enabled identification of clinically useful biomarkers, particularly, when heterogeneity in complex tumor tissue was reduced using laser capture microdissection (LCM). In the current study, we performed high resolution proteomic analysis on two cohorts of ER positive breast tumors derived from patients who either manifested good or poor outcome to tamoxifen treatment upon recurrence. A total of 112 fresh frozen tumors were collected from multiple medical centers and divided into two sets: an in-house training and a multi-center test set. Epithelial tumor cells were enriched with LCM and analyzed by nano-LC Orbitrap mass spectrometry (MS), which yielded >3000 and >4000 quantified proteins in the training and test sets, respectively. Raw data are available via ProteomeXchange with identifiers PXD000484 and PXD000485. Statistical analysis showed differential abundance of 99 proteins, of which a subset of 4 proteins was selected through a multivariate step-down to develop a predictor for tamoxifen treatment outcome. The 4-protein signature significantly predicted poor outcome patients in the test set, independent of predictive histopathological characteristics (hazard ratio [HR] = 2.17; 95% confidence interval [CI] = 1.15 to 4.17; multivariate Cox regression p value = 0.017). Immunohistochemical (IHC) staining of PDCD4, one of the signature proteins, on an independent set of formalin-fixed paraffin-embedded tumor tissues provided and independent technical validation (HR = 0.72; 95% CI = 0.57 to 0.92; multivariate Cox regression p value = 0.009). We hereby report the first validated protein predictor for tamoxifen treatment outcome in recurrent ER-positive breast cancer. IHC further showed that PDCD4 is an independent marker. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Targeted MS Assay Predicting Tamoxifen Resistance in Estrogen-Receptor-Positive Breast Cancer Tissues and Sera.

PubMed

De Marchi, Tommaso; Kuhn, Erik; Dekker, Lennard J; Stingl, Christoph; Braakman, Rene B H; Opdam, Mark; Linn, Sabine C; Sweep, Fred C G J; Span, Paul N; Luider, Theo M; Foekens, John A; Martens, John W M; Carr, Steven A; Umar, Arzu

2016-04-01

We recently reported on the development of a 4-protein-based classifier (PDCD4, CGN, G3BP2, and OCIAD1) capable of predicting outcome to tamoxifen treatment in recurrent, estrogen-receptor-positive breast cancer based on high-resolution MS data. A precise and high-throughput assay to measure these proteins in a multiplexed, targeted fashion would be favorable to measure large numbers of patient samples to move these findings toward a clinical setting. By coupling immunoprecipitation to multiple reaction monitoring (MRM) MS and stable isotope dilution, we developed a high-precision assay to measure the 4-protein signature in 38 primary breast cancer whole tissue lysates (WTLs). Furthermore, we evaluated the presence and patient stratification capabilities of our signature in an independent set of 24 matched (pre- and post-therapy) sera. We compared the performance of immuno-MRM (iMRM) with direct MRM in the absence of fractionation and shotgun proteomics in combination with label-free quantification (LFQ) on both WTL and laser capture microdissected (LCM) tissues. Measurement of the 4-proteins by iMRM showed not only higher accuracy in measuring proteotypic peptides (Spearman r: 0.74 to 0.93) when compared with MRM (Spearman r: 0.0 to 0.76) but also significantly discriminated patient groups based on treatment outcome (hazard ratio [HR]: 10.96; 95% confidence interval [CI]: 4.33 to 27.76; Log-rank P < 0.001) when compared with LCM (HR: 2.85; 95% CI: 1.24 to 6.54; Log-rank P = 0.013) and WTL (HR: 1.16; 95% CI: 0.57 to 2.33; Log-rank P = 0.680) LFQ-based predictors. Serum sample analysis by iMRM confirmed the detection of the four proteins in these samples. We hereby report that iMRM outperformed regular MRM, confirmed our previous high-resolution MS results in tumor tissues, and has shown that the 4-protein signature is measurable in serum samples.

MicroRNA-145 is regulated by DNA methylation and p53 gene mutation in prostate cancer

PubMed Central

Suh, Seong O.; Chen, Yi; Zaman, Mohd Saif; Hirata, Hiroshi; Yamamura, Soichiro; Shahryari, Varahram; Liu, Jan; Tabatabai, Z.Laura; Kakar, Sanjay; Deng, Guoren; Tanaka, Yuichiro; Dahiya, Rajvir

2011-01-01

MiR-145 is downregulated in various cancers including prostate cancer. However, the underlying mechanisms of miR-145 downregulation are not fully understood. Here, we reported that miR-145 was silenced through DNA hypermethylation and p53 mutation status in laser capture microdissected (LCM) prostate cancer and matched adjacent normal tissues. In 22 of 27 (81%) prostate tissues, miR-145 was significantly downregulated in the cancer compared with the normal tissues. Further studies on miR-145 downregulation mechanism showed that miR-145 is methylated at the promoter region in both prostate cancer tissues and 50 different types of cancer cell lines. In seven cancer cell lines with miR-145 hypermethylation, 5-aza-2′-deoxycytidine treatment dramatically induced miR-145 expression. Interestingly, we also found a significant correlation between miR-145 expression and the status of p53 gene in both LCM prostate tissues and 47 cancer cell lines. In 29 cell lines with mutant p53, miR-145 levels were downregulated in 28 lines (97%), whereas in 18 cell lines with wild-type p53 (WT p53), miR-145 levels were downregulated in only 6 lines (33%, P < 0.001). Electrophoretic mobility shift assay showed that p53 binds to the p53 response element upstream of miR-145, but the binding was inhibited by hypermethylation. To further confirm that p53 binding to miR-145 could regulate miR-145 expression, we transfected WT p53 and MUT p53 into PC-3 cells and found that miR-145 is upregulated by WT p53 but not with MUTp53. The apoptotic cells are increased after WT p53 transfection. In summary, this is the first report documenting that downregulation of miR-145 is through DNA methylation and p53 mutation pathways in prostate cancer. PMID:21349819

Biomarkers of anhedonic-like behavior, antidepressant drug refraction, and stress resilience in a rat model of depression.

PubMed

Christensen, T; Bisgaard, C F; Wiborg, O

2011-11-24

The aim of the present study was to identify potential biomarkers for depression in the search for novel disease targets and treatment regimens. Furthermore, the study includes a search for biomarkers involved in treatment resistance and stress resilience in order to investigate mechanisms underlying antidepressant drug refraction and stress-coping strategies. Depression-related transcriptomic changes in gene expression profiles were investigated in laser-captured microdissected (LCM) rat hippocampal granular cell layers (GCL) using the chronic mild stress (CMS) rat model of depression and chronic administration of two selective serotonin reuptake inhibitors (SSRIs), escitalopram and sertraline. CMS rats were segregated into diverging groups according to behavioral readouts, and under stringent constraints, the associated differential gene regulations were analyzed. Accordingly, we identified four genes associated with recovery, two genes implicated in treatment resistance, and three genes involved in stress resilience. The identified genes associated with mechanisms of cellular plasticity, including signal transduction, cell proliferation, cell differentiation, and synaptic release. Hierarchical clustering analysis confirmed the subgroup segregation pattern in the CMS model. Thus antidepressant treatment refractors cluster with anhedonic-like rats, and, interestingly, stress-resilient rats cluster with rats undergoing antidepressant-mediated recovery from anhedonia, suggesting antidepressant mechanisms of action to emulate endogenous stress-coping strategies. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

EB1 protein alteration characterizes sporadic but not ulcerative colitis associated colorectal cancer.

PubMed

Gemoll, Timo; Kollbeck, Sophie L; Karstens, Karl F; Hò, Gia G; Hartwig, Sonja; Strohkamp, Sarah; Schillo, Katharina; Thorns, Christoph; Oberländer, Martina; Kalies, Kathrin; Lehr, Stefan; Habermann, Jens K

2017-08-15

While carcinogenesis in Sporadic Colorectal Cancer (SCC) has been thoroughly studied, less is known about Ulcerative Colitis associated Colorectal Cancer (UCC). This study aimed to identify and validate differentially expressed proteins between clinical samples of SCC and UCC to elucidate new insights of UCC/SCC carcinogenesis and progression. Multiplex-fluorescence two-dimensional gel electrophoresis (2-D DIGE) and mass spectrometry identified 67 proteoforms representing 43 distinct proteins. After analysis by Ingenuity Pathway Analysis ® (IPA), subsequent Western blot validation proofed the differential expression of Heat shock 27 kDA protein 1 (HSPB1) and Microtubule-associated protein R/EB family, member 1 (EB1) while the latter one showed also expression differences by immunohistochemistry. Fresh frozen tissue of UCC ( n = 10) matched with SCC ( n = 10) was investigated. Proteins of cancerous intestinal mucosal cells were obtained by Laser Capture Microdissection (LCM) and compared by 2-D DIGE. Significant spots were identified by mass spectrometry. After IPA, three proteins [EB1, HSPB1, and Annexin 5 (ANXA5)] were chosen for further validation by Western blotting and tissue microarray-based immunohistochemistry. This study identified significant differences in protein expression of colorectal carcinoma cells from UCC patients compared to patients with SCC. Particularly, EB1 was validated in an independent clinical cohort.

Apple polyphenol phloretin potentiates the anticancer actions of paclitaxel through induction of apoptosis in human hep G2 cells.

PubMed

Yang, Kuo-Ching; Tsai, Chia-Yi; Wang, Ying-Jan; Wei, Po-Li; Lee, Chia-Hwa; Chen, Jui-Hao; Wu, Chih-Hsiung; Ho, Yuan-Soon

2009-05-01

Phloretin (Ph), which can be obtained from apples, apple juice, and cider, is a known inhibitor of the type II glucose transporter (GLUT2). In this study, real-time PCR analysis of laser-capture microdissected (LCM) human hepatoma cells showed elevated expression (>5-fold) of GLUT2 mRNA in comparison with nonmalignant hepatocytes. In vitro and in vivo studies were performed to assess Ph antitumor activity when combined with paclitaxel (PTX) for treatment of human liver cancer cells. Inhibition of GLUT2 by Ph potentiated the anticancer effects of PTX, resensitizing human liver cancer cells to drugs. These results demonstrate that 50-150 microM Ph significantly potentiates DNA laddering induced in Hep G2 cells by 10 nM PTX. Activity assays showed that caspases 3, 8, and 9 are involved in this apoptosis. The antitumor therapeutic efficacy of Ph (10 mg/kg body weight) was determined in cells of the SCID mouse model that were treated in parallel with PTX (1 mg/kg body weight). The Hep G2-xenografted tumor volume was reduced more than fivefold in the Ph + PTX-treated mice compared to the PTX-treated group. These results suggest that Ph may be useful for cancer chemotherapy and chemoprevention.

Site-specific programming of the host epithelial transcriptome by the gut microbiota.

PubMed

Sommer, Felix; Nookaew, Intawat; Sommer, Nina; Fogelstrand, Per; Bäckhed, Fredrik

2015-03-28

The intestinal epithelium separates us from the microbiota but also interacts with it and thus affects host immune status and physiology. Previous studies investigated microbiota-induced responses in the gut using intact tissues or unfractionated epithelial cells, thereby limiting conclusions about regional differences in the epithelium. Here, we sought to investigate microbiota-induced transcriptional responses in specific fractions of intestinal epithelial cells. To this end, we used microarray analysis of laser capture microdissection (LCM)-harvested ileal and colonic tip and crypt epithelial fractions from germ-free and conventionally raised mice and from mice during the time course of colonization. We found that about 10% of the host's transcriptome was microbially regulated, mainly including genes annotated with functions in immunity, cell proliferation, and metabolism. The microbial impact on host gene expression was highly site specific, as epithelial responses to the microbiota differed between cell fractions. Specific transcriptional regulators were enriched in each fraction. In general, the gut microbiota induced a more rapid response in the colon than in the ileum. Our study indicates that the microbiota engage different regulatory networks to alter host gene expression in a particular niche. Understanding host-microbiota interactions on a cellular level may facilitate signaling pathways that contribute to health and disease and thus provide new therapeutic strategies.

Selective decline of neurotrophin and neurotrophin receptor genes within CA1 pyramidal neurons and hippocampus proper: Correlation with cognitive performance and neuropathology in mild cognitive impairment and Alzheimer's disease.

PubMed

Ginsberg, Stephen D; Malek-Ahmadi, Michael H; Alldred, Melissa J; Che, Shaoli; Elarova, Irina; Chen, Yinghua; Jeanneteau, Freddy; Kranz, Thorsten M; Chao, Moses V; Counts, Scott E; Mufson, Elliott J

2017-09-09

Hippocampal CA1 pyramidal neurons, a major component of the medial temporal lobe memory circuit, are selectively vulnerable during the progression of Alzheimer's disease (AD). The cellular mechanism(s) underlying degeneration of these neurons and the relationship to cognitive performance remains largely undefined. Here, we profiled neurotrophin and neurotrophin receptor gene expression within microdissected CA1 neurons along with regional hippocampal dissections from subjects who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI), or AD using laser capture microdissection (LCM), custom-designed microarray analysis, and qPCR of CA1 subregional dissections. Gene expression levels were correlated with cognitive test scores and AD neuropathology criteria. We found a significant downregulation of several neurotrophin genes (e.g., Gdnf, Ngfb, and Ntf4) in CA1 pyramidal neurons in MCI compared to NCI and AD subjects. In addition, the neurotrophin receptor transcripts TrkB and TrkC were decreased in MCI and AD compared to NCI. Regional hippocampal dissections also revealed select neurotrophic gene dysfunction providing evidence for vulnerability within the hippocampus proper during the progression of dementia. Downregulation of several neurotrophins of the NGF family and cognate neurotrophin receptor (TrkA, TrkB, and TrkC) genes correlated with antemortem cognitive measures including the Mini-Mental State Exam (MMSE), a composite global cognitive score (GCS), and Episodic, Semantic, and Working Memory, Perceptual Speed, and Visuospatial domains. Significant correlations were found between select neurotrophic expression downregulation and neuritic plaques (NPs) and neurofibrillary tangles (NFTs), but not diffuse plaques (DPs). These data suggest that dysfunction of neurotrophin signaling complexes have profound negative sequelae within vulnerable hippocampal cell types, which play a role in mnemonic and executive dysfunction during the progression of AD. © 2017 Wiley Periodicals, Inc.

Spatially Directed Proteomics of the Human Lens Outer Cortex Reveals an Intermediate Filament Switch Associated With the Remodeling Zone

PubMed Central

Wenke, Jamie L.; McDonald, W. Hayes; Schey, Kevin L.

2016-01-01

Purpose To quantify protein changes in the morphologically distinct remodeling zone (RZ) and adjacent regions of the human lens outer cortex using spatially directed quantitative proteomics. Methods Lightly fixed human lens sections were deparaffinized and membranes labeled with fluorescent wheat germ agglutinin (WGA-TRITC). Morphology directed laser capture microdissection (LCM) was used to isolate tissue from four distinct regions of human lens outer cortex: differentiating zone (DF), RZ, transition zone (TZ), and inner cortex (IC). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the plasma membrane fraction from three lenses (21-, 22-, and 27-year) revealed changes in major cytoskeletal proteins including vimentin, filensin, and phakinin. Peptides from proteins of interest were quantified using multiple reaction monitoring (MRM) mass spectrometry and isotopically-labeled internal peptide standards. Results Results revealed an intermediate filament switch from vimentin to beaded filament proteins filensin and phakinin that occurred at the RZ. Several other cytoskeletal proteins showed significant changes between regions, while most crystallins remained unchanged. Targeted proteomics provided accurate, absolute quantification of these proteins and confirmed vimentin, periplakin, and periaxin decrease from the DF to the IC, while filensin, phakinin, and brain acid soluble protein 1 (BASP1) increase significantly at the RZ. Conclusions Mass spectrometry-compatible fixation and morphology directed laser capture enabled proteomic analysis of narrow regions in the human lens outer cortex. Results reveal dramatic cytoskeletal protein changes associated with the RZ, suggesting that one role of these proteins is in membrane deformation and/or the establishment of ball and socket joints in the human RZ. PMID:27537260

Alterations of Dermal Connective Tissue Collagen in Diabetes: Molecular Basis of Aged-Appearing Skin

PubMed Central

Argyropoulos, Angela J.; Robichaud, Patrick; Balimunkwe, Rebecca Mutesi; Fisher, Gary J.; Hammerberg, Craig; Yan, Yan

2016-01-01

Alterations of the collagen, the major structural protein in skin, contribute significantly to human skin connective tissue aging. As aged-appearing skin is more common in diabetes, here we investigated the molecular basis of aged-appearing skin in diabetes. Among all known human matrix metalloproteinases (MMPs), diabetic skin shows elevated levels of MMP-1 and MMP-2. Laser capture microdissection (LCM) coupled real-time PCR indicated that elevated MMPs in diabetic skin were primarily expressed in the dermis. Furthermore, diabetic skin shows increased lysyl oxidase (LOX) expression and higher cross-linked collagens. Atomic force microscopy (AFM) further indicated that collagen fibrils were fragmented/disorganized, and key mechanical properties of traction force and tensile strength were increased in diabetic skin, compared to intact/well-organized collagen fibrils in non-diabetic skin. In in vitro tissue culture system, multiple MMPs including MMP-1 and MM-2 were induced by high glucose (25 mM) exposure to isolated primary human skin dermal fibroblasts, the major cells responsible for collagen homeostasis in skin. The elevation of MMPs and LOX over the years is thought to result in the accumulation of fragmented and cross-linked collagen, and thus impairs dermal collagen structural integrity and mechanical properties in diabetes. Our data partially explain why old-looking skin is more common in diabetic patients. PMID:27104752

Transcriptional analysis of phloem-associated cells of potato.

PubMed

Lin, Tian; Lashbrook, Coralie C; Cho, Sung Ki; Butler, Nathaniel M; Sharma, Pooja; Muppirala, Usha; Severin, Andrew J; Hannapel, David J

2015-09-03

Numerous signal molecules, including proteins and mRNAs, are transported through the architecture of plants via the vascular system. As the connection between leaves and other organs, the petiole and stem are especially important in their transport function, which is carried out by the phloem and xylem, especially by the sieve elements in the phloem system. The phloem is an important conduit for transporting photosynthate and signal molecules like metabolites, proteins, small RNAs, and full-length mRNAs. Phloem sap has been used as an unadulterated source to profile phloem proteins and RNAs, but unfortunately, pure phloem sap cannot be obtained in most plant species. Here we make use of laser capture microdissection (LCM) and RNA-seq for an in-depth transcriptional profile of phloem-associated cells of both petioles and stems of potato. To expedite our analysis, we have taken advantage of the potato genome that has recently been fully sequenced and annotated. Out of the 27 k transcripts assembled that we identified, approximately 15 k were present in phloem-associated cells of petiole and stem with greater than ten reads. Among these genes, roughly 10 k are affected by photoperiod. Several RNAs from this day length-regulated group are also abundant in phloem cells of petioles and encode for proteins involved in signaling or transcriptional control. Approximately 22 % of the transcripts in phloem cells contained at least one binding motif for Pumilio, Nova, or polypyrimidine tract-binding proteins in their downstream sequences. Highlighting the predominance of binding processes identified in the gene ontology analysis of active genes from phloem cells, 78 % of the 464 RNA-binding proteins present in the potato genome were detected in our phloem transcriptome. As a reasonable alternative when phloem sap collection is not possible, LCM can be used to isolate RNA from specific cell types, and along with RNA-seq, provides practical access to expression profiles of phloem tissue. The combination of these techniques provides a useful approach to the study of phloem and a comprehensive picture of the mechanisms associated with long-distance signaling. The data presented here provide valuable insights into potentially novel phloem-mobile mRNAs and phloem-associated RNA-binding proteins.

Simultaneous genomic identification and profiling of a single cell using semiconductor-based next generation sequencing.

PubMed

Watanabe, Manabu; Kusano, Junko; Ohtaki, Shinsaku; Ishikura, Takashi; Katayama, Jin; Koguchi, Akira; Paumen, Michael; Hayashi, Yoshiharu

2014-09-01

Combining single-cell methods and next-generation sequencing should provide a powerful means to understand single-cell biology and obviate the effects of sample heterogeneity. Here we report a single-cell identification method and seamless cancer gene profiling using semiconductor-based massively parallel sequencing. A549 cells (adenocarcinomic human alveolar basal epithelial cell line) were used as a model. Single-cell capture was performed using laser capture microdissection (LCM) with an Arcturus® XT system, and a captured single cell and a bulk population of A549 cells (≈ 10(6) cells) were subjected to whole genome amplification (WGA). For cell identification, a multiplex PCR method (AmpliSeq™ SNP HID panel) was used to enrich 136 highly discriminatory SNPs with a genotype concordance probability of 10(31-35). For cancer gene profiling, we used mutation profiling that was performed in parallel using a hotspot panel for 50 cancer-related genes. Sequencing was performed using a semiconductor-based bench top sequencer. The distribution of sequence reads for both HID and Cancer panel amplicons was consistent across these samples. For the bulk population of cells, the percentages of sequence covered at coverage of more than 100 × were 99.04% for the HID panel and 98.83% for the Cancer panel, while for the single cell percentages of sequence covered at coverage of more than 100 × were 55.93% for the HID panel and 65.96% for the Cancer panel. Partial amplification failure or randomly distributed non-amplified regions across samples from single cells during the WGA procedures or random allele drop out probably caused these differences. However, comparative analyses showed that this method successfully discriminated a single A549 cancer cell from a bulk population of A549 cells. Thus, our approach provides a powerful means to overcome tumor sample heterogeneity when searching for somatic mutations.

Laser Capture Microdissection Assisted Identification of Epithelial MicroRNA Expression Signatures for Prognosis of Stage I NSCLC

DTIC Science & Technology

2011-10-01

SiC  relativ stains (B) are p tandard error 0 ng of RN on of a muc , a finding s measured crease in t dissection l, given tha of total RN ummary o...the yield and quality of microRNAs from LMD microdissectates of FFPE tissues for downstream analysis. Materials and Methods Ethics statement

Mapping mitochondrial heteroplasmy in a Leydig tumor by laser capture micro-dissection and cycling temperature capillary electrophoresis.

PubMed

Refinetti, Paulo; Arstad, Christian; Thilly, William G; Morgenthaler, Stephan; Ekstrøm, Per Olaf

2017-01-01

The growth of tumor cells is accompanied by mutations in nuclear and mitochondrial genomes creating marked genetic heterogeneity. Tumors also contain non-tumor cells of various origins. An observed somatic mitochondrial mutation would have occurred in a founding cell and spread through cell division. Micro-anatomical dissection of a tumor coupled with assays for mitochondrial point mutations permits new insights into this growth process. More generally, the ability to detect and trace, at a histological level, somatic mitochondrial mutations in human tissues and tumors, makes these mutations into markers for lineage tracing. A tumor was first sampled by a large punch biopsy and scanned for any significant degree of heteroplasmy in a set of sequences containing known mutational hotspots of the mitochondrial genome. A heteroplasmic tumor was sliced at a 12 μm thickness and placed on membranes. Laser capture micro-dissection was used to take 25000 μm 2 subsamples or spots. After DNA amplification, cycling temperature capillary electrophoresis (CTCE) was used on the laser captured samples to quantify mitochondrial mutant fractions. Of six testicular tumors studied, one, a Leydig tumor, was discovered to carry a detectable degree of heteroplasmy for two separate point mutations: a C → T mutation at bp 64 and a T → C mutation found at bp 152. From this tumor, 381 spots were sampled with laser capture micro-dissection. The ordered distribution of spots exhibited a wide range of fractions of the mutant sequences from 0 to 100% mutant copies. The two mutations co-distributed in the growing tumor indicating they were present on the same genome copies in the founding cell. Laser capture microdissection of sliced tumor samples coupled with CTCE-based point mutation assays provides an effective and practical means to obtain maps of mitochondrial mutational heteroplasmy within human tumors.

LCM-seq reveals the crucial role of LsSOC1 in heat-promoted bolting of lettuce (Lactuca sativa L.).

PubMed

Chen, Zijing; Zhao, Wensheng; Ge, Danfeng; Han, Yingyan; Ning, Kang; Luo, Chen; Wang, Shenglin; Liu, Renyi; Zhang, Xiaolan; Wang, Qian

2018-05-17

Lettuce (Lactuca sativa L.) is one of the most economically important vegetables. The floral transition in lettuce is accelerated under high temperatures, which can significantly decrease yields. However, the molecular mechanism underlying the floral tranition in lettuce is poorly known. Using laser capture microdissection coupled with RNA sequencing, we isolated shoot apical meristem cells from the bolting-sensitive lettuce line S39 at four critical stages of development. Subsequently, we screened specifically for the flowering-related gene LsSOC1 during the floral transition through comparative transcriptomic analysis. Molecular biology, developmental biology, and biochemical tools were combined to investigate the biological function of LsSOC1 in lettuce. LsSOC1 knockdown by RNA interference resulted in a significant delay in the timing of bolting and insensitivity to high temperature, which indicated that LsSOC1 functions as an activator during heat-promoted bolting in lettuce. We determined that two heat-shock transcription factors, HsfA1e and HsfA4c, bound to the promoter of LsSOC1 to confirm that LsSOC1 played an important role in heat-promoted bolting. This study indicates that LsSOC1 plays a crucial role in the heat-promoted bolting process in lettuce. Further investigation of LsSOC1 may be useful for clarification of the bolting mechanism in lettuce. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Prediction of the in planta Phakopsora pachyrhizi secretome and potential effector families.

PubMed

de Carvalho, Mayra C da C G; Costa Nascimento, Leandro; Darben, Luana M; Polizel-Podanosqui, Adriana M; Lopes-Caitar, Valéria S; Qi, Mingsheng; Rocha, Carolina S; Carazzolle, Marcelo Falsarella; Kuwahara, Márcia K; Pereira, Goncalo A G; Abdelnoor, Ricardo V; Whitham, Steven A; Marcelino-Guimarães, Francismar C

2017-04-01

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, can cause losses greater than 80%. Despite its economic importance, there is no soybean cultivar with durable ASR resistance. In addition, the P. pachyrhizi genome is not yet available. However, the availability of other rust genomes, as well as the development of sample enrichment strategies and bioinformatics tools, has improved our knowledge of the ASR secretome and its potential effectors. In this context, we used a combination of laser capture microdissection (LCM), RNAseq and a bioinformatics pipeline to identify a total of 36 350 P. pachyrhizi contigs expressed in planta and a predicted secretome of 851 proteins. Some of the predicted secreted proteins had characteristics of candidate effectors: small size, cysteine rich, do not contain PFAM domains (except those associated with pathogenicity) and strongly expressed in planta. A comparative analysis of the predicted secreted proteins present in Pucciniales species identified new members of soybean rust and new Pucciniales- or P. pachyrhizi-specific families (tribes). Members of some families were strongly up-regulated during early infection, starting with initial infection through haustorium formation. Effector candidates selected from two of these families were able to suppress immunity in transient assays, and were localized in the plant cytoplasm and nuclei. These experiments support our bioinformatics predictions and show that these families contain members that have functions consistent with P. pachyrhizi effectors. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Laser Microdissection and Atmospheric Pressure Chemical Ionization Mass Spectrometry Coupled for Multimodal Imaging

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

Lorenz, Matthias; Ovchinnikova, Olga S; Kertesz, Vilmos

2013-01-01

This paper describes the coupling of ambient laser ablation surface sampling, accomplished using a laser capture microdissection system, with atmospheric pressure chemical ionization mass spectrometry for high spatial resolution multimodal imaging. A commercial laser capture microdissection system was placed in close proximity to a modified ion source of a mass spectrometer designed to allow for sampling of laser ablated material via a transfer tube directly into the ionization region. Rhodamine 6G dye of red sharpie ink in a laser etched pattern as well as cholesterol and phosphatidylcholine in a cerebellum mouse brain thin tissue section were identified and imaged frommore » full scan mass spectra. A minimal spot diameter of 8 m was achieved using the 10X microscope cutting objective with a lateral oversampling pixel resolution of about 3.7 m. Distinguishing between features approximately 13 m apart in a cerebellum mouse brain thin tissue section was demonstrated in a multimodal fashion including co-registered optical and mass spectral chemical images.« less

Making sense of sparse rating data in collaborative filtering via topographic organization of user preference patterns.

PubMed

Polcicová, Gabriela; Tino, Peter

2004-01-01

We introduce topographic versions of two latent class models (LCM) for collaborative filtering. Latent classes are topologically organized on a square grid. Topographic organization of latent classes makes orientation in rating/preference patterns captured by the latent classes easier and more systematic. The variation in film rating patterns is modelled by multinomial and binomial distributions with varying independence assumptions. In the first stage of topographic LCM construction, self-organizing maps with neural field organized according to the LCM topology are employed. We apply our system to a large collection of user ratings for films. The system can provide useful visualization plots unveiling user preference patterns buried in the data, without loosing potential to be a good recommender model. It appears that multinomial distribution is most adequate if the model is regularized by tight grid topologies. Since we deal with probabilistic models of the data, we can readily use tools from probability and information theories to interpret and visualize information extracted by our system.

Optimizing Frozen Sample Preparation for Laser Microdissection: Assessment of CryoJane Tape-Transfer System®

PubMed Central

Golubeva, Yelena G.; Smith, Roberta M.; Sternberg, Lawrence R.

2013-01-01

Laser microdissection is an invaluable tool in medical research that facilitates collecting specific cell populations for molecular analysis. Diversity of research targets (e.g., cancerous and precancerous lesions in clinical and animal research, cell pellets, rodent embryos, etc.) and varied scientific objectives, however, present challenges toward establishing standard laser microdissection protocols. Sample preparation is crucial for quality RNA, DNA and protein retrieval, where it often determines the feasibility of a laser microdissection project. The majority of microdissection studies in clinical and animal model research are conducted on frozen tissues containing native nucleic acids, unmodified by fixation. However, the variable morphological quality of frozen sections from tissues containing fat, collagen or delicate cell structures can limit or prevent successful harvest of the desired cell population via laser dissection. The CryoJane Tape-Transfer System®, a commercial device that improves cryosectioning outcomes on glass slides has been reported superior for slide preparation and isolation of high quality osteocyte RNA (frozen bone) during laser dissection. Considering the reported advantages of CryoJane for laser dissection on glass slides, we asked whether the system could also work with the plastic membrane slides used by UV laser based microdissection instruments, as these are better suited for collection of larger target areas. In an attempt to optimize laser microdissection slide preparation for tissues of different RNA stability and cryosectioning difficulty, we evaluated the CryoJane system for use with both glass (laser capture microdissection) and membrane (laser cutting microdissection) slides. We have established a sample preparation protocol for glass and membrane slides including manual coating of membrane slides with CryoJane solutions, cryosectioning, slide staining and dissection procedure, lysis and RNA extraction that facilitated efficient dissection and high quality RNA retrieval from CryoJane preparations. CryoJane technology therefore has the potential to facilitate standardization of laser microdissection slide preparation from frozen tissues. PMID:23805281

Proteomic Analysis of Matched Formalin-Fixed, Paraffin-Embedded Specimens in Patients with Advanced Serous Ovarian Carcinoma

PubMed Central

Smith, Ashlee L.; Sun, Mai; Bhargava, Rohit; Stewart, Nicolas A.; Flint, Melanie S.; Bigbee, William L.; Krivak, Thomas C.; Strange, Mary A.; Cooper, Kristine L.; Zorn, Kristin K.

2013-01-01

Objective: The biology of high grade serous ovarian carcinoma (HGSOC) is poorly understood. Little has been reported on intratumoral homogeneity or heterogeneity of primary HGSOC tumors and their metastases. We evaluated the global protein expression profiles of paired primary and metastatic HGSOC from formalin-fixed, paraffin-embedded (FFPE) tissue samples. Methods: After IRB approval, six patients with advanced HGSOC were identified with tumor in both ovaries at initial surgery. Laser capture microdissection (LCM) was used to extract tumor for protein digestion. Peptides were extracted and analyzed by reversed-phase liquid chromatography coupled to a linear ion trap mass spectrometer. Tandem mass spectra were searched against the UniProt human protein database. Differences in protein abundance between samples were assessed and analyzed by Ingenuity Pathway Analysis software. Immunohistochemistry (IHC) for select proteins from the original and an additional validation set of five patients was performed. Results: Unsupervised clustering of the abundance profiles placed the paired specimens adjacent to each other. IHC H-score analysis of the validation set revealed a strong correlation between paired samples for all proteins. For the similarly expressed proteins, the estimated correlation coefficients in two of three experimental samples and all validation samples were statistically significant (p < 0.05). The estimated correlation coefficients in the experimental sample proteins classified as differentially expressed were not statistically significant. Conclusion: A global proteomic screen of primary HGSOC tumors and their metastatic lesions identifies tumoral homogeneity and heterogeneity and provides preliminary insight into these protein profiles and the cellular pathways they constitute. PMID:28250404

Evidence for the Complexity of MicroRNA-Mediated Regulation in Ovarian Cancer: A Systems Approach

PubMed Central

Shahab, Shubin W.; Matyunina, Lilya V.; Mezencev, Roman; Walker, L. DeEtte; Bowen, Nathan J.; Benigno, Benedict B.; McDonald, John F.

2011-01-01

MicroRNAs (miRNAs) are short (∼22 nucleotides) regulatory RNAs that can modulate gene expression and are aberrantly expressed in many diseases including cancer. Previous studies have shown that miRNAs inhibit the translation and facilitate the degradation of their targeted messenger RNAs (mRNAs) making them attractive candidates for use in cancer therapy. However, the potential clinical utility of miRNAs in cancer therapy rests heavily upon our ability to understand and accurately predict the consequences of fluctuations in levels of miRNAs within the context of complex tumor cells. To evaluate the predictive power of current models, levels of miRNAs and their targeted mRNAs were measured in laser captured micro-dissected (LCM) ovarian cancer epithelial cells (CEPI) and compared with levels present in ovarian surface epithelial cells (OSE). We found that the predicted inverse correlation between changes in levels of miRNAs and levels of their mRNA targets held for only ∼11% of predicted target mRNAs. We demonstrate that this low inverse correlation between changes in levels of miRNAs and their target mRNAs in vivo is not merely an artifact of inaccurate miRNA target predictions but the likely consequence of indirect cellular processes that modulate the regulatory effects of miRNAs in vivo. Our findings underscore the complexities of miRNA-mediated regulation in vivo and the necessity of understanding the basis of these complexities in cancer cells before the therapeutic potential of miRNAs can be fully realized. PMID:21811625

Regulation of apoptosis by peroxisome proliferators.

PubMed

Roberts, Ruth A; Michel, Cecile; Coyle, Beth; Freathy, Caroline; Cain, Kelvin; Boitier, Eric

2004-04-01

Peroxisome proliferators (PPs) constitute a large and chemically diverse family of non-genotoxic rodent hepatocarcinogens that activate the PP-activated receptor alpha (PPARalpha). In order to investigate the hypothesis that PPs elicit their carcinogenic effects through the suppression of apoptosis, we established an in vitro assay for apoptosis using both primary rat hepatocytes and the FaO rat hepatoma cell line. Apoptosis was induced by transforming growth factor beta1 (TGFbeta1), the physiological negative regulator of liver growth. In this system, PPs could suppress both spontaneous and TGFbeta1-induced apoptosis. In order to understand the mechanisms of this regulation of apoptosis, we conducted microarray analysis followed by pathway-specific gene clustering in TGFbeta1-treated cells. After treatment, 76 genes were up-regulated and 185 were down-regulated more than 1.5-fold. Cluster analysis of up-regulated genes revealed three clusters, A-C. Cluster A (4h) was associated with 12% apoptosis and consisted of genes mainly of the cytoskeleton and extracellular matrix such as troponin and the proteoglycan SDC4. In cluster B (8h; 25% apoptosis), there were many pro- and anti-apoptotic genes such as XIAP, BAK1 and BAD, whereas at 16h (40% apoptosis) the regulated genes were mainly those of the cellular stress pathways such as the genes implicated in the activation of the transcription factor NFkappab. Genes found down-regulated in response to TGFbeta1 were mainly those associated with oxidative stress and several genes implicated in glutathione production and maintenance. Thus, TGFbeta1 may induce apoptosis via a down regulation of oxidant defence leading to the generation of reactive oxygen species. The ability of PPs to impact on these apoptosis pathways remains to be determined. To approach this question, we have developed a technique using laser capture microdissection of livers treated with the PP, clofibric acid coupled with gene expression array analysis. Results show that some of the key steps of the LCM process had an impact on the gene profiles generated. However, this did not preclude accurate determination of a PP-specific molecular signature. Thus, the choice of appropriate controls will ensure that meaningful gene expression analyses can be performed on tissue microdissected from the foci generated in clofibric acid treated livers. These data will allow the identification of specific genes that are regulated by PPs leading to changes in apoptosis and ultimately to tumours.

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

Molecular analysis of HER2 signaling in human breast cancer by functional protein pathway activation mapping

PubMed Central

Wulfkuhle, Julia D.; Berg, Daniela; Wolff, Claudia; Langer, Rupert; Tran, Kai; Illi, Julie; Espina, Virginia; Pierobon, Mariaelena; Deng, Jianghong; DeMichele, Angela; Walch, Axel; Bronger, Holger; Becker, Ingrid; Waldhör, Christine; Höfler, Heinz; Esserman, Laura; Liotta, Lance A.; Becker, Karl-Friedrich; Petricoin, Emanuel F.

2017-01-01

Purpose Targeting of the HER2 protein in human breast cancer represents a major advance in oncology, but relies on measurements of total HER2 protein and not HER2 signaling network activation. We utilized reverse phase protein microarrays (RPMAs) to measure total and phosphorylated HER2 in the context of HER family signaling to understand correlations between phosphorylated and total levels of HER2 and downstream signaling activity. Experimental Design Three independent study sets, comprising a total of 415 individual patient samples from flash frozen core biopsy samples and FFPE surgical and core samples, were analyzed via RPMA. The phosphorylation and total levels of the HER receptor family proteins and downstream signaling molecules were measured in laser capture microdissected (LCM) enriched tumor epithelium from 127 frozen pre-treatment core biopsy samples and whole tissue lysates from 288 FFPE samples and these results were compared to FISH and IHC. Results RPMA measurements of total HER2 were highly concordant (> 90% all sets) with FISH and/or IHC data, as was phosphorylation of HER2 in the FISH/IHC+ population. Phosphorylation analysis of HER family signaling identified HER2 activation in some FISH/IHC- tumors and, identical to that seen with FISH/IHC+ tumors, the HER2 activation was concordant with EGFR and HER3 phosphorylation and downstream signaling endpoint activation. Conclusions Molecular profiling of HER2 signaling of a large cohort of human breast cancer specimens using a quantitative and sensitive functional pathway activation mapping technique reveals IHC-/FISH-/pHER2+ tumors with HER2 pathway activation independent of total HER2 levels and functional signaling through HER3 and EGFR. PMID:23045247

Developmental Exposure to Estrogen Alters Differentiation and Epigenetic Programming in a Human Fetal Prostate Xenograft Model

PubMed Central

Saffarini, Camelia M.; McDonnell-Clark, Elizabeth V.; Amin, Ali; Huse, Susan M.; Boekelheide, Kim

2015-01-01

Prostate cancer is the most frequent non-cutaneous malignancy in men. There is strong evidence in rodents that neonatal estrogen exposure plays a role in the development of this disease. However, there is little information regarding the effects of estrogen in human fetal prostate tissue. This study explored early life estrogen exposure, with and without a secondary estrogen and testosterone treatment in a human fetal prostate xenograft model. Histopathological lesions, proliferation, and serum hormone levels were evaluated at 7, 30, 90, and 200-day time-points after xenografting. The expression of 40 key genes involved in prostatic glandular and stromal growth, cell-cycle progression, apoptosis, hormone receptors and tumor suppressors was evaluated using a custom PCR array. Epigenome-wide analysis of DNA methylation was performed on whole tissue, and laser capture-microdissection (LCM) isolated epithelial and stromal compartments of 200-day prostate xenografts. Combined initial plus secondary estrogenic exposures had the most severe tissue changes as revealed by the presence of hyperplastic glands at day 200. Gene expression changes corresponded with the cellular events in the KEGG prostate cancer pathway, indicating that initial plus secondary exposure to estrogen altered the PI3K-Akt signaling pathway, ultimately resulting in apoptosis inhibition and an increase in cell cycle progression. DNA methylation revealed that differentially methylated CpG sites significantly predominate in the stromal compartment as a result of estrogen-treatment, thereby providing new targets for future investigation. By using human fetal prostate tissue and eliminating the need for species extrapolation, this study provides novel insights into the gene expression and epigenetic effects related to prostate carcinogenesis following early life estrogen exposure. PMID:25799167

Interrogation of transcriptomic changes associated with drug-induced hepatic sinusoidal dilatation in colorectal cancer.

PubMed

Jarzabek, Monika A; Proctor, William R; Vogt, Jennifer; Desai, Rupal; Dicker, Patrick; Cain, Gary; Raja, Rajiv; Brodbeck, Jens; Stevens, Dale; van der Stok, Eric P; Martens, John W M; Verhoef, Cornelis; Hegde, Priti S; Byrne, Annette T; Tarrant, Jacqueline M

2018-01-01

Drug-related sinusoidal dilatation (SD) is a common form of hepatotoxicity associated with oxaliplatin-based chemotherapy used prior to resection of colorectal liver metastases (CRLM). Recently, hepatic SD has also been associated with anti-delta like 4 (DLL4) cancer therapies targeting the NOTCH pathway. To investigate the hypothesis that NOTCH signaling plays an important role in drug-induced SD, gene expression changes were examined in livers from anti-DLL4 and oxaliplatin-induced SD in non-human primate (NHP) and patients, respectively. Putative mechanistic biomarkers of bevacizumab (bev)-mediated protection against oxaliplatin-induced SD were also investigated. RNA was extracted from whole liver sections or centrilobular regions by laser-capture microdissection (LCM) obtained from NHP administered anti-DLL4 fragment antigen-binding (F(ab')2 or patients with CRLM receiving oxaliplatin-based chemotherapy with or without bev. mRNA expression was quantified using high-throughput real-time quantitative PCR. Significance analysis was used to identify genes with differential expression patterns (false discovery rate (FDR) < 0.05). Eleven (CCL2, CCND1, EFNB2, ERG, ICAM1, IL16, LFNG, NOTCH1, NOTCH4, PRDX1, and TGFB1) and six (CDH5, EFNB2, HES1, IL16, MIK67, HES1 and VWF) candidate genes were differentially expressed in the liver of anti-DLL4- and oxaliplatin-induced SD, respectively. Addition of bev to oxaliplatin-based chemotherapy resulted in differential changes in hepatic CDH5, HEY1, IL16, JAG1, MMP9, NOTCH4 and TIMP1 expression. This work implicates NOTCH and IL16 pathways in the pathogenesis of drug-induced SD and further explains the hepato-protective effect of bev in oxaliplatin-induced SD observed in CRLM patients.

Spatial-Resolution Cell Type Proteome Profiling of Cancer Tissue by Fully Integrated Proteomics Technology.

PubMed

Xu, Ruilian; Tang, Jun; Deng, Quantong; He, Wan; Sun, Xiujie; Xia, Ligang; Cheng, Zhiqiang; He, Lisheng; You, Shuyuan; Hu, Jintao; Fu, Yuxiang; Zhu, Jian; Chen, Yixin; Gao, Weina; He, An; Guo, Zhengyu; Lin, Lin; Li, Hua; Hu, Chaofeng; Tian, Ruijun

2018-05-01

Increasing attention has been focused on cell type proteome profiling for understanding the heterogeneous multicellular microenvironment in tissue samples. However, current cell type proteome profiling methods need large amounts of starting materials which preclude their application to clinical tumor specimens with limited access. Here, by seamlessly combining laser capture microdissection and integrated proteomics sample preparation technology SISPROT, specific cell types in tumor samples could be precisely dissected with single cell resolution and processed for high-sensitivity proteome profiling. Sample loss and contamination due to the multiple transfer steps are significantly reduced by the full integration and noncontact design. H&E staining dyes which are necessary for cell type investigation could be selectively removed by the unique two-stage design of the spintip device. This easy-to-use proteome profiling technology achieved high sensitivity with the identification of more than 500 proteins from only 0.1 mm 2 and 10 μm thickness colon cancer tissue section. The first cell type proteome profiling of four cell types from one colon tumor and surrounding normal tissue, including cancer cells, enterocytes, lymphocytes, and smooth muscle cells, was obtained. 5271, 4691, 4876, and 2140 protein groups were identified, respectively, from tissue section of only 5 mm 2 and 10 μm thickness. Furthermore, spatially resolved proteome distribution profiles of enterocytes, lymphocytes, and smooth muscle cells on the same tissue slices and across four consecutive sections with micrometer distance were successfully achieved. This fully integrated proteomics technology, termed LCM-SISPROT, is therefore promising for spatial-resolution cell type proteome profiling of tumor microenvironment with a minute amount of clinical starting materials.

Divergence between motoneurons: gene expression profiling provides a molecular characterization of functionally discrete somatic and autonomic motoneurons

PubMed Central

Cui, Dapeng; Dougherty, Kimberly J.; Machacek, David W.; Sawchuk, Michael; Hochman, Shawn; Baro, Deborah J.

2009-01-01

Studies in the developing spinal cord suggest that different motoneuron (MN) cell types express very different genetic programs, but the degree to which adult programs differ is unknown. To compare genetic programs between adult MN columnar cell types, we used laser capture micro-dissection (LCM) and Affymetrix microarrays to create expression profiles for three columnar cell types: lateral and medial MNs from lumbar segments and sympathetic preganglionic motoneurons located in the thoracic intermediolateral nucleus. A comparison of the three expression profiles indicated that ~7% (813/11,552) of the genes showed significant differences in their expression levels. The largest differences were observed between sympathetic preganglionic MNs and the lateral motor column, with 6% (706/11,552) of the genes being differentially expressed. Significant differences in expression were observed for 1.8% (207/11,552) of the genes when comparing sympathetic preganglionic MNs with the medial motor column. Lateral and medial MNs showed the least divergence, with 1.3% (150/11,552) of the genes being differentially expressed. These data indicate that the amount of divergence in expression profiles between identified columnar MNs does not strictly correlate with divergence of function as defined by innervation patterns (somatic/muscle vs. autonomic/viscera). Classification of the differentially expressed genes with regard to function showed that they underpin all fundamental cell systems and processes, although most differentially expressed genes encode proteins involved in signal transduction. Mining the expression profiles to examine transcription factors essential for MN development suggested that many of the same transcription factors participatein combinatorial codes in embryonic and adult neurons, but patterns of expression change significantly. PMID:16317082

ROLES OF ADIPOCYTES AND FIBROBLASTS IN ACTIVATION OF THE ALTERNATIVE PATHWAY OF COMPLEMENT IN INFLAMMATORY ARTHRITIS IN MICE

PubMed Central

Arend, William P.; Mehta, Gaurav; Antonioli, Alexandra H.; Takahashi, Minoru; Takahashi, Kazue; Stahl, Gregory L.; Holers, V. Michael; Banda, Nirmal K.

2013-01-01

The complement system is involved in mediation of joint damage in rheumatoid arthritis, with evidence suggesting activation of both the classical and alternative pathways (AP). The AP is both necessary and sufficient to mediate collagen antibody-induced arthritis (CAIA), an experimental animal model of immune complex (IC)-induced joint disease. The AP in mice is dependent on MASP-1/3 cleavage of pro-factor D (pro-FD) into mature FD. The objectives of the present study were to determine the cells synthesizing MASP-1/3 and pro-FD in synovial tissue. CAIA was studied in wild-type C57BL/6 mice, and the localization of mRNA and protein for FD and MASP-1/3 in synovial adipose tissue (SAT) and fibroblast-like synoviocytes (FLS) was determined using various techniques, including laser capture micro-dissection (LCM). SAT was the sole source of mRNA for pro-FD. Cultured differentiated 3T3 adipocytes, a surrogate for SAT, produced pro-FD but no mature FD. FLS were the main source of MASP-1/3 mRNA and protein. Using cartilage micro-particles (CMP) coated with anti-collagen mAb and serum from MASP-1/3−/− mice as a source of factor B, pro-FD in 3T3 supernatants was cleaved into mature FD by MASP-1/3 in FLS supernatants. The mature FD was eluted from the CMP, and was not present in the supernatants from the incubation with CMP, indicating that cleavage of pro-FD into mature FD by MASP-1 occurred on the CMP. These results demonstrate that pathogenic activation of the AP may occur in the joint through IC adherent to cartilage and the local production of necessary AP proteins by adipocytes and FLS. PMID:23650618

Preterm infant gut microbiota affects intestinal epithelial development in a humanized microbiome gnotobiotic mouse model.

PubMed

Yu, Yueyue; Lu, Lei; Sun, Jun; Petrof, Elaine O; Claud, Erika C

2016-09-01

Development of the infant small intestine is influenced by bacterial colonization. To promote establishment of optimal microbial communities in preterm infants, knowledge of the beneficial functions of the early gut microbiota on intestinal development is needed. The purpose of this study was to investigate the impact of early preterm infant microbiota on host gut development using a gnotobiotic mouse model. Histological assessment of intestinal development was performed. The differentiation of four epithelial cell lineages (enterocytes, goblet cells, Paneth cells, enteroendocrine cells) and tight junction (TJ) formation was examined. Using weight gain as a surrogate marker for health, we found that early microbiota from a preterm infant with normal weight gain (MPI-H) induced increased villus height and crypt depth, increased cell proliferation, increased numbers of goblet cells and Paneth cells, and enhanced TJs compared with the changes induced by early microbiota from a poor weight gain preterm infant (MPI-L). Laser capture microdissection (LCM) plus qRT-PCR further revealed, in MPI-H mice, a higher expression of stem cell marker Lgr5 and Paneth cell markers Lyz1 and Cryptdin5 in crypt populations, along with higher expression of the goblet cell and mature enterocyte marker Muc3 in villus populations. In contrast, MPI-L microbiota failed to induce the aforementioned changes and presented intestinal characteristics comparable to a germ-free host. Our data demonstrate that microbial communities have differential effects on intestinal development. Future studies to identify pioneer settlers in neonatal microbial communities necessary to induce maturation may provide new insights for preterm infant microbial ecosystem therapeutics. Copyright © 2016 the American Physiological Society.

Transcriptional Profiling of Foam Cells Reveals Induction of Guanylate-Binding Proteins Following Western Diet Acceleration of Atherosclerosis in the Absence of Global Changes in Inflammation.

PubMed

Goo, Young-Hwa; Son, Se-Hee; Yechoor, Vijay K; Paul, Antoni

2016-04-18

Foam cells are central to two major pathogenic processes in atherogenesis: cholesterol buildup in arteries and inflammation. The main underlying cause of cholesterol deposition in arteries is hypercholesterolemia. This study aimed to assess, in vivo, whether elevated plasma cholesterol also alters the inflammatory balance of foam cells. Apolipoprotein E-deficient mice were fed regular mouse chow through the study or were switched to a Western-type diet (WD) 2 or 14 weeks before death. Consecutive sections of the aortic sinus were used for lesion quantification or to isolate RNA from foam cells by laser-capture microdissection (LCM) for microarray and quantitative polymerase chain reaction analyses. WD feeding for 2 or 14 weeks significantly increased plasma cholesterol, but the size of atherosclerotic lesions increased only in the 14-week WD group. Expression of more genes was affected in foam cells of mice under prolonged hypercholesterolemia than in mice fed WD for 2 weeks. However, most transcripts coding for inflammatory mediators remained unchanged in both WD groups. Among the main players in inflammatory or immune responses, chemokine (C-X-C motif) ligand 13 was induced in foam cells of mice under WD for 2 weeks. The interferon-inducible GTPases, guanylate-binding proteins (GBP)3 and GBP6, were induced in the 14-week WD group, and other GBP family members were moderately increased. Our results indicate that acceleration of atherosclerosis by hypercholesterolemia is not linked to global changes in the inflammatory balance of foam cells. However, induction of GBPs uncovers a novel family of immune modulators with a potential role in atherogenesis. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Expression and distribution of TRPV2 in rat brain.

PubMed

Nedungadi, Thekkethil Prashant; Dutta, Mayurika; Bathina, Chandra Sekhar; Caterina, Michael J; Cunningham, J Thomas

2012-09-01

Transient receptor potential (TRP) proteins are non-selective cation channels that mediate sensory transduction. The neuroanatomical localization and the physiological roles of isoform TRPV2 in the rodent brain are largely unknown. We report here the neuroanatomical distribution of TRPV2 in the adult male rat brain focusing on the hypothalamus and hindbrain regions involved in osmoregulation, autonomic function and energy metabolism. For this we utilized immunohistochemistry combined with brightfield microscopy. In the forebrain, the densest immunostaining was seen in both the supraoptic nucleus (SON) and the magnocellular division of the paraventricular nucleus (PVN) of the hypothalamus. TRPV2 immunoreactivity was also seen in the organum vasculosum of the lamina terminalis, the median preoptic nucleus and the subfornical organ, in addition to the arcuate nucleus of the hypothalamus (ARH), the medial forebrain bundle, the cingulate cortex and the globus pallidus to name a few. In the hindbrain, intense staining was seen in the nucleus of the solitary tract, hypoglossal nucleus, nucleus ambiguous, and the rostral division of the ventrolateral medulla (RVLM) and some mild staining in the area prostrema. To ascertain the specificity of the TRPV2 antibody used in this paper, we compared the TRPV2 immunoreactivity of wildtype (WT) and knockout (KO) mouse brain tissue. Double immunostaining with arginine vasopressin (AVP) using confocal microscopy showed a high degree of colocalization of TRPV2 in the magnocellular SON and PVN. Using laser capture microdissection (LCM) we also show that AVP neurons in the SON contain TRPV2 mRNA. TRPV2 was also co-localized with dopamine beta hydroxylase (DBH) in the NTS and the RVLM of the hindbrain. Based on our results, TRPV2 may play an important role in several CNS networks that regulate body fluid homeostasis, autonomic function, and metabolism. Copyright © 2012 Elsevier Inc. All rights reserved.

Expression and Distribution of TRPV2 in Rat Brain

PubMed Central

Nedungadi, Thekkethil Prashant; Dutta, Mayurika; Bathina, Chandra Sekhar; Caterina, Michael J; Cunningham, J. Thomas

2012-01-01

Transient receptor potential (TRP) proteins are non-selective cation channels that mediate sensory transduction. The neuroanatomical localization and the physiological roles of isoform TRPV2 in the rodent brain are largely unknown. We report here the neuroanatomical distribution of TRPV2 in the adult male rat brain focusing on hypothalamus and hindbrain regions involved in osmoregulation, autonomic function and energy metabolism. For this we utilized immunohistochemistry combined with brighfield microscopy. In the forebrain, the densest immunostaining was seen in both the supraoptic nucleus (SON) and the magnocellular division of the paraventricular nucleus (PVN) of the hypothalamus. TRPV2 immunoreactivity was also seen in the organum vasculosum of the lamina terminalis, the median preoptic nucleus and the subfornical organ, in addition to the arcuate nucleus of the hypothalamus (ARH), the medial forebrain bundle, the cingulate cortex and the globus pallidus to name a few. In the hindbrain, intense staining was seen in the nucleus of the solitary tract, hypoglossal nucleus, nucleus ambiguous, and the rostral division of the ventrolateral medulla (RVLM) and some mild staining in the area prostrema. To ascertain the specificity of the TRPV2 antibody used in this paper, we compared the TRPV2 immunoreactivity of wildtype (WT) and knockout (KO) mouse brain tissue. Double immunostaining with arginine vasopressin (AVP) using confocal microscopy showed a high degree of colocalization of TRPV2 in the magnocellular SON and PVN. Using laser capture microdissection (LCM) we also show that AVP neurons in the SON contain TRPV2 mRNA. TRPV2 was also co-localized with dopamine beta hydroxylase (DBH) in the NTS and the RVLM of the hindbrain. Based on our results, TRPV2 may play an important role in several CNS networks that regulate body fluid homeostasis, autonomic function, and metabolism. PMID:22750329

Robust optical properties of sandwiched lateral composition modulation GaInP structure grown by molecular beam epitaxy

DOE PAGES

Park, Kwangwook; Kang, Seokjin; Ravindran, Sooraj; ...

2016-12-26

Double-hetero structure lateral composition modulated (LCM) GaInP and sandwiched LCM GaInP having the same active layer thickness were grown and their optical properties were compared. Sandwiched LCM GaInP showed robust optical properties due to periodic potential nature of the LCM structure, and the periodicity was undistorted even for thickness far beyond the critical layer thickness. A thick LCM GaInP structure with undistorted potential that could preserve the properties of native LCM structure was possible by stacking thin LCM GaInP structures interspaced with strain compensating GaInP layers. Furthermore, the sandwiched structure could be beneficial in realizing the LCM structure embedded highmore » efficiency solar cells.« less

Proteomic analysis of laser capture microscopy purified myotendinous junction regions from muscle sections

PubMed Central

2014-01-01

The myotendinous junction is a specialized structure of the muscle fibre enriched in mechanosensing complexes, including costameric proteins and core elements of the z-disc. Here, laser capture microdissection was applied to purify membrane regions from the myotendinous junctions of mouse skeletal muscles, which were then processed for proteomic analysis. Sarcolemma sections from the longitudinal axis of the muscle fibre were used as control for the specificity of the junctional preparation. Gene ontology term analysis of the combined lists indicated a statistically significant enrichment in membrane-associated proteins. The myotendinous junction preparation contained previously uncharacterized proteins, a number of z-disc costameric ligands (e.g., actinins, capZ, αB cristallin, filamin C, cypher, calsarcin, desmin, FHL1, telethonin, nebulin, titin and an enigma-like protein) and other proposed players of sarcomeric stretch sensing and signalling, such as myotilin and the three myomesin homologs. A subset were confirmed by immunofluorescence analysis as enriched at the myotendinous junction, suggesting that laser capture microdissection from muscle sections is a valid approach to identify novel myotendinous junction players potentially involved in mechanotransduction pathways. PMID:25071420

Circadian Profiling of Amino Acids in the SCN and Cerebral Cortex by Laser Capture Microdissection-Mass Spectrometry.

PubMed

Fustin, Jean-Michel; Karakawa, Sachise; Okamura, Hitoshi

2017-12-01

The suprachiasmatic nucleus (SCN) is an extremely robust self-sustained oscillator, containing virtually the same molecular clock present in other tissues in the body but, in addition, endowed with tight intercellular coupling dependent on multiple neurotransmitter systems that allow the SCN to function as the "master clock." Several studies on the circadian SCN transcriptome have been published and compared with the transcriptome of other tissues, but the recent focus shift toward the circadian metabolome and the importance of small molecules for circadian timekeeping has so far been limited to macroscopic tissues such as the liver. Here, we report the successful use of laser capture microdissection coupled with liquid chromatography/tandem mass spectrometry for the circadian profiling of SCN amino acids. Among 18 amino acids detected, 10 (55.5%) showed significant variations, particularly marked for proline, lysine, and histidine, with higher levels during the subjective day. Moreover, we compared SCN and cortical amino acid levels between wild-type and Bmal1-deficient animals, either in the whole body or specifically in the liver. Interestingly, lack of Bmal1 in the whole body led to a significant increase in most amino acids in the SCN but not in the cerebral cortex. In contrast, deletion of Bmal1 in the liver mostly affected cortical amino acid levels during the subjective day. This study demonstrates that laser capture microdissection can be used for the isolation of microscopic brain structures for metabolomic purposes and reveals interactions between liver and SCN amino acid metabolism.

Optical band gap determination of calcium doped lanthanum manganite nano particle tailored with polypyrrole

NASA Astrophysics Data System (ADS)

Gopalakrishna, Smitha Mysore; Murugendrappa, Malalkere Veerappa

2018-05-01

In this paper we bring forth the effect of La0.7Ca0.3MnO3 (LCM) perovskite nano particle on the optical band gap in composition with conducting Polypyrrole (PPy) prepared by chemical oxidation method. The morphology and crystalline phase were determined by SEM, TEM and X-Ray diffraction studies. The Optical band gap studies were analyzed using the UV-VIS spectrometer scanned in the range 200 nm to 600 nm for pure PPy and PPy/LCM composites. There is a characteristic peak observed for the composites situated around 315 nm for pure PPy, PPy/LCM10 and PPy/LCM50. But for higher compositions of LCM weight percentage like 30%, 40% and 50% the peak shift slightly to higher wavelength side. The peak shifts to 320 nm, 325 nm and 335 nm respectively. The optical band gap increased for Pure PPy, PPy/LCM10 and PPy/LCM20 and found to decrease gradually for PPy/LCM30, PPy/LCM40 and PPy/LCM50. The studies suggest that LCM composition in the PPy chain has a role in modifying the wavelength and in turn its band gap. The study may find application in organic devices working at high frequency and voltage.

Cell differentiation in cardiac myxomas: confocal microscopy and gene expression analysis after laser capture microdissection.

PubMed

Pucci, Angela; Mattioli, Claudia; Matteucci, Marco; Lorenzini, Daniele; Panvini, Francesca; Pacini, Simone; Ippolito, Chiara; Celiento, Michele; De Martino, Andrea; Dolfi, Amelio; Belgio, Beatrice; Bortolotti, Uberto; Basolo, Fulvio; Bartoloni, Giovanni

2018-05-22

Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.

mRNA expression profiling of laser microbeam microdissected cells from slender embryonic structures.

PubMed

Scheidl, Stefan J; Nilsson, Sven; Kalén, Mattias; Hellström, Mats; Takemoto, Minoru; Håkansson, Joakim; Lindahl, Per

2002-03-01

Microarray hybridization has rapidly evolved as an important tool for genomic studies and studies of gene regulation at the transcriptome level. Expression profiles from homogenous samples such as yeast and mammalian cell cultures are currently extending our understanding of biology, whereas analyses of multicellular organisms are more difficult because of tissue complexity. The combination of laser microdissection, RNA amplification, and microarray hybridization has the potential to provide expression profiles from selected populations of cells in vivo. In this article, we present and evaluate an experimental procedure for global gene expression analysis of slender embryonic structures using laser microbeam microdissection and laser pressure catapulting. As a proof of principle, expression profiles from 1000 cells in the mouse embryonic (E9.5) dorsal aorta were generated and compared with profiles for captured mesenchymal cells located one cell diameter further away from the aortic lumen. A number of genes were overexpressed in the aorta, including 11 previously known markers for blood vessels. Among the blood vessel markers were endoglin, tie-2, PDGFB, and integrin-beta1, that are important regulators of blood vessel formation. This demonstrates that microarray analysis of laser microbeam micro-dissected cells is sufficiently sensitive for identifying genes with regulative functions.

Pesticide residue concentration in soil following conventional and Low-Input Crop Management in a Mediterranean agro-ecosystem, in Central Greece.

PubMed

Karasali, Helen; Marousopoulou, Anna; Machera, Kyriaki

2016-01-15

The present study was focused on the comparative evaluation of pesticide residues, determined in soil samples from Kopaida region, Greece before and after the implementation of Low-Input Crop Management (LCM) protocols. LCM has been suggested as an environmental friendly plant protection approach to be applied on crops growing in vulnerable to pollution ecosystems, with special focus on the site specific problems. In the case of the specific pilot area, the vulnerability was mainly related to the pollution of water bodies from agrochemicals attributed to diffuse pollution primarily from herbicides and secondarily from insecticides. A total of sixty-six soil samples, were collected and analyzed during a three-year monitoring study and the results of the determined pesticide residues were considered for the impact evaluation of applied plant protection methodology. The LCM was developed and applied in the main crops growing in the pilot area i.e. cotton, maize and industrial tomato. Herbicides active ingredients such as ethalfluralin, trifluralin, pendimethalin, S-metolachlor and fluometuron were detected in most samples at various concentrations. Ethalfluralin, which was the active ingredient present in the majority of the samples ranged from 0.01 μg g(-1) to 0.26 μg g(-1) soil dry weight. However, the amount of herbicides measured after the implementation of LCM for two cropping periods, was reduced by more than 75% in all cases. The method of analysis was based on the simultaneous extraction of the target compounds by mechanical shaking, followed by liquid chromatography mass spectrometric and gas chromatography electron capture (LC-MS/MS and GC-ECD) analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

Online, absolute quantitation of propranolol from spatially distinct 20-μm and 40-μm dissections of brain, liver, and kidney thin tissue sections by laser microdissection – liquid vortex capture – mass spectrometry

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

Kertesz, Vilmos; Vavrek, Marissa; Freddo, Carol

Here, spatial resolved quantitation of chemical species in thin tissue sections by mass spectrometric methods has been constrained by the need for matrix-matched standards or other arduous calibration protocols and procedures to mitigate matrix effects (e.g., spatially varying ionization suppression). Reported here is the use of laser cut and drop sampling with a laser microdissection-liquid vortex capture electrospray ionization tandem mass spectrometry (LMD-LVC/ESI-MS/MS) system for online and absolute quantitation of propranolol in mouse brain, kidney, and liver thin tissue sections of mice administered with the drug at a 7.5 mg/kg dose, intravenously. In this procedure either 20 μm x 20more » μm or 40 μm x 40 μm tissue microdissections were cut and dropped into the flowing solvent of the capture probe. During transport to the ESI source drug related material was completely extracted from the tissue into the solvent, which contained a known concentration of propranolol-d 7 as an internal standard. This allowed absolute quantitation to be achieved with an external calibration curve generated from standards containing the same fixed concentration of propranolold-d 7 and varied concentrations of propranolol. Average propranolol concentrations determined with the laser cut and drop sampling method closely agreed with concentration values obtained from 2.3 mm diameter tissue punches from serial sections that were extracted and quantified by HPLC/ESI-MS/MS measurements. In addition, the relative abundance of hydroxypropranolol glucuronide metabolites were recorded and found to be consistent with previous findings.« less

Laser Capture Microdissection and Multiplex-Tandem PCR Analysis of Proximal Tubular Epithelial Cell Signaling in Human Kidney Disease

PubMed Central

Wilkinson, Ray; Wang, Xiangju; Kassianos, Andrew J.; Zuryn, Steven; Roper, Kathrein E.; Osborne, Andrew; Sampangi, Sandeep; Francis, Leo; Raghunath, Vishwas; Healy, Helen

2014-01-01

Interstitial fibrosis, a histological process common to many kidney diseases, is the precursor state to end stage kidney disease, a devastating and costly outcome for the patient and the health system. Fibrosis is historically associated with chronic kidney disease (CKD) but emerging evidence is now linking many forms of acute kidney disease (AKD) with the development of CKD. Indeed, we and others have observed at least some degree of fibrosis in up to 50% of clinically defined cases of AKD. Epithelial cells of the proximal tubule (PTEC) are central in the development of kidney interstitial fibrosis. We combine the novel techniques of laser capture microdissection and multiplex-tandem PCR to identify and quantitate “real time” gene transcription profiles of purified PTEC isolated from human kidney biopsies that describe signaling pathways associated with this pathological fibrotic process. Our results: (i) confirm previous in-vitro and animal model studies; kidney injury molecule-1 is up-regulated in patients with acute tubular injury, inflammation, neutrophil infiltration and a range of chronic disease diagnoses, (ii) provide data to inform treatment; complement component 3 expression correlates with inflammation and acute tubular injury, (iii) identify potential new biomarkers; proline 4-hydroxylase transcription is down-regulated and vimentin is up-regulated across kidney diseases, (iv) describe previously unrecognized feedback mechanisms within PTEC; Smad-3 is down-regulated in many kidney diseases suggesting a possible negative feedback loop for TGF-β in the disease state, whilst tight junction protein-1 is up-regulated in many kidney diseases, suggesting feedback interactions with vimentin expression. These data demonstrate that the combined techniques of laser capture microdissection and multiplex-tandem PCR have the power to study molecular signaling within single cell populations derived from clinically sourced tissue. PMID:24475278

Online, absolute quantitation of propranolol from spatially distinct 20-μm and 40-μm dissections of brain, liver, and kidney thin tissue sections by laser microdissection – liquid vortex capture – mass spectrometry

DOE PAGES

Kertesz, Vilmos; Vavrek, Marissa; Freddo, Carol; ...

2016-05-23

Here, spatial resolved quantitation of chemical species in thin tissue sections by mass spectrometric methods has been constrained by the need for matrix-matched standards or other arduous calibration protocols and procedures to mitigate matrix effects (e.g., spatially varying ionization suppression). Reported here is the use of laser cut and drop sampling with a laser microdissection-liquid vortex capture electrospray ionization tandem mass spectrometry (LMD-LVC/ESI-MS/MS) system for online and absolute quantitation of propranolol in mouse brain, kidney, and liver thin tissue sections of mice administered with the drug at a 7.5 mg/kg dose, intravenously. In this procedure either 20 μm x 20more » μm or 40 μm x 40 μm tissue microdissections were cut and dropped into the flowing solvent of the capture probe. During transport to the ESI source drug related material was completely extracted from the tissue into the solvent, which contained a known concentration of propranolol-d 7 as an internal standard. This allowed absolute quantitation to be achieved with an external calibration curve generated from standards containing the same fixed concentration of propranolold-d 7 and varied concentrations of propranolol. Average propranolol concentrations determined with the laser cut and drop sampling method closely agreed with concentration values obtained from 2.3 mm diameter tissue punches from serial sections that were extracted and quantified by HPLC/ESI-MS/MS measurements. In addition, the relative abundance of hydroxypropranolol glucuronide metabolites were recorded and found to be consistent with previous findings.« less

Intravenous lacosamide in seizure emergencies: Observations from a hospitalized in-patient adult population.

PubMed

d'Orsi, Giuseppe; Pascarella, Maria Grazia; Martino, Tommaso; Carapelle, Elena; Pacillo, Francesca; Di Claudio, Maria Teresa; Mancini, Daniela; Trivisano, Marina; Avolio, Carlo; Specchio, Luigi M

2016-11-01

to evaluate the efficacy and safety of intravenous (IV) lacosamide (LCM) in the treatment of seizure clusters (SC) and status epilepticus (SE) in hospitalized adult patients. we prospectively analyzed treatment response, seizure outcome, and adverse effects of IV LCM in 38 patients with seizure emergencies (15 with SC, 23 with SE) during a hospital stay. The loading dose of IV LCM was 200-400mg and the maintenance dose was 200-400mg daily. Response to IV LCM was evaluated within 20min, 4h and 24h of LCM infusion. an acute anti-seizure effect after IV LCM was especially evident when it was first used - (SC) or second line (established SE) treatment. In particular, 87% of SC patients (13/15) and 80% of established SE (8/10) demonstrated response to LCM treatment, while no patients with super-refractory SE (0/8) responded to IV LCM according to our criteria. The loading of IV LCM was well tolerated, with mild adverse effects (2/38 temporary dizziness). In most patients, during and after administration of the loading dose of IV LCM a temporary (30min-1h) sedation was observed. No ECG and laboratory values-changes were documented in any of the patients. LCM is an effective and well-tolerated treatment when used to treat SC in hospitalized adult patients. As add-on therapy, it may be useful to stop seizure activity in patients with focal SE not responding to first/second-line intravenous AEDs. Copyright © 2016 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

The Use of Laser Microdissection in Forensic Sexual Assault Casework: Pros and Cons Compared to Standard Methods.

PubMed

Costa, Sergio; Correia-de-Sá, Paulo; Porto, Maria J; Cainé, Laura

2017-07-01

Sexual assault samples are among the most frequently analyzed in a forensic laboratory. These account for almost half of all samples processed routinely, and a large portion of these cases remain unsolved. These samples often pose problems to traditional analytic methods of identification because they consist most frequently of cell mixtures from at least two contributors: the victim (usually female) and the perpetrator (usually male). In this study, we propose the use of current preliminary testing for sperm detection in order to determine the chances of success when faced with samples which can be good candidates to undergo analysis with the laser microdissection technology. Also, we used laser microdissection technology to capture fluorescently stained cells of interest differentiated by gender. Collected materials were then used for DNA genotyping with commercially available amplification kits such as Minifiler, Identifiler Plus, NGM, and Y-Filer. Both the methodology and the quality of the results were evaluated to assess the pros and cons of laser microdissection compared with standard methods. Overall, the combination of fluorescent staining combined with the Minifiler amplification kit provided the best results for autosomal markers, whereas the Y-Filer kit returned the expected results regardless of the used method. © 2017 American Academy of Forensic Sciences.

mRNA Expression Profiling of Laser Microbeam Microdissected Cells from Slender Embryonic Structures

PubMed Central

Scheidl, Stefan J.; Nilsson, Sven; Kalén, Mattias; Hellström, Mats; Takemoto, Minoru; Håkansson, Joakim; Lindahl, Per

2002-01-01

Microarray hybridization has rapidly evolved as an important tool for genomic studies and studies of gene regulation at the transcriptome level. Expression profiles from homogenous samples such as yeast and mammalian cell cultures are currently extending our understanding of biology, whereas analyses of multicellular organisms are more difficult because of tissue complexity. The combination of laser microdissection, RNA amplification, and microarray hybridization has the potential to provide expression profiles from selected populations of cells in vivo. In this article, we present and evaluate an experimental procedure for global gene expression analysis of slender embryonic structures using laser microbeam microdissection and laser pressure catapulting. As a proof of principle, expression profiles from 1000 cells in the mouse embryonic (E9.5) dorsal aorta were generated and compared with profiles for captured mesenchymal cells located one cell diameter further away from the aortic lumen. A number of genes were overexpressed in the aorta, including 11 previously known markers for blood vessels. Among the blood vessel markers were endoglin, tie-2, PDGFB, and integrin-β1, that are important regulators of blood vessel formation. This demonstrates that microarray analysis of laser microbeam micro-dissected cells is sufficiently sensitive for identifying genes with regulative functions. PMID:11891179

In Silico Docking and Electrophysiological Characterization of Lacosamide Binding Sites on Collapsin Response Mediator Protein-2 Identifies a Pocket Important in Modulating Sodium Channel Slow Inactivation*

PubMed Central

Wang, Yuying; Brittain, Joel M.; Jarecki, Brian W.; Park, Ki Duk; Wilson, Sarah M.; Wang, Bo; Hale, Rachel; Meroueh, Samy O.; Cummins, Theodore R.; Khanna, Rajesh

2010-01-01

The anti-epileptic drug (R)-lacosamide ((2R)-2-(acetylamino)-N-benzyl-3-methoxypropanamide (LCM)) modulates voltage-gated sodium channels (VGSCs) by preferentially interacting with slow inactivated sodium channels, but the observation that LCM binds to collapsin response mediator protein 2 (CRMP-2) suggests additional mechanisms of action for LCM. We postulated that CRMP-2 levels affects the actions of LCM on VGSCs. CRMP-2 labeling by LCM analogs was competitively displaced by excess LCM in rat brain lysates. Manipulation of CRMP-2 levels in the neuronal model system CAD cells affected slow inactivation of VGSCs without any effects on other voltage-dependent properties. In silico docking was performed to identify putative binding sites in CRMP-2 that may modulate the effects of LCM on VGSCs. These studies identified five cavities in CRMP-2 that can accommodate LCM. CRMP-2 alanine mutants of key residues within these cavities were functionally similar to wild-type CRMP-2 as assessed by similar levels of enhancement in dendritic complexity of cortical neurons. Next, we examined the effects of expression of wild-type and mutant CRMP-2 constructs on voltage-sensitive properties of VGSCs in CAD cells: 1) steady-state voltage-dependent activation and fast-inactivation properties were not affected by LCM, 2) CRMP-2 single alanine mutants reduced the LCM-mediated effects on the ability of endogenous Na+ channels to transition to a slow inactivated state, and 3) a quintuplicate CRMP-2 alanine mutant further decreased this slow inactivated fraction. Collectively, these results identify key CRMP-2 residues that can coordinate LCM binding thus making it more effective on its primary clinical target. PMID:20538611

Virtual Manufacturing of Composite Structures for Ground Platforms, A DARPA Instant Foundry Adaptive Through Bits (iFAB) Program

DTIC Science & Technology

2012-08-01

This document contains color. 14. ABSTRACT This effort focused specifically on the Liquid Composite Molding (LCM) class of processes as they...SUBJECT TERMS Liquid Composite Molding (LCM), fabrication, manufacturability assessment 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF... Molding (LCM) .......................................................................... 2 1.1.1 LCM Process Variations

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

PubMed

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

2017-10-28

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

Prevention of posttraumatic axon sprouting by blocking CRMP2-mediated neurite outgrowth and tubulin polymerization

PubMed Central

Wilson, Sarah M.; Xiong, Wenhui; Wang, Yuying; Ping, Xingjie; Head, Jessica D.; Brittain, Joel M.; Gagare, Pravin D.; Ramachandran, P. Veeraraghavan; Jin, Xiaoming; Khanna, Rajesh

2012-01-01

Epileptogenesis following traumatic brain injury (TBI) is likely due to a combination of increased excitability, disinhibition, and increased excitatory connectivity via aberrant axon sprouting. Targeting these pathways could be beneficial in the prevention and treatment of posttraumatic epilepsy. Here, we tested this possibility using the novel anticonvulsant (R)-N-benzyl 2-acetamido-3-methoxypropionamide ((R)-lacosamide (LCM) which acts on both voltage-gated sodium channels and collapsin response mediator protein 2 (CRMP2), an axonal growth/guidance protein. LCM inhibited CRMP2-mediated neurite outgrowth, an effect phenocopied by CRMP2 knockdown. Mutation of LCM binding sites in CRMP2 reduced the neurite inhibitory effect of LCM by ~8-fold. LCM also reduced CRMP2-mediated tubulin polymerization. Thus, LCM selectively impairs CRMP2-mediated microtubule polymerization which underlies its neurite outgrowth and branching. To determine whether LCM inhibits axon sprouting in vivo, LCM was injected into rats subjected to partial cortical isolation, an animal model of posttraumatic epileptogenesis that exhibits axon sprouting in cortical pyramidal neurons. Two weeks following injury, excitatory synaptic connectivity of cortical layer V pyramidal neurons was mapped using patch clamp recordings and laser scanning photostimulation of caged glutamate. In comparison to injured control animals, there was a significant decrease in the map size of excitatory synaptic connectivity in LCM-treated rats, suggesting that LCM treatment prevented enhanced excitatory synaptic connectivity due to posttraumatic axon sprouting. These findings suggest, for the first time, that LCM’s mode of action involves interactions with CRMP2 to inhibit posttraumatic axon sprouting. PMID:22433297

APPLICATION OF LASER CAPTURE MICRODISSECTION AND PROTEIN MICROARRAY TECHNOLOGIES IN THE MOLECULAR ANALYSIS OF AIRWAY INJURY FOLLOWING POLLUTION PARTICLE EXPOSURE

EPA Science Inventory

Understanding the mechanisms by which various types of air pollution particles (PM) mediate adverse health effects would provide biological plausibility to epidemiological associations of increased rates of morbidity and mortality. The majority of information regarding the means ...

Add-on lacosamide: a retrospective study on the relationship between serum concentration, dosage, and adverse events.

PubMed

Hillenbrand, Barbara; Wisniewski, Ilona; Jürges, Uta; Steinhoff, Bernhard J

2011-11-01

We performed a retrospective study in patients with poorly controlled epilepsy treated with add-on lacosamide (LCM) to investigate the relationship of LCM-related adverse events with LCM serum concentration and weight-dependent dosage. We collected serum concentrations, weight-related dosages, and occurrences of the seven most frequent adverse events according to the randomized double-blind, placebo-controlled trials. Seventy of 131 patients could be sufficiently evaluated. LCM serum concentrations and weight-related dosages in patients with and without typical adverse events did not differ significantly. Closer analysis of the data suggested that dizziness as the leading adverse event occurred significantly more often if LCM was combined with classic sodium channel blockers. There was a significant correlation between LCM serum concentrations and co-medication, so there is still evidence for dependent variables that might have a relevant impact in individual cases. However, our data do not allow definition of a safety range for LCM. Copyright © 2011 Elsevier Inc. All rights reserved.

Chronic treatment with the new anticonvulsant drug lacosamide impairs learning and memory processes in rats: A possible role of BDNF/TrkB ligand receptor system.

PubMed

Shishmanova-Doseva, Michaela; Peychev, Lyudmil; Koeva, Yvetta; Terzieva, Dora; Georgieva, Katerina; Peychev, Zhivko

2018-06-01

Cognitive impairment is considered a frequent side effect in the drug treatment of epilepsy. The objective of the present study was to investigate the effects of lacosamide (LCM) on learning and memory processes in rats, on the serum level of brain-derived neurotrophic factor (BDNF) and BDNF/TrkB ligand receptor system expression in the hippocampal formation. Male Wistar rats underwent long-term treatment with three different doses of lacosamide - 3 mg/kg (LCM 3), 10 mg/kg (LCM 10) and 30 mg/kg (LCM 30). All rats were subjected to one active and one passive avoidance tests. The BDNF/TrkB immunohistochemical expression in the hippocampus was measured and serum BDNF was determined. The LCM-treated rats made fewer avoidance responses than controls during acquisition training and in the memory retention test. The number of escapes in the LCM 10 and LCM 30 groups decreased throughout the test, while the rats in the LCM 3 group showed fewer escapes only in the memory test in the active avoidance task. In the step-down test, the latency time of the LCM-30 treated rats was reduced as compared with the controls during the learning session and the short- and long-term memory retention tests. Lacosamide induced a dose-dependent reduction of the hippocampal expression of BDNF and its receptor TrkB. We found no significant difference between BDNF serum levels in the test animals and controls. The results of the study suggest that LCM suppresses the learning and memory processes in rats, with the inhibition of hippocampal BDNF/TrkB ligand receptor system being one of the possible mechanisms causing this effect. Copyright © 2018 Elsevier Inc. All rights reserved.

Thermal Rate Coefficients for the Astrochemical Process C + CH+ → C2+ + H by Ring Polymer Molecular Dynamics.

PubMed

Rampino, Sergio; Suleimanov, Yury V

2016-12-22

Thermal rate coefficients for the astrochemical reaction C + CH + → C 2 + + H were computed in the temperature range 20-300 K by using novel rate theory based on ring polymer molecular dynamics (RPMD) on a recently published bond-order based potential energy surface and compared with previous Langevin capture model (LCM) and quasi-classical trajectory (QCT) calculations. Results show that there is a significant discrepancy between the RPMD rate coefficients and the previous theoretical results that can lead to overestimation of the rate coefficients for the title reaction by several orders of magnitude at very low temperatures. We argue that this can be attributed to a very challenging energy profile along the reaction coordinate for the title reaction, not taken into account in extenso by either the LCM or QCT approximation. In the absence of any rigorous quantum mechanical or experimental results, the computed RPMD rate coefficients represent state-of-the-art estimates to be included in astrochemical databases and kinetic networks.

Clinical management of elderly patients with epilepsy; the use of lacosamide in a single center setting.

PubMed

Rainesalo, Sirpa; Mäkinen, Jussi; Raitanen, Jani; Peltola, Jukka

2017-10-01

Lacosamide (LCM) is a third-generation antiepileptic drug (AED) for which there is limited experience in the treatment of elderly patients with epilepsy. This study was performed to evaluate the use of LCM in this particular patient group, focusing on its tolerability and effectiveness. This is a retrospective, single-center study, in patients over 60years old treated with LCM between 1/2010 and 5/2015. Altogether, 233 elderly patients receiving LCM were identified; of these, 67 fulfilled the inclusion criteria, i.e., LCM administered for at least 2weeks. Lacosamide was initiated for acute seizure disorders (prolonged complex partial seizures, recurrent seizures, or status epilepticus) in 54 patients (81%) and for chronic epilepsy in 13 patients in an outpatient setting. The mean follow-up period for LCM treatment was 14months. The mean daily dose of LCM at the end of follow-up was 368mg (range: 100-600) for those 57 patients that continued treatment. Ten patients (15%) stopped LCM treatment but none because of lack of efficacy and only three patients (4%) because of side effects. The most frequent side effects were dizziness, fatigue, and tremor. Lacosamide was well tolerated even at relatively high doses and in combination therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

Solvent effects on differentiation of mouse brain tissue using laser microdissection ‘cut and drop’ sampling with direct mass spectral analysis

DOE PAGES

Cahill, John F.; Kertesz, Vilmos; Porta, Tiffany; ...

2018-02-08

Rationale: Laser microdissection-liquid vortex capture/electrospray ionization mass spectrometry (LMD-LVC/ESI-MS) has potential for on-line classification of tissue but an investigation into what analytical conditions provide best spectral differentiation has not been conducted. The effects of solvent, ionization polarity, and spectral acquisition parameters on differentiation of mouse brain tissue regions are described.Methods: Individual 40 × 40 μm microdissections from cortex, white, grey, granular, and nucleus regions of mouse brain tissue were analyzed using different capture/ESI solvents, in positive and negative ion mode ESI, using time-of-flight (TOF)-MS and sequential window acquisitions of all theoretical spectra (SWATH)-MS (a permutation of tandem-MS), and combinations thereof.more » Principal component analysis-linear discriminant analysis (PCA-LDA), applied to each mass spectral dataset, was used to determine the accuracy of differentiation of mouse brain tissue regions. Results: Mass spectral differences associated with capture/ESI solvent composition manifested as altered relative distributions of ions rather than the presence or absence of unique ions. In negative ion mode ESI, 80/20 (v/v) methanol/water yielded spectra with low signal/noise ratios relative to other solvents. PCA-LDA models acquired using 90/10 (v/v) methanol/chloroform differentiated tissue regions with 100% accuracy while data collected using methanol misclassified some samples. The combination of SWATH-MS and TOF-MS data improved differentiation accuracy.Conclusions: Combined TOF-MS and SWATH-MS data differentiated white, grey, granular, and nucleus mouse tissue regions with greater accuracy than when solely using TOF-MS data. Using 90/10 (v/v) methanol/chloroform, tissue regions were perfectly differentiated. Lastly, these results will guide future studies looking to utilize the potential of LMD-LVC/ESI-MS for tissue and disease differentiation.« less

Solvent effects on differentiation of mouse brain tissue using laser microdissection ‘cut and drop’ sampling with direct mass spectral analysis

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

Cahill, John F.; Kertesz, Vilmos; Porta, Tiffany

Rationale: Laser microdissection-liquid vortex capture/electrospray ionization mass spectrometry (LMD-LVC/ESI-MS) has potential for on-line classification of tissue but an investigation into what analytical conditions provide best spectral differentiation has not been conducted. The effects of solvent, ionization polarity, and spectral acquisition parameters on differentiation of mouse brain tissue regions are described.Methods: Individual 40 × 40 μm microdissections from cortex, white, grey, granular, and nucleus regions of mouse brain tissue were analyzed using different capture/ESI solvents, in positive and negative ion mode ESI, using time-of-flight (TOF)-MS and sequential window acquisitions of all theoretical spectra (SWATH)-MS (a permutation of tandem-MS), and combinations thereof.more » Principal component analysis-linear discriminant analysis (PCA-LDA), applied to each mass spectral dataset, was used to determine the accuracy of differentiation of mouse brain tissue regions. Results: Mass spectral differences associated with capture/ESI solvent composition manifested as altered relative distributions of ions rather than the presence or absence of unique ions. In negative ion mode ESI, 80/20 (v/v) methanol/water yielded spectra with low signal/noise ratios relative to other solvents. PCA-LDA models acquired using 90/10 (v/v) methanol/chloroform differentiated tissue regions with 100% accuracy while data collected using methanol misclassified some samples. The combination of SWATH-MS and TOF-MS data improved differentiation accuracy.Conclusions: Combined TOF-MS and SWATH-MS data differentiated white, grey, granular, and nucleus mouse tissue regions with greater accuracy than when solely using TOF-MS data. Using 90/10 (v/v) methanol/chloroform, tissue regions were perfectly differentiated. Lastly, these results will guide future studies looking to utilize the potential of LMD-LVC/ESI-MS for tissue and disease differentiation.« less

Double Knockdown of Prolyyl Hydroxylase and Factor Inhibiting HIF with Non-Viral Minicircle Gene Therapy Enhances Stem Cell Mobilization and Angiogenesis After Myocardial Infarction

PubMed Central

Huang, Mei; Nguyen, Patricia; Jia, Fangjun; Hu, Shijun; Gong, Yongquan; de Almeida, Patricia E.; Wang, Li; Nag, Divya; Kay, Mark A.; Giaccia, Amato J; Robbins, Robert C.; Wu, Joseph C.

2011-01-01

Background Under normoxic conditions, hypoxia inducible factor-1 alpha (HIF-1α) is rapidly degraded by two hydroxylases, prolyl hydroxylase (PHD) and factor inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its up-regulation may be an effective therapeutic option for ischemic heart failure. Methods and Results PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin sequences for inhibiting PHD isoenzyme 2 (shPHD2) and FIH (shFIH) were inserted into novel non-viral minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit+ cardiac progenitor cells (CPCs) demonstrated higher expression of angiogenesis factors in the double knockdown group compared to the single knockdown and shScramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially following LAD ligation in adult FVB mice (n=60). Functional studies using magnetic resonance imaging (MRI), echocardiography, and pressure-volume (PV) loops showed greater improvement in cardiac function in the double knockdown group. To assess mechanism(s) of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double knockdown group. Fluorescence activated cell sorting (FACS) showed significantly higher activation of endogenous c-kit+ cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser capture microdissection (LCM) analysis confirmed up-regulation of HIF-1α protein and angiogenesis genes, respectively. Conclusions We demonstrated that HIF-1α up-regulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function. PMID:21911818

EBM regeneration and changes in EBM component mRNA expression in stromal cells after corneal injury

PubMed Central

Santhanam, Abirami; Marino, Gustavo K.; Torricelli, Andre A. M.

2017-01-01

Purpose To investigate the production of the epithelial basement membrane (EBM) component mRNAs at time points before lamina lucida and lamina densa regeneration in anterior stromal cells after corneal injury that would heal with and without fibrosis. Methods Rabbit corneas were removed from 2 to 19 days after −4.5D or −9.0D photorefractive keratectomy (PRK) with the VISX S4 IR laser. Corneas were evaluated with transmission electron microscopy (TEM) for full regeneration of the lamina lucida and the lamina densa. Laser capture microdissection (LCM) based quantitative real-time (RT)–PCR was used to quantitate the expression of mRNAs for laminin α-3 (LAMA3), perlecan, nidogen-1, and nidogen-2 in the anterior stroma. Results After −4.5D PRK, EBM was found to be fully regenerated at 8 to 10 days after surgery. At 4 days after PRK, the nidogen-2 and LAMA3 mRNAs levels were detected at statistically significantly lower levels in the anterior stroma of the −9.0D PRK corneas (where the EBM would not fully regenerate) compared to the −4.5D PRK corneas (where the EBM was destined to fully regenerate). At 7 days after PRK, nidogen-2 and LAMA3 mRNAs continued to be statistically significantly lower in the anterior stroma of the −9.0D PRK corneas compared to their expression in the anterior stroma of the −4.5D PRK corneas. Conclusions Key EBM components LAMA3 and nidogen-2 mRNAs are expressed at higher levels in the anterior stroma during EBM regeneration in the −4.5D PRK corneas where the EBM is destined to fully regenerate and no haze developed compared to the −9.0D PRK corneas where the EBM will not fully regenerate and myofibroblast-related stromal fibrosis (haze) will develop. PMID:28275314

Spatial regulation of bone morphogenetic proteins (BMPs) in postnatal articular and growth plate cartilage

PubMed Central

Garrison, Presley; Yue, Shanna; Hanson, Jeffrey; Baron, Jeffrey; Lui, Julian C.

2017-01-01

Articular and growth plate cartilage both arise from condensations of mesenchymal cells, but ultimately develop important histological and functional differences. Each is composed of three layers—the superficial, mid and deep zones of articular cartilage and the resting, proliferative and hypertrophic zones of growth plate cartilage. The bone morphogenetic protein (BMP) system plays an important role in cartilage development. A gradient in expression of BMP-related genes has been observed across growth plate cartilage, likely playing a role in zonal differentiation. To investigate the presence of a similar expression gradient in articular cartilage, we used laser capture microdissection (LCM) to separate murine growth plate and articular cartilage from the proximal tibia into their six constituent zones, and used a solution hybridization assay with color-coded probes (nCounter) to quantify mRNAs for 30 different BMP-related genes in each zone. In situ hybridization and immunohistochemistry were then used to confirm spatial expression patterns. Expression gradients for Bmp2 and 6 were observed across growth plate cartilage with highest expression in hypertrophic zone. However, intracellular BMP signaling, assessed by phospho-Smad1/5/8 immunohistochemical staining, appeared to be higher in the proliferative zone and prehypertrophic area than in hypertrophic zone, possibly due to high expression of Smad7, an inhibitory Smad, in the hypertrophic zone. We also found BMP expression gradients across the articular cartilage with BMP agonists primarily expressed in the superficial zone and BMP functional antagonists primarily expressed in the deep zone. Phospho-Smad1/5/8 immunohistochemical staining showed a similar gradient. In combination with previous evidence that BMPs regulate chondrocyte proliferation and differentiation, the current findings suggest that BMP signaling gradients exist across both growth plate and articular cartilage and that these gradients may contribute to the spatial differentiation of chondrocytes in the postnatal endochondral skeleton. PMID:28467498

Molecular Signature and Mechanisms of Hepatitis D Virus-Associated Hepatocellular Carcinoma.

PubMed

Diaz, Giacomo; Engle, Ronald E; Tice, Ashley; Melis, Marta; Montenegro, Stephanie; Rodriguez-Canales, Jaime; Hanson, Jeffrey; Emmert-Buck, Michael R; Bock, Kevin W; Moore, Ian N; Zamboni, Fausto; Govindarajan, Sugantha; Kleiner, David; Farci, Patrizia

2018-06-01

There is limited data on the molecular mechanisms whereby hepatitis D virus (HDV) promotes liver cancer. Therefore, serum and liver specimens obtained at the time of liver transplantation from well-characterized patients with HDV-HCC (n-5) and with non-HCC HDV cirrhosis (n=7) were studied using an integrated genomic approach. Transcriptomic profiling was performed using laser capture-microdissected (LCM) malignant and non-malignant hepatocytes, tumorous and non-tumorous liver tissue from patients with HDV-HCC, and liver tissue from patients with non-HCC HDV cirrhosis. HDV-HCC was also compared with hepatitis B virus (HBV) HBV-HCC alone and hepatitis C virus (HCV) HCV-HCC. HDV malignant hepatocytes were characterized by an enrichment of up-regulated transcripts associated with pathways involved in cell cycle/DNA replication, damage and repair (sonic hedgehog, GADD45, DNA-damage-induced 14-3-3σ, cyclins and cell cycle regulation, cell cycle: G2/M DNA-damage checkpoint regulation, and hereditary breast cancer). Moreover, a large network of genes identified functionally relate to DNA repair, cell cycle, mitotic apparatus and cell division, including 4 cancer testis antigen genes, attesting to the critical role of genetic instability in this tumor. Besides being over-expressed, these genes were also strongly co-regulated. Gene co-regulation was high not only when compared to non-malignant hepatocytes, but also to malignant hepatocytes from HBV-HCC alone or HCV-HCC. Activation and co-regulation of genes critically associated with DNA replication, damage, and repair point to genetic instability as an important mechanism of HDV hepatocarcinogenesis. This specific HDV-HCC trait emerged also from the comparison of the molecular pathways identified for each hepatitis virus-associated HCC. Despite the dependence of HDV on HBV, these findings suggest that HDV and HBV promote carcinogenesis by distinct molecular mechanisms. This study identifies a molecular signature of HDV-associated hepatocellular carcinoma and suggests the potential for new biomarkers for early diagnostics. Copyright ©2018, American Association for Cancer Research.

Inverted-U response of lacosamide on pilocarpine-induced status epilepticus and oxidative stress in C57BL/6 mice is independent of hippocampal collapsin response mediator protein-2.

PubMed

Nirwan, Nikita; Siraj, Fouzia; Vohora, Divya

2018-06-18

Currently, lacosamide (LCM) is not approved for use in status epilepticus (SE) but several shreds of evidence are available to support its use. The present study was, therefore, undertaken to evaluate the effect of LCM on pilocarpine (PILO) induced SE and neurodegeneration in C57BL/6 mice and to ascertain the involvement of CRMP-2 in mediating above effect. Pilocarpine-induced SE model was developed to explore the effect of LCM 20, 40 and 80 mg/kg in mice. We assessed the seizure severity, seizure latency, spontaneous alternation behavior (SAB) and motor coordination by behavioral observation. Histopathological evaluation and measurement of the levels of CRMP-2, reduced glutathione (GSH) and malondialdehyde (MDA) were carried out in mice hippocampus. LCM exhibited a biphasic effect i.e., protection against SE at 20 mg/kg and 40 mg/kg dose whilst aggravated seizure-like behavior and mortality at 80 mg/kg. Further, it increased percentage alternation (i.e., restored spatial memory) in SAB and elevated motor impairment with increasing dose. Histologically, LCM 20 mg/kg and 40 mg/kg (but not 80 mg/kg) reduced neurodegeneration. LCM 20 mg/kg and 40 mg/kg reversed the elevated MDA and GSH levels while 80 mg/kg showed a tendency to increase oxidative stress. In contrast, LCM (at all doses) reversed the pilocarpine-induced elevation of collapsin response mediator protein-2 (CRMP-2). LCM protected against pilocarpine-induced SE, associated neurodegeneration and improved pilocarpine-associated impairment of spatial memory. The study reveals that CRMP-2 may not be mediating the inverted-U-response of LCM at least in pilocarpine model. Therefore, the anti-oxidant effect of LCM (and not its ability to modulate CRMP-2) was anticipated as the mechanism underlying neuroprotection. Copyright © 2018 Elsevier B.V. All rights reserved.

A common reference-based indirect comparison meta-analysis of eslicarbazepine versus lacosamide as add on treatments for focal epilepsy.

PubMed

Brigo, Francesco; Trinka, Eugen; Bragazzi, Nicola Luigi; Nardone, Raffaele; Milan, Alberto; Grillo, Elisabetta

2016-11-01

Eslicarbazepine acetate (ESL) and lacosamide (LCM) have recently emerged as add-on treatments in patients with focal epilepsy experiencing seizures despite adequate monotherapy. Both drugs enhance slow inactivation of voltage-gated sodium channels. To date no randomized controlled trial (RCT) has directly compared ESL with LCM as add-on treatments for focal epilepsy. Our aim was to indirectly compare the efficacy of ESL and LCM used as add-on treatments in patients with focal epilepsy using common reference-based indirect comparison meta-analysis. We systematically searched RCTs in which ESL or LCM has been used as add-on treatment in patients with focal epilepsy and compared with placebo. Following outcomes were considered: ≥50% reduction in seizure frequency; seizure freedom; treatment withdrawal for any reason; ≥25% increase in seizure frequency. Random-effects Mantel-Haenszel meta-analyses were performed to obtain odds ratios (ORs) for the efficacy of ESL or LCM versus placebo. Adjusted indirect comparisons were then made between ESL and LCM using the obtained results, and comparing the minimum and the highest effective recommended daily dose of each drug. Eight studies were included. Indirect comparisons adjusted for dose-effect showed no difference between ESL and LCM for responder rate, seizure freedom, and withdrawal rates. We could not assess increase in seizure frequency due to lack of data. Indirect comparisons failed to find a significant difference in efficacy between add-on ESL and LCM in patients with focal epilepsy. Direct head-to-head clinical trials comparing ESL with LCM as add-on antiepileptic treatment are required to confirm these results. Copyright © 2016 Elsevier B.V. All rights reserved.

LASER CAPTURE MICRODISSECTION AND GENE ARRAY ANALYSIS OF PALATAL EPITHELIAL AND MESENCHYMAL CELLS EXPOSED TO TCDD

EPA Science Inventory

Palatal shelves from embryos exposed on gestation day (GD) 12 to either retinoic acid (RA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) contact but fail to fuse. It is of interest to know if diverse agents that induce clefting via the same etiology also activate the same biochem...

Development of environmental friendly lost circulation material from banana peel

NASA Astrophysics Data System (ADS)

Sauki, Arina; Hasan, Nur â.€˜Izzati; Naimi, Fardelen Binti Md; Othman, Nur Hidayati

2017-12-01

Loss of expensive mud could lead to major financial problem in executing a drilling project and is one of the biggest problems that need to be tackled during drilling. Synthetic Based Mud (SBM) is the most stable state of the art drilling mud used in current drilling technologies. However, the problem with lost circulation is still inevitable. The focus of this project is to develop a new potential waste material from banana peel in order to combat lost circulation in SBM. Standard industrial Lost Circulation Material (LCM) is used to compare the performance of banana peel as LCM in SBM. The effects of different sizing of banana peels (600 micron, 300 micron and 100 micron) were studied on the rheological and filtration properties of SBM and the bridging performance of banana peel as LCM additive. The tests were conducted using viscometer, HTHP filter press and sand bed tester. Thermal analysis of banana peel was also studied using TGA. According to the results obtained, 300 and 100 micron size of banana peel LCM exhibited an improved bridging performance by 65% as compared to industrial LCM. However, banana peel LCM with the size of 600 micron failed to act as LCM due to the total invasion of mud into the sand bed.

Long-term exposure and safety of lacosamide monotherapy for the treatment of partial-onset (focal) seizures: Results from a multicenter, open-label trial.

PubMed

Vossler, David G; Wechsler, Robert T; Williams, Paulette; Byrnes, William; Therriault, Sheila

2016-10-01

To assess long-term use and safety of lacosamide (LCM) ≤800 mg/day monotherapy in patients with partial-onset seizures (POS) enrolled previously in a historical-controlled, conversion-to-monotherapy study (SP902; NCT00520741). Patients completing or exiting SP902 with LCM as monotherapy or as adjunctive therapy were eligible to enter this 2-year open-label extension (OLE) trial (SP904; NCT00530855) at a starting dose ±100 mg/day of their final SP902 dose. Investigators could adjust the LCM dose to 100-800 mg/day and add up to two antiepileptic drugs to optimize tolerability and seizure reduction. Three hundred twenty-two patients received LCM: 210 patients (65.2%) completed and 112 (34.8%) discontinued, most commonly owing to withdrawal of consent (9.3%). Two hundred fifty-eight patients (80.1%) had ≥1 year of and 216 (67.1%) had ≥2 years of LCM exposure, of whom 179/258 (69.4%) achieved LCM monotherapy lasting for any 12-month period, and 126/216 (58.3%) patients exposed for ≥24 months achieved LCM monotherapy for any 24-month period. Total exposure = 525.5 patient-years. The median modal dose was 500 mg/day. Two hundred ninety-two patients (90.7%) achieved LCM monotherapy at some point during the study. Sixty-five of 87 patients who exited and 193/235 who completed SP902 were exposed for ≥12 months, and 43.1% and 78.2%, respectively, achieved LCM monotherapy for ≥12 months. Median LCM monotherapy duration was 587.0 days (2-791 days); 91.0% of patients reported treatment-emergent adverse events, of which the most common were dizziness (27.3%), headache (17.1%), and nausea (14.3%). Compared with the SP902 study baseline, 74.2% of patients had a ≥50% seizure reduction and 5.6% were seizure-free at 24 months. The majority of patients were receiving LCM monotherapy at 0, 12, and 24 months in this OLE. Lacosamide monotherapy (median dose of 500 mg/day) had a safety profile similar to that of adjunctive therapy studies. These results support the use of lacosamide as long-term monotherapy treatment for adults with POS. © 2016 The Authors. Epilepsia published by Wiley Periodicals, Inc. on behalf of International League Against Epilepsy.

Fish consumption is inversely associated with male lung cancer mortality in countries with high levels of cigarette smoking or animal fat consumption.

PubMed

Zhang, J; Temme, E H; Kesteloot, H

2000-08-01

A striking difference in fish consumption and lung cancer mortality (LCM) exists among populations worldwide. This study investigated the relation between fish consumption and LCM at the population level. Sex-specific LCM data, mostly around 1993 and fish consumption data for 10 periods 1961-1994 in 36 countries were obtained from WHO and FAO, respectively. A significant inverse correlation exists between log fish consumption and LCM rate in 9 out of the 10 time periods (r = -0.34 to r = -0.46, P = 0.044 to P = 0.005). After adjusting for smoking and other confounders, log fish consumption (% of total energy [% E]) was inversely and significantly associated with LCM rate (per 100 000 per year) in all 10 time periods (beta = -26.3 to beta = -36.7; P = 0.0039 to P < 0.0001). The stratified analysis showed that this inverse relation was significant only in countries with above median level of smoking (>2437 cigarettes/adult/year) or animal fat minus fish fat consumption (22.4% E). An increase in fish consumption by 1% E was calculated to reduce mean male LCM rate of the populations examined in the age class of 45-74 years by 8.4%. In women, no significant relation between fish consumption and LCM could be established. Fish consumption is associated with a reduced risk from LCM, but this possible protective effect is clear-cut only in men and in countries with high levels of cigarette smoking or animal fat consumption.

The Function of Neuroendocrine Cells in Prostate Cancer

DTIC Science & Technology

2013-04-01

integration site. We then performed deep sequencing and aligned reads to the genome. Our analysis revealed that both histological phenotypes are derived from...lentiviral integration site analysis . (B) Laser capture microdissection was performed on individual glands containing both squamous and...lentiviral integration site analysis . LTR: long terminal repeat (viral DNA), PCR: polymerase chain reaction. (D) Venn diagrams depict shared lentiviral

Inhibition of sympathetic sprouting in CCD rats by lacosamide.

PubMed

Wang, Yuying; Huo, Fuquan

2018-05-14

Early hyperexcitability activity of injured nerve/neuron is critical for developing sympathetic nerve sprouting within dorsal root ganglia (DRG). Since lacosamide (LCM), an anticonvulsant, inhibits Na + channel. The present study tried to test the potential effect of LCM on inhibiting sympathetic sprouting in vivo. LCM (50 mg/kg) was daily injected intraperitoneally into rats subjected to chronic compression DRG (CCD), an animal model of neuropathic pain that exhibits sympathetic nerve sprouting, for the 1st 7 days after injury. Mechanical sensitivity was tested from day 3 to day 18 after injury, and then DRGs were removed off. Immunohistochemical staining for tyrosine hydroxylase (TH) was examined to observe sympathetic sprouting, and patch-clamp recording was performed to test the excitability and Na + current of DRG neurons. Early systemic LCM treatment significantly reduced TH immunoreactivity density in injured DRG, lowered the excitability level of injured DRG neurons, and increased paw withdrawal threshold (PWT). These effects on reducing sympathetic sprouting, inhibiting excitability and suppressing pain behavior were observed 10 days after the end of early LCM injection. In vitro 100 μM LCM instantly reduced the excitability of CCD neurons via inhibiting Na + current and reducing the amplitude of AP. All the findings suggest, for the first time, that early administration of LCM inhibited sympathetic sprouting and then alleviated neuropathic pain. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Beneficial Combination of Lacosamide with Retigabine in Experimental Animals: An Isobolographic Analysis.

PubMed

Luszczki, Jarogniew J; Zagaja, Mirosław; Miziak, Barbara; Kondrat-Wrobel, Maria W; Zaluska, Katarzyna; Wroblewska-Luczka, Paula; Adamczuk, Piotr; Czuczwar, Stanislaw J; Florek-Luszczki, Magdalena

2018-01-01

To isobolographically determine the types of interactions that occur between retigabine and lacosamide (LCM; two third-generation antiepileptic drugs) with respect to their anticonvulsant activity and acute adverse effects (sedation) in the maximal electroshock-induced seizures (MES) and chimney test (motor performance) in adult male Swiss mice. Type I isobolographic analysis for nonparallel dose-response effects for the combination of retigabine with LCM (at the fixed-ratio of 1:1) in both the MES and chimney test in mice was performed. Brain concentrations of retigabine and LCM were measured by high-pressure liquid chromatography (HPLC) to characterize any pharmacokinetic interactions occurring when combining these drugs. Linear regression analysis revealed that retigabine had its dose-response effect line nonparallel to that of LCM in both the MES and chimney tests. The type I isobolographic analysis illustrated that retigabine combined with LCM (fixed-ratio of 1:1) exerted an additive interaction in the mouse MES model and sub-additivity (antagonism) in the chimney test. With HPLC, retigabine and LCM did not mutually change their total brain concentrations, thereby confirming the pharmacodynamic nature of the interaction. LCM combined with retigabine possesses a beneficial preclinical profile (benefit index ranged from 2.07 to 2.50) and this 2-drug combination is worth recommending as treatment plan to patients with pharmacoresistant epilepsy. © 2017 S. Karger AG, Basel.

Lacosamide in the treatment of patients with epilepsy and intellectual disabilities: A long-term study of 136 patients.

PubMed

Böttcher, Stefan; Lutz, Martin T; Mayer, Thomas

2017-10-01

This study aimed to analyze the retention rate of lacosamide (LCM) in patients with epilepsy and intellectual disabilities (IDs), to identify factors influencing retention rate, and to investigate the LCM retention rate with and without concomitant sodium channel blocker (SCB). We hypothesized that the retention rate of LCM with concomitant SCB would be lower than without SCB. Using the Kaplan-Meier estimator, we conducted a monocentric, retrospective, observational, open-label study to evaluate LCM retention rates in patients with IDs and drug-resistant epilepsy. In addition, the impact of therapy-related variables on the long-term retention of LCM was evaluated. One hundred thirty-six subjects with IDs and drug-resistant epilepsy were included (age 2-66 years); most patients had focal epilepsy. Long-term retention rates were 62.0% at 1 year, 43.7% at 2 years, and 29.1% at 3 and 4 years. Reasons for LCM discontinuation included insufficient therapeutic benefits (69%), adverse events (11%), or a combination of both factors (8%). The LCM retention rate was influenced by the number of background antiepileptic drugs (AEDs). An additional and independent influence of concomitant therapy with SCB on retention rate could not be confirmed. One of the major challenges in medically caring for patients with epilepsy and IDs is the high rate of drug resistance. However, there is a lack of evidence-based information about the efficacy and tolerability of AEDs in this population. It has been shown that concomitant SCB use is a key factor in increasing the risk of LCM failure in children with epilepsy. This finding has not been replicated in our predominantly adult sample of patients with IDs. Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.

Eslicarbazepine and the enhancement of slow inactivation of voltage-gated sodium channels: a comparison with carbamazepine, oxcarbazepine and lacosamide.

PubMed

Hebeisen, Simon; Pires, Nuno; Loureiro, Ana I; Bonifácio, Maria João; Palma, Nuno; Whyment, Andrew; Spanswick, David; Soares-da-Silva, Patrício

2015-02-01

This study aimed at evaluating the effects of eslicarbazepine, carbamazepine (CBZ), oxcarbazepine (OXC) and lacosamide (LCM) on the fast and slow inactivated states of voltage-gated sodium channels (VGSC). The anti-epileptiform activity was evaluated in mouse isolated hippocampal slices. The anticonvulsant effects were evaluated in MES and the 6-Hz psychomotor tests. The whole-cell patch-clamp technique was used to investigate the effects of eslicarbazepine, CBZ, OXC and LCM on sodium channels endogenously expressed in N1E-115 mouse neuroblastoma cells. CBZ and eslicarbazepine exhibit similar concentration dependent suppression of epileptiform activity in hippocampal slices. In N1E-115 mouse neuroblastoma cells, at a concentration of 250 μM, the voltage dependence of the fast inactivation was not influenced by eslicarbazepine, whereas LCM, CBZ and OXC shifted the V0.5 value (mV) by -4.8, -12.0 and -16.6, respectively. Eslicarbazepine- and LCM-treated fast-inactivated channels recovered similarly to control conditions, whereas CBZ- and OXC-treated channels required longer pulses to recover. CBZ, eslicarbazepine and LCM shifted the voltage dependence of the slow inactivation (V0.5, mV) by -4.6, -31.2 and -53.3, respectively. For eslicarbazepine, LCM, CBZ and OXC, the affinity to the slow inactivated state was 5.9, 10.4, 1.7 and 1.8 times higher than to the channels in the resting state, respectively. In conclusion, eslicarbazepine did not share with CBZ and OXC the ability to alter fast inactivation of VGSC. Both eslicarbazepine and LCM reduce VGSC availability through enhancement of slow inactivation, but LCM demonstrated higher interaction with VGSC in the resting state and with fast inactivation gating. Copyright © 2014 Elsevier Ltd. All rights reserved.

[Efficacy of Lactose-free Milk in Korean Adults with Lactose Intolerance].

PubMed

Park, Sun Hee; Chang, Young Woon; Kim, Soo Jung; Lee, Min Hye; Nam, Ji Hyeok; Oh, Chi Hyuk; Kim, Jung-Wook; Jang, Jae-Young; Yang, Jin Oh; Yoo, Jin Ah; Chung, Jin Young

2016-01-25

Lactose-free milk (LFM) is available for nutrient supply for those with lactose intolerance (LI). However, there are no consistent results of the efficacy of LFM in LI subjects. We aimed to examine the changes of gastrointestinal (GI) symptoms and hydrogen breath test (HBT) values after ingestion of lactose contained milk (LCM) vs. LFM. From May 2015 to September 2015, thirty-five healthy adults with history of LCM-induced GI symptoms were recruited at a tertiary hospital. For the diagnosis of LI, HBT with LCM 550 mL (lactose 25 g) was performed every 20 minutes for 3 hours. The test was defined as "positive" when H2 peak exceeded 20 ppm above baseline values (ΔH2>20 ppm). When the subjects are diagnosed as LI, the second HBT using LFM 550 mL (lactose 0 g) was performed 7 days later. Subjects were asked to complete a questionnaire about the occurrence and severity of GI symptoms. Among a total of 35 subjects, 31 were diagnosed with LI at first visit, and their LCM-related symptoms were abdominal pain (98.6%), borborygmus (96.8%), diarrhea (90.3%), and flatus (87.1%). The ΔH2 value in subjects taking LCM (103.7±66.3ppm) significantly decreased to 6.3±4.9 ppm after ingesting LFM (p<0.0001). There were also significant reduction in total symptom scores and the severity of each symptom when LCM was changed to LFM (p<0.0001). This is the first report that LFM reduce LCM-related GI symptoms and H2 production in Korean adults. LFM can be an effective alternative for LCM in adults with LI.

Status epilepticus developing during lacosamide monotherapy

PubMed Central

Papacostas, Savvas S

2015-01-01

Two cases with partial onset epilepsy who developed status epilepticus (SE) on lacosamide (LCM) monotherapy are reported. LCM is an effective adjunctive antiepileptic drug (AED) for partial-onset epilepsy and as infusion in SE. It has also shown efficacy in monotherapy. The reported cases achieved control of seizures with adjunctive LCM treatment and were afterwards converted to monotherapy. Both patients subsequently developed SE while on LCM monotherapy. They were on monotherapy for at least 2 months after withdrawal of concomitant AEDs precluding the possibility of withdrawal-induced SE. Pharmacovigilance is indicated when LCM is administered in monotherapy in order to assess its proper therapeutic potential and its putative limitations especially in cases where it may prove ineffective. Moreover, vigilance is necessary whenever any concomitant antiepileptic is tapered regardless of the substances used. Higher doses may be needed when an AED is used in monotherapy. PMID:25628098

Effects of lacosamide and carbamazepine on human motor cortex excitability: a double-blind, placebo-controlled transcranial magnetic stimulation study.

PubMed

Lang, Nicolas; Rothkegel, Holger; Peckolt, Hannes; Deuschl, Günther

2013-11-01

Lacosamide (LCM) and carbamazepine (CBZ) are antiepileptic drugs both acting on neuronal voltage-gated sodium channels. Patch-clamp studies demonstrated significant differences in how LCM and CBZ affect neuronal membrane excitability. Despite valuable information patch-clamp studies provide, they also comprise some constraints. For example, little is known about effects of LCM on intracortical synaptic excitability. In contrast, transcranial magnetic stimulation (TMS) can describe drug-induced changes at the system level of the human cerebral cortex. The present study was designed to explore dose-depended effects of LCM and effects of CBZ on motor cortex excitability with TMS in a randomized, double-blind, placebo-controlled crossover trial in healthy human subjects. Subjects received 600 mg CBZ, 200 mg LCM, 400 mg LCM or placebo preceding TMS measurements. Compared to placebo, TMS motor thresholds were significantly increased after carbamazepine and lacosamide, with a trend for a dose dependent effect of lacosamide. Both, carbamazepine and lacosamide did not affect TMS parameters of intracortical synaptic excitability. TMS measurements suggest that lacosamide and carbamazepine predominantly act on neuronal membrane excitability. Copyright © 2013 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

Comparison of cell-type-specific vs transmural aortic gene expression in experimental aneurysms.

PubMed

Sho, Eiketsu; Sho, Mien; Nanjo, Hiroshi; Kawamura, Koichi; Masuda, Hirotake; Dalman, Ronald L

2005-05-01

Abdominal aortic aneurysm (AAA) progression and disease resistance are related to mural cellularity; adventitial macrophages and neocapillaries predominate in larger, advanced aneurysms, whereas smaller AAAs have fewer macrophages and retain more medial smooth muscle cells (SMCs). Expression analysis of mRNA derived from the entire aorta may mask the role that specific cell types play in modulating disease progression. We used laser capture microdissection (LCM) to isolate SMC and macrophage-predominant mural cell populations for gene expression analysis in variable-flow AAA. Rat AAAs were created via porcine pancreatic elastase (PPE) infusion. Aortic flow was increased via femoral arteriovenous fistula creation (HF-AAA) or reduced via unilateral iliac ligation (LF-AAA) in selected cohorts. SMC and macrophage-predominant cell populations were isolated via LCM and analyzed for expression of pro-inflammatory transcription factors and chemokines, cytokines, and proteolytic enzymes via real-time polymerase chain reaction. Aortic PPE infusion precipitated endothelial cell (EC) denudation, SMC apoptosis, and elastic lamellar degeneration. Increased aortic flow (HF > NF > LF) stimulated restorative EC and SMC proliferation (45.8 +/- 6.6 > 30.5 +/- 2.1 > 21 +/- 3.6 and 212.2 +/- 9.8 > 136.5 +/- 8.9 > 110 +/- 13.5, respectively, for both cell types; P < .05) at 5 days after PPE infusion, while simultaneously reducing medial SMC apoptosis and transmural macrophage infiltration. Expression of nuclear factor kappa B (NF-kappab), granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage migration inhibitory (MIF), heparin-binding EGF-like factor (HB-EGF) and inducible nitric oxide synthase (iNOS) varied between cell types and flow conditions at all time points examined. Gelatinolytic protease expression varied by cell type in response to flow loading (eg, increased in SMCs, decreased in macrophages), consistent with observed patterns of elastolysis and SMC proliferation reported in prior experiments. Flow differentially regulates cell-specific AAA gene expression. Whole-organ analysis of AAA tissue lysates obscures important cellular responses to inflammation and flow, and may explain previous seemingly contradictory observations regarding proteolysis and cell proliferation. Cell-type specific expression and functional analyses may substantially clarify the pathophysiology of AAA disease. Understanding aneurysmal aortic degeneration at the most fundamental level is a critical precursor to the development of next-generation therapies such as drug-eluting endografts and/or medical therapies to limit expansion of preclinical AAA in high-risk or elderly patients. Although animal modeling is necessary to gain insight into the early initiating events of AAA disease, the methods used in such analyses have critical bearing on the conclusions drawn regarding pathogenesis and potential therapeutic derivations. By analyzing cell-type-specific gene expression rather than whole-organ tissue lysates, the precise roles of important mediators such as metalloproteinases can be placed in the appropriate context. Further refinement of these techniques may allow cell-specific therapies to be applied at defined time points in disease progression with improved patient outcome and reduced procedural morbidity.

Lacosamide in status epilepticus: Systematic review of current evidence.

PubMed

Strzelczyk, Adam; Zöllner, Johann Philipp; Willems, Laurent M; Jost, Julie; Paule, Esther; Schubert-Bast, Susanne; Rosenow, Felix; Bauer, Sebastian

2017-06-01

The intravenous formulation of lacosamide (LCM) and its good overall tolerability and safety favor the use in status epilepticus (SE). The aim of this systematic review was to identify and evaluate studies reporting on the use of LCM in SE. We performed a systematic literature search of electronic databases using a combined search strategy from 2008 until October 2016. Using a standardized assessment form, information on the study design, methodologic framework, data sources, efficacy, and adverse events attributed to LCM were extracted from each publication and systematically reported. In total, 522 SE episodes (51.7% female) in 486 adults and 36 children and adolescents were evaluated with an overall LCM efficacy of 57%. Efficacy was comparable between use in nonconvulsive (57%; 82/145) and generalized-convulsive (61%; 30/49; p = 0.68) SE, whereas overall success rate was better in focal motor SE (92%; 34/39, p = 0.013; p < 0.001). The efficacy with later positioning of LCM decreased from 100% to 20%. The main adverse events during treatment of SE are dizziness, abnormal vision, diplopia, and ataxia. Overall, lacosamide is well tolerated and has no clinically relevant drug-drug interactions. The available data regarding the use of LCM in SE are promising, with a success rate of 57%. The strength of LCM is the lack of interaction potential and the option for intravenous use in emergency situations requiring rapid uptitration. Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.

The Luminous Convolution Model-The light side of dark matter

NASA Astrophysics Data System (ADS)

Cisneros, Sophia; Oblath, Noah; Formaggio, Joe; Goedecke, George; Chester, David; Ott, Richard; Ashley, Aaron; Rodriguez, Adrianna

2014-03-01

We present a heuristic model for predicting the rotation curves of spiral galaxies. The Luminous Convolution Model (LCM) utilizes Lorentz-type transformations of very small changes in the photon's frequencies from curved space-times to construct a dynamic mass model of galaxies. These frequency changes are derived using the exact solution to the exterior Kerr wave equation, as opposed to a linearized treatment. The LCM Lorentz-type transformations map between the emitter and the receiver rotating galactic frames, and then to the associated flat frames in each galaxy where the photons are emitted and received. This treatment necessarily rests upon estimates of the luminous matter in both the emitter and the receiver galaxies. The LCM is tested on a sample of 22 randomly chosen galaxies, represented in 33 different data sets. LCM fits are compared to the Navarro, Frenk & White (NFW) Dark Matter Model and to the Modified Newtonian Dynamics (MOND) model when possible. The high degree of sensitivity of the LCM to the initial assumption of a luminous mass to light ratios (M/L), of the given galaxy, is demonstrated. We demonstrate that the LCM is successful across a wide range of spiral galaxies for predicting the observed rotation curves. Through the generous support of the MIT Dr. Martin Luther King Jr. Fellowship program.

Potential role for human P-glycoprotein in the transport of lacosamide.

PubMed

Zhang, Chunbo; Chanteux, Hugues; Zuo, Zhong; Kwan, Patrick; Baum, Larry

2013-07-01

Antiepileptic drugs (AEDs) do not effectively treat 30-40% of patients with epilepsy. Export of AEDs by P-glycoprotein (Pgp, ABCB1, or MDR1), which is overexpressed in the blood-brain barrier in drug-resistant patients, may be a mechanism for resistance to AEDs. For most recently approved AEDs, whether they are transported by Pgp is unknown. We investigated whether a new AED, lacosamide (LCM), is a substrate of human Pgp. LLC-PK1 and MDCKII cells transfected with the human MDR1 gene were used to determine the substrate status of LCM in concentration equilibrium transport assays (CETAs). An equal concentration of drug was initially loaded in both the apical and basal chambers, and the concentration in both chambers was measured up to 4 h. The experiments were repeated in the presence of the Pgp inhibitors verapamil and tariquidar. Caco-2 assays were used to determine the intrinsic permeability and efflux ratio of LCM as well as its potential to inhibit digoxin, a Pgp substrate. Lacosamide was transported by MDR1-transfected cells from basolateral to apical sides. The efflux of LCM could be completely blocked by verapamil or tariquidar. In Caco-2 assays, LCM showed high permeability without a significant efflux ratio; it did not inhibit digoxin, a Pgp substrate. Although LCM is a substrate of Pgp in CETA, Caco-2 data demonstrated that passive diffusion should play a major role in the overall disposition of LCM. The critical role of Pgp should be addressed in vivo. Wiley Periodicals, Inc. © 2013 International League Against Epilepsy.

Simulation Based Low-Cost Composite Process Development at the US Air Force Research Laboratory

NASA Technical Reports Server (NTRS)

Rice, Brian P.; Lee, C. William; Curliss, David B.

2003-01-01

Low-cost composite research in the US Air Force Research Laboratory, Materials and Manufacturing Directorate, Organic Matrix Composites Branch has focused on the theme of affordable performance. Practically, this means that we use a very broad view when considering the affordability of composites. Factors such as material costs, labor costs, recurring and nonrecurring manufacturing costs are balanced against performance to arrive at the relative affordability vs. performance measure of merit. The research efforts discussed here are two projects focused on affordable processing of composites. The first topic is the use of a neural network scheme to model cure reaction kinetics, then utilize the kinetics coupled with simple heat transport models to predict, in real-time, future exotherms and control them. The neural network scheme is demonstrated to be very robust and a much more efficient method that mechanistic cure modeling approach. This enables very practical low-cost processing of thick composite parts. The second project is liquid composite molding (LCM) process simulation. LCM processing of large 3D integrated composite parts has been demonstrated to be a very cost effective way to produce large integrated aerospace components specific examples of LCM processes are resin transfer molding (RTM), vacuum assisted resin transfer molding (VARTM), and other similar approaches. LCM process simulation is a critical part of developing an LCM process approach. Flow simulation enables the development of the most robust approach to introducing resin into complex preforms. Furthermore, LCM simulation can be used in conjunction with flow front sensors to control the LCM process in real-time to account for preform or resin variability.

Lacosamide and sodium channel-blocking antiepileptic drug cross-titration against levetiracetam background therapy.

PubMed

Baulac, M; Byrnes, W; Williams, P; Borghs, S; Webster, E; De Backer, M; Dedeken, P

2017-04-01

To assess prospectively the effectiveness of lacosamide (LCM) added to levetiracetam (LEV) after down-titration of a concomitant sodium channel blocker (SCB) among patients with focal epilepsy not adequately controlled on LEV and SCB. In this open-label trial, LCM was initiated at 100 mg/day and up-titrated to 200-600 mg/day over 9 weeks; SCB down-titration started when LCM dose reached 200 mg/day. Patients remained on stable LCM/LEV doses for 12 weeks' maintenance (21-week treatment period). The primary outcome was retention rate on LCM. Due to recruitment challenges, fewer than the planned 300 patients participated in the trial, resulting in the trial being underpowered. Overall, 120 patients (mean age 39.7 years) started and 93 completed the trial. The most frequently used SCBs were lamotrigine (39.2%), carbamazepine (30.8%) and oxcarbazepine (27.5%). Eighty-four patients adhered to protocol and discontinued their SCB after cross-titration, but there was insufficient evidence for 36 patients. Retention rate was 73.3% (88/120) for all patients and 83.3% (70/84) for those with evidence of SCB discontinuation. Seizure freedom for patients completing maintenance was 14.0% (13/93). Discontinuation due to adverse events (6.7%) and lack of efficacy (3.3%) occurred primarily during cross-titration. Most frequently reported adverse events during treatment were dizziness (23.3%), headache (15.0%) and fatigue (8.3%). In patients with uncontrolled seizures on LEV/SCB, the LCM/LEV combination appeared to be effective and well tolerated. A cross-titration schedule-flexible LCM up-titration, concomitant SCB down-titration and stable background LEV-could present a feasible and practical approach to initiating LCM while minimizing pharmacodynamic interactions with a SCB. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Laser capture microdissection-microarray analysis of focal segmental glomerulosclerosis glomeruli.

PubMed

Bennett, Michael R; Czech, Kimberly A; Arend, Lois J; Witte, David P; Devarajan, Prasad; Potter, S Steven

2007-01-01

Focal segmental glomerulosclerosis (FSGS) is a major cause of end-stage renal disease. In this report we used laser capture microdissection to purify diseased glomeruli, and microarrays to provide universal gene expression profiles. The results provide a deeper understanding of the molecular mechanisms of the disease process and suggest novel therapeutic strategies. Consistent with earlier studies, molecular markers of the differentiated podocyte, including WT1, nephrin, and VEGF, were dramatically downregulated in the diseased glomerulus. We also observed multiple changes consistent with increased TGF-beta signaling, including elevated expression of TGF-beta(2), TGF-beta(3), SMAD2, TGF-beta(1) receptor, and thrombospondin. In addition, there was relatively low level expression of Csf1r, a marker of macrophages, but elevated expression of the chemokines CXCL1, CXCL2, CCL3, and CXCL14. We also observed strongly upregulated expression of Sox9, a transcription factor that can drive a genetic program of chondrogenesis and fibrosis. Further, the gene with the greatest fold increase in expression in the diseased glomerulus was osteopontin, which has been previously strongly implicated in kidney fibrosis in the unilateral ureteral obstruction mouse model. These results confirm old findings, and indicate the involvement of new genetic pathways in the cause and progression of FSGS. Copyright 2007 S. Karger AG, Basel.

Discovery of Transcription Factors Novel to Mouse Cerebellar Granule Cell Development Through Laser-Capture Microdissection.

PubMed

Zhang, Peter G Y; Yeung, Joanna; Gupta, Ishita; Ramirez, Miguel; Ha, Thomas; Swanson, Douglas J; Nagao-Sato, Sayaka; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Daub, Carsten O; Arner, Erik; de Hoon, Michiel; Carninci, Piero; Forrest, Alistair R R; Hayashizaki, Yoshihide; Goldowitz, Dan

2018-06-01

Laser-capture microdissection was used to isolate external germinal layer tissue from three developmental periods of mouse cerebellar development: embryonic days 13, 15, and 18. The cerebellar granule cell-enriched mRNA library was generated with next-generation sequencing using the Helicos technology. Our objective was to discover transcriptional regulators that could be important for the development of cerebellar granule cells-the most numerous neuron in the central nervous system. Through differential expression analysis, we have identified 82 differentially expressed transcription factors (TFs) from a total of 1311 differentially expressed genes. In addition, with TF-binding sequence analysis, we have identified 46 TF candidates that could be key regulators responsible for the variation in the granule cell transcriptome between developmental stages. Altogether, we identified 125 potential TFs (82 from differential expression analysis, 46 from motif analysis with 3 overlaps in the two sets). From this gene set, 37 TFs are considered novel due to the lack of previous knowledge about their roles in cerebellar development. The results from transcriptome-wide analyses were validated with existing online databases, qRT-PCR, and in situ hybridization. This study provides an initial insight into the TFs of cerebellar granule cells that might be important for development and provide valuable information for further functional studies on these transcriptional regulators.

Characterization of myocardial lesions associated with cardiomyopathy syndrome in Atlantic salmon, Salmo salar L., using laser capture microdissection.

PubMed

Wiik-Nielsen, J; Løvoll, M; Fritsvold, C; Kristoffersen, A B; Haugland, Ø; Hordvik, I; Aamelfot, M; Jirillo, E; Koppang, E O; Grove, S

2012-12-01

Cardiomyopathy syndrome (CMS) in Atlantic salmon, Salmo salar L., is characterized by focal infiltration in the spongy myocardium and endocardium of the heart. The origin of the mononuclear infiltrate is unknown. Using experimentally infected fish, we investigated localization of the causative agent, piscine myocarditis virus (PMCV), within the heart and characterized the cell population associated with myocardial lesions. Cellular and transcriptional characteristics in the lesions were compared with adjacent non-infiltrated tissues using laser capture microdissection, RT-qPCR and immunohistochemistry. Our results reveal that PMCV is almost exclusively present in myocardial lesions. The inflammatory infiltrate comprises a variety of leucocyte populations, including T cells, B cells, MHC class II(+) and CD83(+) cells, most likely of the macrophage line. Correlation analyses demonstrated co-ordinated leucocyte activity at the site of the virus infection. Cellular proliferation and/or DNA repair was demonstrated within the myocardial lesions. Different cell populations, mainly myocytes, stained positive for proliferating cell nuclear antigen (PCNA). Densities of endothelial cells and fibroblasts were not significantly increased. The simultaneous presence of PMCV and various inflammatory cells in all myocardial lesions analysed may indicate that both viral lytic and immunopathological effects may contribute to the pathogenesis of CMS. © 2012 Blackwell Publishing Ltd.

Dlx1 and Rgs5 in the Ductus Arteriosus: Vessel-Specific Genes Identified by Transcriptional Profiling of Laser-Capture Microdissected Endothelial and Smooth Muscle Cells

PubMed Central

Bökenkamp, Regina; van Brempt, Ronald; van Munsteren, Jacoba Cornelia; van den Wijngaert, Ilse; de Hoogt, Ronald; Finos, Livio; Goeman, Jelle; Groot, Adriana Cornelia Gittenberger-de; Poelmann, Robert Eugen; Blom, Nicolaas Andreas; DeRuiter, Marcus Cornelis

2014-01-01

Closure of the ductus arteriosus (DA) is a crucial step in the transition from fetal to postnatal life. Patent DA is one of the most common cardiovascular anomalies in children with significant clinical consequences especially in premature infants. We aimed to identify genes that specify the DA in the fetus and differentiate it from the aorta. Comparative microarray analysis of laser-captured microdissected endothelial (ECs) and vascular smooth muscle cells (SMCs) from the DA and aorta of fetal rats (embryonic day 18 and 21) identified vessel-specific transcriptional profiles. We found a strong age-dependency of gene expression. Among the genes that were upregulated in the DA the regulator of the G-protein coupled receptor 5 (Rgs5) and the transcription factor distal-less homeobox 1 (Dlx1) exhibited the highest and most significant level of differential expression. The aorta showed a significant preferential expression of the Purkinje cell protein 4 (Pcp4) gene. The results of the microarray analysis were validated by real-time quantitative PCR and immunohistochemistry. Our study confirms vessel-specific transcriptional profiles in ECs and SMCs of rat DA and aorta. Rgs5 and Dlx1 represent novel molecular targets for the regulation of DA maturation and closure. PMID:24489801

Lacosamide efficacy and tolerability in clinical practice - Post marketing analysis from a single dedicated epilepsy center.

PubMed

Baker, Mark D; Abou-Khalil, Bassel W; Arain, Amir; Tummala, Pavan; Azar, Nabil J; Haas, Kevin F; Sonmezturk, Hasan H

2018-06-13

Post marketing analysis of anti-epileptic drug (AED) efficacy and tolerability is of great value to the clinician since it is more representative of clinical practice than clinical trial data. We analyzed our experience with lacosamide (LCM) in patients treated after marketing. We identified all patients who were treated with LCM during the four year period after marketing, excluding patients who were in clinical trials. We recorded demographic data and analyzed efficacy and tolerability in patients who had at least one follow up visit or telephone call 3 months after the initiation of LCM. A total of 165 patients met our inclusion criteria. The mean age was 41 years. The majority of the cohort had focal epilepsy (146 patients) (88.4%). The mean duration of treatment was 31.2 months. Eighty one patients (49.1%) were continuing LCM at last follow up. Adverse effects (AEs) and discontinuation were significantly more common when LCM was added to one or more Na-channel blocking agents (NCB) (p = 0.0003 and 0.17). The 50% responder rate was 26% at 3 months and increased to 49% at 36 months. Patients were more likely to continue the drug and less likely to have AEs with slower titration over >4 weeks (p = 0.02 for each). Four or more previously failed AEDs predicted poorer response rate compared to three or less AEDs (p = 0.001). LCM use in clinical practice was associated with greater rate of seizure freedom than in clinical trials. Discontinuation and occurrence of AEs were significantly more likely with faster titration and adding LCM to NCB agents. Copyright © 2018 Elsevier B.V. All rights reserved.

Evaluating the longer-term cognitive effects of adjunctive perampanel compared to lacosamide in a naturalistic outpatient setting.

PubMed

Meschede, Carolin; Witt, Juri-Alexander; Rademacher, Michael; von Wrede, Randi D; Elger, Christian E; Helmstaedter, Christoph

2018-04-22

This retrospective longitudinal study aims to compare the longer-term cognitive and behavioral side effects of adjunctive antiepileptic treatment with perampanel (PER) and lacosamide (LCM), two third generation antiepileptic drugs with suggested favorable cognitive profiles. The two drugs were monitored according to a previously established routine diagnostic protocol (Helmstaedter et al. E&B 2013;26:182-7) which facilitates the retrospective comparison of antiepileptic drug tolerability in a naturalistic outpatient setting. Records from 94 patients were evaluated who underwent neuropsychological assessment before and under adjunctive treatment with either PER (n = 57) or LCM (n = 37). Cognition was assessed using the EpiTrack screening for executive functions and a VLMT short form for verbal memory. Subjective assessments included a German QOLIE-10 adaptation (quality of life) and an extended Adverse Events Profile (AEP). The median follow-up interval was 36 weeks. Multivariate repeated measures statistics revealed a non-significant trend towards an interaction effect "time - treatment arm" on both executive function and memory. When analyzed separately executive functions and memory scores significantly improved under LCM (t = -2.76 p < 0.01 and t = -2.44 p < 0.05 respectively). Subjectively, PER was associated with improvements in 2/18 physiological domains and in the LCM group 1/9 cognitive domains deteriorated. Seizure freedom was achieved for five patients treated with LCM (14%) and 15 treated with PER (26%, χ 2  = 2.2, n.s.). In a naturalistic outpatient setting, chronic adjunctive treatment with PER and LCM did not negatively affect cognition and LCM may even improve cognition. Neither drug increased self-reported irritability or aggression. This suggests favorable longer-term tolerability. Copyright © 2018 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

Treatment with lacosamide impedes generalized seizures in a rodent model of cortical dysplasia.

PubMed

Nemes, Ashley D; O'Dwyer, Rebecca; Najm, Imad M; Ying, Zhong; Gonzalez-Martinez, Jorge; Alexopoulos, Andreas V

2017-10-01

Epilepsy is a common neurologic disorder resulting in spontaneous, recurrent seizures. About 30-40% of patients are not responsive to pharmacologic therapies. This may be due to the differences between individual patients such as etiology, underlying pathophysiology, and seizure focus, and it highlights the importance of new drug discovery and testing in this field. Our goal was to determine the efficacy of lacosamide (LCM), a drug approved for the treatment of focal seizures, in a model of generalized epilepsy with cortical dysplasia (CD). We sought to compare LCM to levetiracetam (LEV), a drug that is currently used for the treatment of both partial and generalized epilepsy and to test its proficiency. Pregnant rats were irradiated to produce pups with malformed cortices in a model of CD, which will be referred to as the "first hit." Adult animals, developed normally (NL) and irradiated (XRT), were surgically implanted with electroencephalography (EEG) electrodes. Baseline EEG was recorded on all rats prior to pretreatments with either LCM, LEV, or placebo (PBO). After 30 min, all rats were injected with a subconvulsive dose of pentylenetetrazole (PTZ), a γ-aminobutyric acid receptor A (GABA A ) antagonist used to provoke generalized seizures as a "second hit." LCM and LEV were both effective against seizures induced by PTZ. XRT rats had a higher seizure incidence with longer and more severe seizures than NL rats. Seizure duration was decreased with both LCM and LEV in all animals. In XRT rats, there was a significant reduction in acute seizure incidence and severity with both LCM and LEV after PTZ injection. Our results suggest that LCM could be used as a potential treatment option for generalized epilepsy with CD as the underlying pathology. Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.

[Research status and prospects of DNA test on difficult specimens].

PubMed

Dang, Hua-Wei; Mao, Jiong; Wang, Hui; Huang, Jiang-Ping; Bai, Xiao-Gang

2012-02-01

This paper reviews the advances of DNA detection on three types of difficult biological specimens including degraded samples, trace evidences and mixed samples. The source of different samples, processing methods and announcements were analyzed. New methods such as mitochondrial test system, changing the original experimental conditions, low-volume PCR amplification and new technologies such as whole genome amplification techniques, laser capture micro-dissection, and mini-STR technology in recent years are introduced.

Laser Capture Microdissection of Pancreatic Acinar Cells to Identify Proteomic Alterations in a Murine Model of Caerulein-Induced Pancreatitis

PubMed Central

Shapiro, John P; Komar, Hannah M; Hancioglu, Baris; Yu, Lianbo; Jin, Ming; Ogata, Yuko; Hart, Phil A; Cruz-Monserrate, Zobeida; Lesinski, Gregory B; Conwell, Darwin L

2017-01-01

Objectives: Chronic pancreatitis (CP) is characterized by inflammation and fibrosis of the pancreas, leading to pain, parenchymal damage, and loss of exocrine and endocrine function. There are currently no curative therapies; diagnosis remains difficult and aspects of pathogenesis remain unclear. Thus, there is a need to identify novel biomarkers to improve diagnosis and understand pathophysiology. We hypothesize that pancreatic acinar regions contain proteomic signatures relevant to disease processes, including secreted proteins that could be detected in biofluids. Methods: Acini from pancreata of mice injected with or without caerulein were collected using laser capture microdissection followed by mass spectrometry analysis. This protocol enabled high-throughput analysis that captured altered protein expression throughout the stages of CP. Results: Over 2,900 proteins were identified, whereas 331 were significantly changed ≥2-fold by mass spectrometry spectral count analysis. Consistent with pathogenesis, we observed increases in proteins related to fibrosis (e.g., collagen, P<0.001), several proteases (e.g., trypsin 1, P<0.001), and altered expression of proteins associated with diminished pancreas function (e.g., lipase, amylase, P<0.05). In comparison with proteomic data from a public data set of CP patients, a significant correlation was observed between proteomic changes in tissue from both the caerulein model and CP patients (r=0.725, P<0.001). CONCLUSIONS: This study illustrates the ability to characterize proteome changes of acinar cells isolated from pancreata of caerulein-treated mice and demonstrates a relationship between signatures from murine and human CP. PMID:28406494

Proteomic analysis of laser-captured paraffin-embedded tissues: a molecular portrait of head and neck cancer progression.

PubMed

Patel, Vyomesh; Hood, Brian L; Molinolo, Alfredo A; Lee, Norman H; Conrads, Thomas P; Braisted, John C; Krizman, David B; Veenstra, Timothy D; Gutkind, J Silvio

2008-02-15

Squamous cell carcinoma of the head and neck (HNSCC), the sixth most prevalent cancer among men worldwide, is associated with poor prognosis, which has improved only marginally over the past three decades. A proteomic analysis of HNSCC lesions may help identify novel molecular targets for the early detection, prevention, and treatment of HNSCC. Laser capture microdissection was combined with recently developed techniques for protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues and a novel proteomics platform. Approximately 20,000 cells procured from FFPE tissue sections of normal oral epithelium and well, moderately, and poorly differentiated HNSCC were processed for mass spectrometry and bioinformatic analysis. A large number of proteins expressed in normal oral epithelium and HNSCC, including cytokeratins, intermediate filaments, differentiation markers, and proteins involved in stem cell maintenance, signal transduction, migration, cell cycle regulation, growth and angiogenesis, matrix degradation, and proteins with tumor suppressive and oncogenic potential, were readily detected. Of interest, the relative expression of many of these molecules followed a distinct pattern in normal squamous epithelia and well, moderately, and poorly differentiated HNSCC tumor tissues. Representative proteins were further validated using immunohistochemical studies in HNSCC tissue sections and tissue microarrays. The ability to combine laser capture microdissection and in-depth proteomic analysis of FFPE tissues provided a wealth of information regarding the nature of the proteins expressed in normal squamous epithelium and during HNSCC progression, which may allow the development of novel biomarkers of diagnostic and prognostic value and the identification of novel targets for therapeutic intervention in HNSCC.

Isolation and genetic analysis of pure cells from forensic biological mixtures: The precision of a digital approach.

PubMed

Fontana, F; Rapone, C; Bregola, G; Aversa, R; de Meo, A; Signorini, G; Sergio, M; Ferrarini, A; Lanzellotto, R; Medoro, G; Giorgini, G; Manaresi, N; Berti, A

2017-07-01

Latest genotyping technologies allow to achieve a reliable genetic profile for the offender identification even from extremely minute biological evidence. The ultimate challenge occurs when genetic profiles need to be retrieved from a mixture, which is composed of biological material from two or more individuals. In this case, DNA profiling will often result in a complex genetic profile, which is then subject matter for statistical analysis. In principle, when more individuals contribute to a mixture with different biological fluids, their single genetic profiles can be obtained by separating the distinct cell types (e.g. epithelial cells, blood cells, sperm), prior to genotyping. Different approaches have been investigated for this purpose, such as fluorescent-activated cell sorting (FACS) or laser capture microdissection (LCM), but currently none of these methods can guarantee the complete separation of different type of cells present in a mixture. In other fields of application, such as oncology, DEPArray™ technology, an image-based, microfluidic digital sorter, has been widely proven to enable the separation of pure cells, with single-cell precision. This study investigates the applicability of DEPArray™ technology to forensic samples analysis, focusing on the resolution of the forensic mixture problem. For the first time, we report here the development of an application-specific DEPArray™ workflow enabling the detection and recovery of pure homogeneous cell pools from simulated blood/saliva and semen/saliva mixtures, providing full genetic match with genetic profiles of corresponding donors. In addition, we assess the performance of standard forensic methods for DNA quantitation and genotyping on low-count, DEPArray™-isolated cells, showing that pure, almost complete profiles can be obtained from as few as ten haploid cells. Finally, we explore the applicability in real casework samples, demonstrating that the described approach provides complete separation of cells with outstanding precision. In all examined cases, DEPArray™ technology proves to be a groundbreaking technology for the resolution of forensic biological mixtures, through the precise isolation of pure cells for an incontrovertible attribution of the obtained genetic profiles. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Matrix vesicles in the fibrous cap of atherosclerotic plaque: possible contribution to plaque rupture.

PubMed

Bobryshev, Y V; Killingsworth, M C; Lord, R S A; Grabs, A J

2008-10-01

Plaque rupture is the most common type of plaque complication and leads to acute ischaemic events such as myocardial infarction and stroke. Calcification has been suggested as a possible indicator of plaque instability. Although the role of matrix vesicles in the initial stages of arterial calcification has been recognized, no studies have yet been carried out to examine a possible role of matrix vesicles in plaque destabilization. Tissue specimens selected for the present study represented carotid specimens obtained from patients undergoing carotid endarterectomy. Serial frozen cross-sections of the tissue specimens were cut and mounted on glass slides. The thickness of the fibrous cap (FCT) in each advanced atherosclerotic lesion, containing a well developed lipid/necrotic core, was measured at its narrowest sites in sets of serial sections. According to established criteria, atherosclerotic plaque specimens were histologically subdivided into two groups: vulnerable plaques with thin fibrous caps (FCT <100 microm) and presumably stable plaques, in which fibrous caps were thicker than 100 microm. Twenty-four carotid plaques (12 vulnerable and 12 presumably stable plaques) were collected for the present analysis of matrix vesicles in fibrous caps. In order to provide a sufficient number of representative areas from each plaque, laser capture microdissection (LCM) was carried out. The quantification of matrix vesicles in ultrathin sections of vulnerable and stable plaques revealed that the numbers of matrix vesicles were significantly higher in fibrous caps of vulnerable plaques than those in stable plaques (8.908+0.544 versus 6.208+0.467 matrix vesicles per 1.92 microm2 standard area; P= 0.0002). Electron microscopy combined with X-ray elemental microanalysis showed that some matrix vesicles in atherosclerotic plaques were undergoing calcification and were characterized by a high content of calcium and phosphorus. The percentage of calcified matrix vesicles/microcalcifications was significantly higher in fibrous caps in vulnerable plaques compared with that in stable plaques (6.705+/-0.436 versus 5.322+/-0494; P= 0.0474). The findings reinforce a view that the texture of the extracellular matrix in the thinning fibrous cap of atherosclerotic plaque is altered and this might contribute to plaque destabilization.

Selective over-expression of fibroblast growth factor receptors 1 and 4 in clinical prostate cancer.

PubMed

Sahadevan, K; Darby, S; Leung, H Y; Mathers, M E; Robson, C N; Gnanapragasam, V J

2007-09-01

Fibroblast growth factor receptors (FGFRs) mediate the tumourigenic effects of FGFs in prostate cancer. These receptors are therefore potential therapeutic targets in the development of inhibitors to this pathway. To identify the most relevant targets, we simultaneously investigated FGFR1-4 expression using a prostate cancer tissue microarray (TMA) and in laser capture microdissected (LCM) prostate epithelial cells. In malignant prostates (n = 138) we observed significant FGFR1 and FGFR4 protein over-expression in comparison with benign prostates (n = 58; p < 0.0001). FGFR1 was expressed at high levels in the majority of tumours (69% of grade 3 or less, 74% of grade 4 and 70% of grade 5), while FGFR4 was strongly expressed in 83% of grade 5 cancers but in only 25% of grade 1-3 cancers (p < 0.0001). At the transcript level we observed a similar pattern, with FGFR1 and FGFR4 mRNA over-expressed in malignant epithelial cells compared to benign cells (p < 0.0005 and p < 0.05, respectively). While total FGFR2 was increased in some cancers, there was no association between expression and tumour grade or stage. Transcript analysis, however, revealed a switch in the predominant isoform expressed from FGFR2IIIb to FGFR2IIIc among malignant epithelial cells. In contrast, protein and transcript expression of FGFR3 was very similar between benign and cancer biopsies. The functional effect of targeting FGFR4 in prostate cancer cells has not previously been investigated. In in vitro experiments, suppression of FGFR4 by RNA interference effectively blocked prostate cancer cell proliferation (p < 0.0001) and invasion (p < 0.001) in response to exogenous stimulation. This effect was evident regardless of whether the cells expressed the FGFR4 Arg388 or Gly388 allele. In parallel experiments, FGFR3 suppression had no discernible effect on cancer cell behaviour. These results suggest evidence of selective over-expression of FGFR1 and FGFR4 in clinical prostate cancer and support the notion of targeted inhibition of these receptors to disrupt FGF signalling.

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

Analysis of microdissected neurons by 18O mass spectrometry reveals altered protein expression in Alzheimer's disease

PubMed Central

Hashimoto, Masakazu; Bogdanovic, Nenad; Nakagawa, Hiroyuki; Volkmann, Inga; Aoki, Mikio; Winblad, Bengt; Sakai, Jun; Tjernberg, Lars O

2012-01-01

Abstract It is evident that the symptoms of Alzheimer's disease (AD) are derived from severe neuronal damage, and especially pyramidal neurons in the hippocampus are affected pathologically. Here, we analysed the proteome of hippocampal neurons, isolated from post-mortem brains by laser capture microdissection. By using 18O labelling and mass spectrometry, the relative expression levels of 150 proteins in AD and controls were estimated. Many of the identified proteins are involved in transcription and nucleotide binding, glycolysis, heat-shock response, microtubule stabilization, axonal transport or inflammation. The proteins showing the most altered expression in AD were selected for immunohistochemical analysis. These analyses confirmed the altered expression levels, and showed in many AD cases a pathological pattern. For comparison, we also analysed hippocampal sections by Western blot. The expression levels found by this method showed poor correlation with the neuron-specific analysis. Hence, we conclude that cell-specific proteome analysis reveals differences in the proteome that cannot be detected by bulk analysis. PMID:21883897

Attitudes of patients with bipolar disorder toward the Life Chart Methodology: a phenomenological study.

PubMed

van Bendegem, Mischa A; van den Heuvel, Silvio C G H; Kramer, Laura J; Goossens, Peter J J

2014-01-01

The Dutch guideline for bipolar disorder (BD) recommends the use of the Life Chart Methodology (LCM) to help patients to monitor fluctuating mood patterns. But in practice patients show ambivalent attitudes toward this instrument. To describe attitudes and motivations of patients with BD for (non-)using the LCM. A phenomenological study with unstructured in-depth interviews of 14 patients with BD. Patient narratives were audio-taped, transcribed verbatim, analyzed, and coded inductively. The results show that despite variability in perceptions and willingness to work with the LCM, the general attitude toward this instrument was a recognized value for using the LCM. However, the emotional impact of daily mood charting was experienced as a substantial burden, particularly during the early stages of diagnosis. The impact of the diagnosis of BD needs to be taken in account when introducing the instrument for the first time to a patient. © The Author(s) 2014.

[Laser microdissection for biology and medicine].

PubMed

Podgornyĭ, O V; Lazarev, V N; Govorun, V M

2012-01-01

For routine extraction of DNA, RNA, proteins and metabolites, small tissue pieces are placed into lysing solution. These tissue pieces in general contain different cell types. For this reason, lysate contains components of different cell types, which complicates the interpretation of molecular analysis results. The laser microdissection allows overcoming this trouble. The laser microdissection is a method to procure tissue samples contained defined cell subpopulations, individual cells and even subsellular components under direct microscopic visualization. Collected samples can be undergone to different downstream molecular assays: DNA analysis, RNA transcript profiling, cDNA library generation and gene expression analysis, proteomic analysis and metabolite profiling. The laser microdissection has wide applications in oncology (research and routine), cellular and molecular biology, biochemistry and forensics. This paper reviews the principles of different laser microdissection instruments, examples of laser microdissection application and problems of sample preparation for laser microdissection.

Lacosamide modulates interictal spiking and high-frequency oscillations in a model of mesial temporal lobe epilepsy

PubMed Central

Behr, Charles; Lévesque, Maxime; Ragsdale, David; Avoli, Massimo

2016-01-01

Objective Nearly one third of patients presenting with mesial temporal lobe epilepsy (MTLE), the most prevalent lesion-related epileptic disorder in adulthood, do not respond to currently available antiepileptic medications. Thus, there is a need to identify and characterize new antiepileptic drugs. In this study, we used the pilocarpine model of MTLE to establish the effects of a third generation drug, lacosamide (LCM), on seizures, interictal spikes and high-frequency oscillations (HFOs, ripples: 80–200 Hz, fast ripples: 250–500 Hz). Methods Sprague–Dawley rats (250–300 g) were injected with pilocarpine to induce a status epilepticus (SE) that was pharmacologically terminated after 1 h. Eight pilocarpine-treated rats were then injected with LCM (30 mg/kg, i.p.) 4 h after SE and daily for 14 days. Eight pilocarpine-treated rats were used as controls and treated with saline. Three days after SE, all rats were implanted with bipolar electrodes in the hippocampal CA3 region, entorhinal cortex (EC), dentate gyrus (DG) and subiculum and EEG-video monitored from day 4 to day 14 after SE. Results LCM-treated animals showed lower rates of seizures (0.21 (±0.11) seizures/day) than controls (2.6 (±0.57), p < 0.05), and a longer latent period (LCM: 11 (±1) days, controls: 6.25 (±1), p < 0.05). Rates of interictal spikes in LCM-treated rats were significantly lower than in controls in CA3 and subiculum (p < 0.05). Rates of ripples and fast ripples associated with interictal spikes in CA3 and subiculum as well as rates of fast ripples occurring outside of interictal spikes in CA3 were also significantly lower in LCM-treated animals. In controls, interictal spikes and associated HFOs correlated to seizure clustering, while this was not the case for isolated HFOs. Significance Our findings show that early treatment with LCM has powerful anti-ictogenic properties in the pilocarpine model of MTLE. These effects are accompanied by decreased rates of interictal spikes and associated HFOs. Isolated HFOs were also modulated by LCM, in a manner that appeared to be unrelated to its antiictogenic effects. These results thus suggest that distinct mechanisms may underlie interictal-associated and isolated HFOs in the pilocarpine model of MTLE. PMID:26220372

Lacosamide protects striatal and hippocampal neurons from in vitro ischemia without altering physiological synaptic plasticity.

PubMed

Mazzocchetti, Petra; Tantucci, Michela; Bastioli, Guendalina; Calabrese, Valeria; Di Filippo, Massimiliano; Tozzi, Alessandro; Calabresi, Paolo; Costa, Cinzia

2018-06-01

Lacosamide ([(R)-2-acetamido-N-benzyl-3-methoxypropanamide], LCM), is an antiepileptic that exerts anticonvulsant activity by selectively enhancing slow sodium channel inactivation. By inhibiting seizures and neuronal excitability it might therefore be a good candidate to stabilize neurons and protect them from energetic insults. Using electrophysiological analyses, we have investigated in mice the possible neuroprotective effect of LCM against in vitro ischemia obtained by oxygen and glucose deprivation (ODG), in striatal and hippocampal tissues, two brain structures particularly susceptible to ischemic injury and of pivotal importance for different form of learning and memory. We also explored in these regions the influence of LCM on firing discharge and on long-term synaptic plasticity. We found that in both areas LCM reduced the neuronal firing activity in a use-dependent manner without influencing the physiological synaptic transmission, confirming its anticonvulsant effects. Moreover, we found that this AED is able to protect, in a dose dependent manner, striatal and hippocampal neurons from energy metabolism failure produced by OGD. This neuroprotective effect does not imply impairment of long-term potentiation of striatal and hippocampal synapses and suggests that LCM might exert additional beneficial therapeutic effects beyond its use as antiepileptic. Copyright © 2018 Elsevier Ltd. All rights reserved.

Efficacy and tolerability of brivaracetam compared to lacosamide, eslicarbazepine acetate, and perampanel as adjunctive treatments in uncontrolled focal epilepsy: Results of an indirect comparison meta-analysis of RCTs.

PubMed

Brigo, Francesco; Bragazzi, Nicola Luigi; Nardone, Raffaele; Trinka, Eugen

2016-11-01

Brivaracetam (BRV), eslicarbazepine acetate (ESL), lacosamide (LCM), and perampanel (PER) have been recently marketed as adjunctive treatments for focal onset seizures. To date, no randomized controlled trial (RCT) has directly compared BRV with ESL, LCM, or PER. To compare BRV with the other add-on AEDs in patients with uncontrolled focal epilepsy, estimating their efficacy and tolerability through an adjusted, common-reference based indirect comparison meta-analysis. We systematically searched RCTs in which add-on treatment with ESL or LCM in patients with focal onset seizures have been compared with placebo. Efficacy and tolerability outcomes were considered. Random-effects Mantel-Haenszel meta-analyses were performed to obtain odds ratios (ORs) for the efficacy of BRV, LCM, ESL, or PER versus placebo. Adjusted indirect comparisons were then made between BRV and the other three AEDs using the obtained results, comparing the minimum and the highest effective recommended daily dose of each drug. Seventeen RCTs, with a total of 4971 patients were included. After adjusting for dose-effects, indirect comparisons showed no difference between BRV and LCM, ESL, or PER for responder rate and seizure freedom. Lower adverse events were observed with high dose BRV compared to high dose ESL or PER, but no difference was found in withdrawing because of adverse events. Indirect comparisons do not demonstrate a significant difference in efficacy between add-on BRV and LCM, ESL, or PER in focal epilepsy, and might suggest a better tolerability of BRV than ESL, and possibly also PER, at the highest effective recommended dose. Copyright © 2016 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

Leiomyoma-derived transforming growth factor-β impairs bone morphogenetic protein-2-mediated endometrial receptivity.

PubMed

Doherty, Leo F; Taylor, Hugh S

2015-03-01

To determine whether transforming growth factor (TGF)-β3 is a paracrine signal secreted by leiomyoma that inhibits bone morphogenetic protein (BMP)-mediated endometrial receptivity and decidualization. Experimental. Laboratory. Women with symptomatic leiomyomas. Endometrial stromal cells (ESCs) and leiomyoma cells were isolated from surgical specimens. Leiomyoma-conditioned media (LCM) was applied to cultured ESC. The TGF-β was blocked by two approaches: TGF-β pan-specific antibody or transfection with a mutant TGF-β receptor type II. Cells were then treated with recombinant human BMP-2 to assess BMP responsiveness. Expression of BMP receptor types 1A, 1B, 2, as well as endometrial receptivity mediators HOXA10 and leukemia inhibitory factor (LIF). Enzyme-linked immunosorbent assay showed elevated TGF-β levels in LCM. LCM treatment of ESC reduced expression of BMP receptor types 1B and 2 to approximately 60% of pretreatment levels. Preincubation of LCM with TGF-β neutralizing antibody or mutant TGF receptor, but not respective controls, prevented repression of BMP receptors. HOXA10 and LIF expression was repressed in recombinant human BMP-2 treated, LCM exposed ESC. Pretreatment of LCM with TGF-β antibody or transfection with mutant TGF receptor prevented HOXA10 and LIF repression. Leiomyoma-derived TGF-β was necessary and sufficient to alter endometrial BMP-2 responsiveness. Blockade of TGF-β prevents repression of BMP-2 receptors and restores BMP-2-stimulated expression of HOXA10 and LIF. Blockade of TGF signaling is a potential strategy to improve infertility and pregnancy loss associated with uterine leiomyoma. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

A low carbohydrate Mediterranean diet improves cardiovascular risk factors and diabetes control among overweight patients with type 2 diabetes mellitus: a 1-year prospective randomized intervention study.

PubMed

Elhayany, A; Lustman, A; Abel, R; Attal-Singer, J; Vinker, S

2010-03-01

The appropriate dietary intervention for overweight persons with type 2 diabetes mellitus (DM2) is unclear. Trials comparing the effectiveness of diets are frequently limited by short follow-up times and high dropout rates. The effects of a low carbohydrate Mediterranean (LCM), a traditional Mediterranean (TM), and the 2003 American Diabetic Association (ADA) diet were compared, on health parameters during a 12-month period. In this 12-month trial, 259 overweight diabetic patients (mean age 55 years, mean body mass index 31.4 kg/m(2)) were randomly assigned to one of the three diets. The primary end-points were reduction of fasting plasma glucose, HbA1c and triglyceride (TG) levels. 194 patients out of 259 (74.9%) completed follow-up. After 12 months, the mean weight loss for all patients was 8.3 kg: 7.7 kg for ADA, 7.4 kg for TM and 10.1 kg for LCM diets. The reduction in HbA1c was significantly greater in the LCM diet than in the ADA diet (-2.0 and -1.6%, respectively, p < 0.022). HDL cholesterol increased (0.1 mmol/l +/- 0.02) only on the LCM (p < 0.002). The reduction in serum TG was greater in the LCM (-1.3 mmol/l) and TM (-1.5 mmol/l) than in the ADA (-0.7 mmol/l), p = 0.001. An intensive 12-month dietary intervention in a community-based setting was effective in improving most modifiable cardiovascular risk factors in all the dietary groups. Only the LCM improved HDL levels and was superior to both the ADA and TM in improving glycaemic control.

Therapeutic Drug Monitoring of Lacosamide in Norway: Focus on Pharmacokinetic Variability, Efficacy and Tolerability.

PubMed

Svendsen, Torleiv; Brodtkorb, Eylert; Baftiu, Arton; Burns, Margrete Larsen; Johannessen, Svein I; Johannessen Landmark, Cecilie

2017-07-01

Lacosamide (LCM) is a new antiepileptic drug (AED). Experience from therapeutic drug monitoring (TDM) in clinical practice is limited. The purpose of this study is to evaluate the pharmacokinetic variability of LCM in relation to efficacy and tolerability in patients with refractory epilepsy in a real-life setting. Variables included age, gender, daily doses and serum concentrations of LCM and other AEDs from the TDM-database at the National Center for Epilepsy in Norway. Clinical data regarding efficacy and tolerability were collected from medical records. The Norwegian Prescription Database (NorPD) was used to include population-based numbers of users. TDM-data from 344 patients were included. The median dose, serum concentration, and concentration/dose (C/D)-ratio of LCM was 350 (range 25-700) mg/day, 19.7 (range 8.1-56.2) µmol/L, and 0.06 (0.02-0.82) µmol/L/mg, respectively. Serum concentrations were reduced by 28% by concomitant use of enzyme inducers and increased by 30% in patients aged >65 years. Efficacy and tolerability were assessed in 227 patients: 29% had >50% seizure reduction (eight seizure free), 30% had no effect, and 44% reported adverse effects. In Norway, there were on average 500 patients per year using LCM in this period based on NorPD. The study demonstrated pharmacokinetic variability and use of TDM of LCM in Norway. Data were collected from multiple sources for improved pharmacovigilance. Serum concentrations were influenced by enzyme inducers and ageing, indicating the usefulness of TDM. Effect and tolerability were favorable within a suggested reference range of 10-40 µmol/L given drug-fasting conditions.

Activity of the anticonvulsant lacosamide in experimental and human epilepsy via selective effects on slow Na+ channel inactivation.

PubMed

Holtkamp, Dominik; Opitz, Thoralf; Niespodziany, Isabelle; Wolff, Christian; Beck, Heinz

2017-01-01

In human epilepsy, pharmacoresistance to antiepileptic drug therapy is a major problem affecting ~30% of patients with epilepsy. Many classical antiepileptic drugs target voltage-gated sodium channels, and their potent activity in inhibiting high-frequency firing has been attributed to their strong use-dependent blocking action. In chronic epilepsy, a loss of use-dependent block has emerged as a potential cellular mechanism of pharmacoresistance for anticonvulsants acting on voltage-gated sodium channels. The anticonvulsant drug lacosamide (LCM) also targets sodium channels, but has been shown to preferentially affect sodium channel slow inactivation processes, in contrast to most other anticonvulsants. We used whole-cell voltage clamp recordings in acutely isolated cells to investigate the effects of LCM on transient Na + currents. Furthermore, we used whole-cell current clamp recordings to assess effects on repetitive action potential firing in hippocampal slices. We show here that LCM exerts its effects primarily via shifting the slow inactivation voltage dependence to more hyperpolarized potentials in hippocampal dentate granule cells from control and epileptic rats, and from patients with epilepsy. It is important to note that this activity of LCM was maintained in chronic experimental and human epilepsy. Furthermore, we demonstrate that the efficacy of LCM in inhibiting high-frequency firing is undiminished in chronic experimental and human epilepsy. Taken together, these results show that LCM exhibits maintained efficacy in chronic epilepsy, in contrast to conventional use-dependent sodium channel blockers such as carbamazepine. They also establish that targeting slow inactivation may be a promising strategy for overcoming target mechanisms of pharmacoresistance. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

Neurological adverse events of new generation sodium blocker antiepileptic drugs. Meta-analysis of randomized, double-blinded studies with eslicarbazepine acetate, lacosamide and oxcarbazepine.

PubMed

Zaccara, Gaetano; Giovannelli, Fabio; Maratea, Dario; Fadda, Valeria; Verrotti, Alberto

2013-09-01

Analysis of overall tolerability and neurological adverse effects (AEs) of eslicarbazepine acetate (ESL), lacosamide (LCM) and oxcarbazepine (OXC) from double-blind, placebo-controlled trials. Indirect comparisons of patients withdrawing because of AEs, and the incidence of some vestibulocerebellar AEs between these three antiepileptic dugs (AEDs). We searched MEDLINE for all randomized, double-blind, placebo-controlled trials investigating therapeutic effects of fixed oral doses of ESL, LCM and OXC in patients with drug resistant epilepsy. Withdrawal rate due to AEs, percentages of patients with serious AEs, and the proportion of patients experiencing any neurological AE, nausea and vomiting were assessed for their association with the experimental drug. Analyses were performed between recommended daily doses of each AED according to the approved summary of product characteristics (SPC). Risk differences were used to evaluate the association of any AE [99% confidence intervals (CIs)] or study withdrawals because of AEs (95% CIs) with the experimental drug. Indirect comparisons between withdrawal rate and AEs dizziness, coordination abnormal/ataxia and diplopia were estimated according to network meta-analysis (Net-MA). Eight randomized, placebo-controlled, double-blind trials (4 with ESL, 3 with LCM, and 1 with OXC) were included in our analysis. At high doses (OXC 1200mg, ESL 1200mg and LCM 400mg) there was an increased risk of AE-related study withdrawals compared to placebo for all drugs. Several AEs were associated with the experimental drug. Both number and frequency of AEs were dose-related. At high recommended doses, patients treated with OXC withdrew from the experimental treatment significantly more frequently than patients treated with ESL and LCM. Furthermore, the AEs coordination abnormal/ataxia and diplopia were significantly more frequently observed in patients treated with OXC compared to patients treated with LCM and ESL. The overall tolerability of AEDs and the incidence of several neurological AEs were clearly dose-dependent. Indirect comparisons between these AEDs, taking into account dose-effect, showed that OXC may be associated with more frequent neurological AEs than LCM and ESL. Copyright © 2013 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

Local collective motion analysis for multi-probe dynamic imaging and microrheology

NASA Astrophysics Data System (ADS)

Khan, Manas; Mason, Thomas G.

2016-08-01

Dynamical artifacts, such as mechanical drift, advection, and hydrodynamic flow, can adversely affect multi-probe dynamic imaging and passive particle-tracking microrheology experiments. Alternatively, active driving by molecular motors can cause interesting non-Brownian motion of probes in local regions. Existing drift-correction techniques, which require large ensembles of probes or fast temporal sampling, are inadequate for handling complex spatio-temporal drifts and non-Brownian motion of localized domains containing relatively few probes. Here, we report an analytical method based on local collective motion (LCM) analysis of as few as two probes for detecting the presence of non-Brownian motion and for accurately eliminating it to reveal the underlying Brownian motion. By calculating an ensemble-average, time-dependent, LCM mean square displacement (MSD) of two or more localized probes and comparing this MSD to constituent single-probe MSDs, we can identify temporal regimes during which either thermal or athermal motion dominates. Single-probe motion, when referenced relative to the moving frame attached to the multi-probe LCM trajectory, provides a true Brownian MSD after scaling by an appropriate correction factor that depends on the number of probes used in LCM analysis. We show that LCM analysis can be used to correct many different dynamical artifacts, including spatially varying drifts, gradient flows, cell motion, time-dependent drift, and temporally varying oscillatory advection, thereby offering a significant improvement over existing approaches.

A novel resistance gene, lnu(H), conferring resistance to lincosamides in Riemerella anatipestifer CH-2.

PubMed

Luo, Hong-Yan; Liu, Ma-Feng; Wang, Ming-Shu; Zhao, Xin-Xin; Jia, Ren-Yong; Chen, Shun; Sun, Kun-Feng; Yang, Qiao; Wu, Ying; Chen, Xiao-Yue; Biville, Francis; Zou, Yuan-Feng; Jing, Bo; Cheng, An-Chun; Zhu, De-Kang

2018-01-01

The Gram-negative bacterium Riemerella anatipestifer CH-2 is resistant to lincosamides, having a lincomycin (LCM) minimum inhibitory concentration (MIC) of 128 µg/mL. The G148_1775 gene of R. anatipestifer CH-2, designated lnu(H), encodes a 260-amino acid protein with ≤41% identity to other reported lincosamide nucleotidylyltransferases. Escherichia coli Rosetta TM (DE3) containing the pBAD24-lnu(H) plasmid showed four- and two-fold increases in the MICs of LCM and clindamycin (CLI), respectively. A kinetic assay of the purified Lnu(H) enzyme for LCM and CLI showed that the protein could inactive lincosamides. Mass spectrometry analysis demonstrated that the Lnu(H) enzyme catalysed adenylylation of lincosamides. In addition, an lnu(H) gene deletion strain exhibited 512- and 32-fold decreases in LCM and CLI MICs, respectively. The wild-type level of lincosamide resistance could be restored by complementation with a shuttle plasmid carrying the lnu(H) gene. The transformant R. anatipestifer ATCC 11845 [lnu(H)] acquired by natural transformation also exhibited high-level lincosamide resistance. Moreover, among 175 R. anatipestifer field isolates, 56 (32.0%) were positive for the lnu(H) gene by PCR. In conclusion, Lnu(H) is a novel lincosamide nucleotidylyltransferase that inactivates LCM and CLI by nucleotidylylation, thus conferring high-level lincosamide resistance to R. anatipestifer CH-2. Copyright © 2017. Published by Elsevier B.V.

Expression of key ion channels in the rat cardiac conduction system by laser capture microdissection and quantitative real-time PCR.

PubMed

Ou, Yan; Niu, Xiao-lin; Ren, Fu-xian

2010-09-01

The objective of this study was to investigate the molecular basis of the inferior nodal extension (INE) in the atrioventricular junctional area that accounts for arrhythmias. The INE was separated from the adult rat heart by laser capture microdissection. The mRNA expression of ion channels was detected by quantitative real-time PCR. Hierarchical clustering was used to demonstrate clustering of expression of genes in sections. The mRNA expression of HCN4, Ca(v)3.1 and Ca(v)3.2 was high in the INE, atrioventricular node and sino-atrial node, and that of Ca(v)3.2 high in Purkinje fibres. Although the expression of HCN1 and Ca(v)1.3 was low in the rat heart, it was relatively higher in the INE, atrioventricular node and sino-atrial node than in right atrial and right ventricular (working) myocytes. Both HCN2 and Ca(v)1.2 were expressed at higher levels in working myocytes than in nodal tissues and in the INE. Hierarchical clustering analysis demonstrated that the expression of the HCN and calcium channels in INE was similar to that in the slow-response automatic cells and different from that in working myocytes and Purkinje fibres. The expression of HCN and calcium channels in the INE of the adult rat heart is similar to that of slow-response automatic cells and provides a substrate for automatic phase 4 depolarization in cells.

Laser Capture and Single Cell Genotyping from Frozen Tissue Sections.

PubMed

Kroneis, Thomas; Ye, Jody; Gillespie, Kathleen

2016-01-01

There is an increasing requirement for genetic analysis of individual cells from tissue sections. This is particularly the case for analysis of tumor cells but is also a requirement for analysis of cells in pancreas from individuals with type 1 diabetes where there is evidence of viral infection or in the analysis of chimerism in pancreas; either post-transplant or as a result of feto-maternal cell transfer.This protocol describes a strategy to isolate cells using laser microdissection and to run a 17plex PCR to discriminate between cells of haplo-identical origin (i.e., fetal and maternal cells) in pancreas tissue but other robust DNA tests could be used. In short, snap-frozen tissues are cryo-sectioned and mounted onto membrane-coated slides. Target cells are harvested from the tissue sections by laser microdissection and pressure catapulting (LMPC) prior to DNA profiling. This is based on amplification of highly repetitive yet stably inherited loci (short tandem repeats, STR) as well as the amelogenin locus for sex determination and separation of PCR products by capillary electrophoresis.

Strategic balance of drug lifecycle management options differs between domestic and foreign companies in Japan.

PubMed

Yamanaka, Takayuki; Kano, Shingo

2016-01-01

Drug approvals and patent protections are critical in drug lifecycle management (LCM) in order to maximize drug discovery investment returns. We analyzed drug LCM activities implemented by 10 top companies in Japan, focusing on drug approvals and patent term extensions. Foreign companies acquired numerous drug approvals primarily for new molecular entities (NMEs), while Japanese companies mainly obtained approvals for improved drugs including new indications, and intensively extended patent terms. Furthermore, we discovered three factors likely responsible for differences in drug LCM strategies of Japanese and foreign companies: research and development capacities for drugs, drug lags of foreign-origin NMEs, and cooperation between Research and Development Departments and Intellectual Property Departments.

LIFE CYCLE ASSESSMENT IN MANAGEMENT, PRODUCT AND PROCESS DESIGN, AND POLICY DECISION MAKING: A CONFERENCE REPORT

EPA Science Inventory

On 24 September 2003, life cycle assessment (LCA) practitioners and decision makers gathered at the InLCA/LCM Conference in Seattle, Washington, USA (see http://www.lcacenter.org/InLCA-LCM03/index.html) to discuss the role of LCA in management, product design, process development...

TVT versus laparoscopic mesh colposuspension: 5-year follow-up results of a randomized clinical trial.

PubMed

Valpas, A; Ala-Nissilä, S; Tomas, E; Nilsson, C G

2015-01-01

Before the introduction of the tension-free vaginal tape (TVT) procedure for the treatment of female stress urinary incontinence, the colposuspension operation was regarded as the "gold standard" procedure. The laparoscopic variant of the colposuspension was introduced as a less invasive operation. The aim of the present trial was to compare the new minimally invasive TVT procedure with laparoscopic mesh colposuspension (LCM). A multicenter randomized clinical trial conducted in six public hospitals in Finland including primary cases of stress incontinence. Objective treatment success criteria were a negative stress test and no retreatment for stress incontinence. Patient satisfaction was assessed by Patients Global Impression of Improvement, a visual analog scale, and the Urinary Incontinence Severity Score. Of 128 randomized patients, 121 underwent the allocated operation. At the 5-year follow-up 77 % in the TVT group and 84 % in the LCM group could be assessed according to the protocol. The objective cure rate was significantly higher in the TVT group (94 %) than in the LCM group (78 %). Subjective treatment satisfaction (completely satisfied with the procedure) was significantly higher in the TVT group (64 %) than in the LCM group (51 %). By per protocol analysis both objective and subjective cure rates were significantly higher in the TVT group than in the LCM group. If cases that were lost to follow-up were regarded as failures, the intension-to-treat analysis found no difference between the groups.

Effect of dairy manure rate and the stabilization time of amended soils on atrazine degradation.

PubMed

Aguilera, Paula; Briceño, Gabriela; Candia, Maribel; Mora, Maria de la Luz; Demanet, Rolando; Palma, Graciela

2009-10-01

The application rate of liquid cow manure (LCM) in the field and the stabilization time of amended soils before application of pre-plant herbicides are factors that determine their efficiency. This study includes evaluation of residual atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) in soil and amended soils with equivalent rate of 100,000; 200,000; and 300,000 L ha(-1) of LCM and the effect of pre-incubation time of amended soils on atrazine degradation. The study was carried out under controlled conditions using an Andisol with previous historical application of atrazine. The respiratory activity and fluorescein diacetate (FDA) studies indicated that the time necessary for stabilization of amended soils is over 20-30 d. During the measurement of respiratory and FDA activity, no significant differences were observed when atrazine was applied. The half-life of atrazine ranged from 5 to 8d and the relative distribution of degradation products seem to be affected by the application of LCM. The pre-incubation time of amended soil and LCM dose would not affect atrazine degradation rate, when the soil has a history of herbicide application. However, repeated applications of LCM in a long period of time could change the soil pH and increase the content of dissolved organic carbon (DOC) which could further contribute to a faster degradation of atrazine. Both effects would reduce the effectiveness of atrazine in weed control.

Energy and charge transfer effects in two-dimensional van der Waals hybrid nanostructures on periodic gold nanopost array

NASA Astrophysics Data System (ADS)

Kim, Jun Young; Kim, Sun Gyu; Youn, Jong Won; Lee, Yongjun; Kim, Jeongyong; Joo, Jinsoo

2018-05-01

Two-dimensional (2D) semiconducting MoS2 and WSe2 flakes grown by chemical vapor deposition were mechanically hybridized. A hexagonal boron nitride (h-BN) dielectric flake was inserted between MoS2 and WSe2 flakes to investigate the nanoscale optical properties of 2D van der Waals hybrid nanostructures. The fabricated MoS2/WSe2 and MoS2/h-BN/WSe2 van der Waals hybrid nanostructures were loaded on a periodic gold nanopost (Au-NPo) array to study energy and charge transfer effects at the surface plasmon resonance (SPR) condition. Nanoscale photoluminescence (PL) spectra of the 2D hybrid nanostructures were measured using a high-resolution laser confocal microscope (LCM). A shift of the LCM PL peak of the MoS2/WSe2 n-p hybrid nanostructures was observed owing to the charge transfer. In contrast, the shift of the LCM PL peak of the MoS2/h-BN/WSe2 n-insulator-p hybrid nanostructure was not considerable, as the inserted h-BN dielectric layer prevented the charge transfer. The intensity of the LCM PL peak of the MoS2/h-BN/WSe2 hybrid nanostructure considerably increased once the nanostructure was loaded on the Au-NPo array, owing to the energy transfer between the 2D materials and the Au-NPo array at the SPR condition, which was confirmed by the increase in the LCM Raman intensity.

Inferring relationships between clinical mastitis, productivity and fertility: a recursive model application including genetics, farm associated herd management, and cow-specific antibiotic treatments.

PubMed

Rehbein, Pia; Brügemann, Kerstin; Yin, Tong; V Borstel, U König; Wu, Xiao-Lin; König, Sven

2013-10-01

A dataset of test-day records, fertility traits, and one health trait including 1275 Brown Swiss cows kept in 46 small-scale organic farms was used to infer relationships among these traits based on recursive Gaussian-threshold models. Test-day records included milk yield (MY), protein percentage (PROT-%), fat percentage (FAT-%), somatic cell score (SCS), the ratio of FAT-% to PROT-% (FPR), lactose percentage (LAC-%), and milk urea nitrogen (MUN). Female fertility traits were defined as the interval from calving to first insemination (CTFS) and success of a first insemination (SFI), and the health trait was clinical mastitis (CM). First, a tri-trait model was used which postulated the recursive effect of a test-day observation in the early period of lactation on liability to CM (LCM), and further the recursive effect of LCM on the following test-day observation. For CM and female fertility traits, a bi-trait recursive Gaussian-threshold model was employed to estimate the effects from CM to CTFS and from CM on SFI. The recursive effects from CTFS and SFI onto CM were not relevant, because CM was recorded prior to the measurements for CTFS and SFI. Results show that the posterior heritability for LCM was 0.05, and for all other traits, heritability estimates were in reasonable ranges, each with a small posterior SD. Lowest heritability estimates were obtained for female reproduction traits, i.e. h(2)=0.02 for SFI, and h(2)≈0 for CTFS. Posterior estimates of genetic correlations between LCM and production traits (MY and MUN), and between LCM and somatic cell score (SCS), were large and positive (0.56-0.68). Results confirm the genetic antagonism between MY and LCM, and the suitability of SCS as an indicator trait for CM. Structural equation coefficients describe the impact of one trait on a second trait on the phenotypic pathway. Higher values for FAT-% and FPR were associated with a higher LCM. The rate of change in FAT-% and in FPR in the ongoing lactation with respect to the previous LCM was close to zero. Estimated recursive effects between SCS and CM were positive, implying strong phenotypic impacts between both traits. Structural equation coefficients explained a detrimental impact of CM on female fertility traits CTFS and SFI. The cow-specific CM treatment had no significant impact on performance traits in the ongoing lactation. For most treatments, beta-lactam-antibiotics were used, but test-day SCS and production traits after the beta-lactam-treatment were comparable to those after other antibiotic as well as homeopathic treatments. Copyright © 2013 Elsevier B.V. All rights reserved.

Latent Curve Models and Latent Change Score Models Estimated in R

ERIC Educational Resources Information Center

Ghisletta, Paolo; McArdle, John J.

2012-01-01

In recent years the use of the latent curve model (LCM) among researchers in social sciences has increased noticeably, probably thanks to contemporary software developments and the availability of specialized literature. Extensions of the LCM, like the the latent change score model (LCSM), have also increased in popularity. At the same time, the R…

Four Cases of a Sociocultural Approach to Mobile Learning in "La Clase Mágica," an Afterschool Technology Club

ERIC Educational Resources Information Center

Prieto, Linda; Arreguín-Anderson, María G.; Yuen, Timothy T.; Ek, Lucila D.; Sánchez, Patricia; Machado-Casas, Margarita; García, Adriana

2016-01-01

This paper presents four projects in which mobile devices are used to support authentic learning in an afterschool technology club, "La Clase Mágica" (LCM@UTSA), designed to motivate underrepresented elementary school children in science, technology, engineering, and mathematics. The implementation of mobile devices into our LCM@UTSA is…

Building unified geospatial data for land-change modeling—A case study in the area of Richmond, Virginia

USGS Publications Warehouse

Donato, David I.; Shapiro, Jason L.

2016-12-13

An effort to build a unified collection of geospatial data for use in land-change modeling (LCM) led to new insights into the requirements and challenges of building an LCM data infrastructure. A case study of data compilation and unification for the Richmond, Va., Metropolitan Statistical Area (MSA) delineated the problems of combining and unifying heterogeneous data from many independent localities such as counties and cities. The study also produced conclusions and recommendations for use by the national LCM community, emphasizing the critical need for simple, practical data standards and conventions for use by localities. This report contributes an uncopyrighted core glossary and a much needed operational definition of data unification.

Matrix vesicles in the fibrous cap of atherosclerotic plaque: possible contribution to plaque rupture

PubMed Central

Bobryshev, Y V; Killingsworth, M C; Lord, R S A; Grabs, A J

2008-01-01

Plaque rupture is the most common type of plaque complication and leads to acute ischaemic events such as myocardial infarction and stroke. Calcification has been suggested as a possible indicator of plaque instability. Although the role of matrix vesicles in the initial stages of arterial calcification has been recognized, no studies have yet been carried out to examine a possible role of matrix vesicles in plaque destabilization. Tissue specimens selected for the present study represented carotid specimens obtained from patients undergoing carotid endarterectomy. Serial frozen cross-sections of the tissue specimens were cut and mounted on glass slides. The thickness of the fibrous cap (FCT) in each advanced atherosclerotic lesion, containing a well developed lipid/necrotic core, was measured at its narrowest sites in sets of serial sections. According to established criteria, atherosclerotic plaque specimens were histologically subdivided into two groups: vulnerable plaques with thin fibrous caps (FCT <100 μm) and presumably stable plaques, in which fibrous caps were thicker than 100 μm. Twenty-four carotid plaques (12 vulnerable and 12 presumably stable plaques) were collected for the present analysis of matrix vesicles in fibrous caps. In order to provide a sufficient number of representative areas from each plaque, laser capture microdissection (LCM) was carried out. The quantification of matrix vesicles in ultrathin sections of vulnerable and stable plaques revealed that the numbers of matrix vesicles were significantly higher in fibrous caps of vulnerable plaques than those in stable plaques (8.908±0.544 versus 6.208±0.467 matrix vesicles per 1.92 μm2 standard area; P= 0.0002). Electron microscopy combined with X-ray elemental microanalysis showed that some matrix vesicles in atherosclerotic plaques were undergoing calcification and were characterized by a high content of calcium and phosphorus. The percentage of calcified matrix vesicles/microcalcifications was significantly higher in fibrous caps in vulnerable plaques compared with that in stable plaques (6.705±0.436 versus 5.322±0A94; P= 0.0474). The findings reinforce a view that the texture of the extracellular matrix in the thinning fibrous cap of atherosclerotic plaque is altered and this might contribute to plaque destabilization. PMID:18194456

Telomere maintenance in laser capture microdissection-purified Barrett's adenocarcinoma cells and effect of telomerase inhibition in vivo.

PubMed

Shammas, Masood A; Qazi, Aamer; Batchu, Ramesh B; Bertheau, Robert C; Wong, Jason Y Y; Rao, Manjula Y; Prasad, Madhu; Chanda, Diptiman; Ponnazhagan, Selvarangan; Anderson, Kenneth C; Steffes, Christopher P; Munshi, Nikhil C; De Vivo, Immaculata; Beer, David G; Gryaznov, Sergei; Weaver, Donald W; Goyal, Raj K

2008-08-01

The aims of this study were to investigate telomere function in normal and Barrett's esophageal adenocarcinoma (BEAC) cells purified by laser capture microdissection and to evaluate the effect of telomerase inhibition in cancer cells in vitro and in vivo. Epithelial cells were purified from surgically resected esophagi. Telomerase activity was measured by modified telomeric repeat amplification protocol and telomere length was determined by real-time PCR assay. To evaluate the effect of telomerase inhibition, adenocarcinoma cell lines were continuously treated with a specific telomerase inhibitor (GRN163L) and live cell number was determined weekly. Apoptosis was evaluated by Annexin labeling and senescence by beta-galactosidase staining. For in vivo studies, severe combined immunodeficient mice were s.c. inoculated with adenocarcinoma cells and following appearance of palpable tumors, injected i.p. with saline or GRN163L. Telomerase activity was significantly elevated whereas telomeres were shorter in BEAC cells relative to normal esophageal epithelial cells. The treatment of adenocarcinoma cells with telomerase inhibitor, GRN163L, led to loss of telomerase activity, reduction in telomere length, and growth arrest through induction of both the senescence and apoptosis. GRN163L-induced cell death could also be expedited by addition of the chemotherapeutic agents doxorubicin and ritonavir. Finally, the treatment with GRN163L led to a significant reduction in tumor volume in a subcutaneous tumor model. We show that telomerase activity is significantly elevated whereas telomeres are shorter in BEAC and suppression of telomerase inhibits proliferation of adenocarcinoma cells both in vitro and in vivo.

Incorporating the life course model into MCH nutrition leadership education and training programs.

PubMed

Haughton, Betsy; Eppig, Kristen; Looney, Shannon M; Cunningham-Sabo, Leslie; Spear, Bonnie A; Spence, Marsha; Stang, Jamie S

2013-01-01

Life course perspective, social determinants of health, and health equity have been combined into one comprehensive model, the life course model (LCM), for strategic planning by US Health Resources and Services Administration's Maternal and Child Health Bureau. The purpose of this project was to describe a faculty development process; identify strategies for incorporation of the LCM into nutrition leadership education and training at the graduate and professional levels; and suggest broader implications for training, research, and practice. Nineteen representatives from 6 MCHB-funded nutrition leadership education and training programs and 10 federal partners participated in a one-day session that began with an overview of the models and concluded with guided small group discussions on how to incorporate them into maternal and child health (MCH) leadership training using obesity as an example. Written notes from group discussions were compiled and coded emergently. Content analysis determined the most salient themes about incorporating the models into training. Four major LCM-related themes emerged, three of which were about training: (1) incorporation by training grants through LCM-framed coursework and experiences for trainees, and similarly framed continuing education and skills development for professionals; (2) incorporation through collaboration with other training programs and state and community partners, and through advocacy; and (3) incorporation by others at the federal and local levels through policy, political, and prevention efforts. The fourth theme focused on anticipated challenges of incorporating the model in training. Multiple methods for incorporating the LCM into MCH training and practice are warranted. Challenges to incorporating include the need for research and related policy development.

Identification of Novel Prostate Cancer-Causitive Gene Mutations by Representational Difference Analysis of Microdissected Prostate Cancer

DTIC Science & Technology

2001-03-01

paired samples of microdissected benign and malignant prostate epithelium. The resulting subtraction products were cloned and screened in Southern blots... benign and malignant human prostate cancer. Data is given to show that microdissected tissue samples retain RNA of sufficient quality to perform gene

Global Combat Support System-Marine Corps Logistics Chain Management Increment 1 (GCSS-MC LCM Inc 1)

DTIC Science & Technology

2016-03-01

Production Document DAE - Defense Acquisition Executive DoD - Department of Defense DoDAF - DoD Architecture Framework FD - Full Deployment FDD ...Jul 2004 Milestone B Jun 2007 Jun 2007 Milestone C May 2010 May 2010 FDD Sep 2014 Mar 2015 FD Dec 2015 Dec 2015 GCSS-MC LCM Inc 1 2016 MAR

Real-time prediction and gating of respiratory motion using an extended Kalman filter and Gaussian process regression

NASA Astrophysics Data System (ADS)

Bukhari, W.; Hong, S.-M.

2015-01-01

Motion-adaptive radiotherapy aims to deliver a conformal dose to the target tumour with minimal normal tissue exposure by compensating for tumour motion in real time. The prediction as well as the gating of respiratory motion have received much attention over the last two decades for reducing the targeting error of the treatment beam due to respiratory motion. In this article, we present a real-time algorithm for predicting and gating respiratory motion that utilizes a model-based and a model-free Bayesian framework by combining them in a cascade structure. The algorithm, named EKF-GPR+, implements a gating function without pre-specifying a particular region of the patient’s breathing cycle. The algorithm first employs an extended Kalman filter (LCM-EKF) to predict the respiratory motion and then uses a model-free Gaussian process regression (GPR) to correct the error of the LCM-EKF prediction. The GPR is a non-parametric Bayesian algorithm that yields predictive variance under Gaussian assumptions. The EKF-GPR+ algorithm utilizes the predictive variance from the GPR component to capture the uncertainty in the LCM-EKF prediction error and systematically identify breathing points with a higher probability of large prediction error in advance. This identification allows us to pause the treatment beam over such instances. EKF-GPR+ implements the gating function by using simple calculations based on the predictive variance with no additional detection mechanism. A sparse approximation of the GPR algorithm is employed to realize EKF-GPR+ in real time. Extensive numerical experiments are performed based on a large database of 304 respiratory motion traces to evaluate EKF-GPR+. The experimental results show that the EKF-GPR+ algorithm effectively reduces the prediction error in a root-mean-square (RMS) sense by employing the gating function, albeit at the cost of a reduced duty cycle. As an example, EKF-GPR+ reduces the patient-wise RMS error to 37%, 39% and 42% in percent ratios relative to no prediction for a duty cycle of 80% at lookahead lengths of 192 ms, 384 ms and 576 ms, respectively. The experiments also confirm that EKF-GPR+ controls the duty cycle with reasonable accuracy.

Laser dissection sampling modes for direct mass spectral analysis [using a hybrid optical microscopy/laser ablation liquid vortex capture/electrospray ionization system

DOE PAGES

Cahill, John F.; Kertesz, Vilmos; Van Berkel, Gary J.

2016-02-01

Here, laser microdissection coupled directly with mass spectrometry provides the capability of on-line analysis of substrates with high spatial resolution, high collection efficiency, and freedom on shape and size of the sampling area. Establishing the merits and capabilities of the different sampling modes that the system provides is necessary in order to select the best sampling mode for characterizing analytically challenging samples. The capabilities of laser ablation spot sampling, laser ablation raster sampling, and laser 'cut and drop' sampling modes of a hybrid optical microscopy/laser ablation liquid vortex capture electrospray ionization mass spectrometry system were compared for the analysis ofmore » single cells and tissue. Single Chlamydomonas reinhardtii cells were monitored for their monogalactosyldiacylglycerol (MGDG) and diacylglyceryltrimethylhomo-Ser (DGTS) lipid content using the laser spot sampling mode, which was capable of ablating individual cells (4-15 m) even when agglomerated together. Turbid Allium Cepa cells (150 m) having unique shapes difficult to precisely measure using the other sampling modes could be ablated in their entirety using laser raster sampling. Intact microdissections of specific regions of a cocaine-dosed mouse brain tissue were compared using laser 'cut and drop' sampling. Since in laser 'cut and drop' sampling whole and otherwise unmodified sections are captured into the probe, 100% collection efficiencies were achieved. Laser ablation spot sampling has the highest spatial resolution of any sampling mode, while laser ablation raster sampling has the highest sampling area adaptability of the sampling modes. In conclusion, laser ablation spot sampling has the highest spatial resolution of any sampling mode, useful in this case for the analysis of single cells. Laser ablation raster sampling was best for sampling regions with unique shapes that are difficult to measure using other sampling modes. Laser 'cut and drop' sampling can be used for cases where the highest sensitivity is needed, for example, monitoring drugs present in trace amounts in tissue.« less

Laser dissection sampling modes for direct mass spectral analysis [using a hybrid optical microscopy/laser ablation liquid vortex capture/electrospray ionization system

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

Cahill, John F.; Kertesz, Vilmos; Van Berkel, Gary J.

Here, laser microdissection coupled directly with mass spectrometry provides the capability of on-line analysis of substrates with high spatial resolution, high collection efficiency, and freedom on shape and size of the sampling area. Establishing the merits and capabilities of the different sampling modes that the system provides is necessary in order to select the best sampling mode for characterizing analytically challenging samples. The capabilities of laser ablation spot sampling, laser ablation raster sampling, and laser 'cut and drop' sampling modes of a hybrid optical microscopy/laser ablation liquid vortex capture electrospray ionization mass spectrometry system were compared for the analysis ofmore » single cells and tissue. Single Chlamydomonas reinhardtii cells were monitored for their monogalactosyldiacylglycerol (MGDG) and diacylglyceryltrimethylhomo-Ser (DGTS) lipid content using the laser spot sampling mode, which was capable of ablating individual cells (4-15 m) even when agglomerated together. Turbid Allium Cepa cells (150 m) having unique shapes difficult to precisely measure using the other sampling modes could be ablated in their entirety using laser raster sampling. Intact microdissections of specific regions of a cocaine-dosed mouse brain tissue were compared using laser 'cut and drop' sampling. Since in laser 'cut and drop' sampling whole and otherwise unmodified sections are captured into the probe, 100% collection efficiencies were achieved. Laser ablation spot sampling has the highest spatial resolution of any sampling mode, while laser ablation raster sampling has the highest sampling area adaptability of the sampling modes. In conclusion, laser ablation spot sampling has the highest spatial resolution of any sampling mode, useful in this case for the analysis of single cells. Laser ablation raster sampling was best for sampling regions with unique shapes that are difficult to measure using other sampling modes. Laser 'cut and drop' sampling can be used for cases where the highest sensitivity is needed, for example, monitoring drugs present in trace amounts in tissue.« less

Preparation and characterization of ultraflat Pt facets by atom-height-resolved differential optical microscopy

NASA Astrophysics Data System (ADS)

Azhagurajan, M.; Wen, R.; Kim, Y. G.; Itoh, T.; Sashikata, K.; Itaya, K.

2015-01-01

We recently demonstrated that improvements to our technique, laser confocal microscopy with differential interference microscopy (LCM-DIM), has rendered it fully capable of resolving monatomic steps with heights of ca. 0.25 nm on Au(111) and Pd(111) surfaces, even as low as 0.14 nm on Si(100), in aqueous solution. In this paper, we describe in detail a method to prepare and characterize, via atomic-layer-resolved LCM-DIM, ultraflat Pt(111) and Pt(100) facets over a wide surface area. The preparation of ultraflat surfaces is important in the characterization at the atomic scale of electrochemical processes under reaction conditions. To showcase the elegance of LCM-DIM, the anodic dissolution of Pt in aqueous HCl is briefly recounted.

Quantitative analysis of fluorouracil-related genes in chronic viral hepatitis using microdissection.

PubMed

Kakinuma, Daisuke; Yoshida, Hiroshi; Mamada, Yasuhiro; Taniai, Nobuhiko; Mizuguchi, Yoshiaki; Takahashi, Tsubasa; Shimizu, Tetsuya; Ishikawa, Yoshinori; Akimaru, Koho; Naito, Zenya; Tajiri, Takashi

2008-01-01

Dihydropyrimidine dehydrogenase is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil. The aim of this study was to determine the levels of messenger RNA for 5-fluorouracil-related metabolic enzymes in cirrhotic liver and to assess the correlation between these mRNA levels and clinicopathological features. The study material consisted of 33 liver samples. The levels of mRNA for the 5- fluorouracil-related metabolic enzymes were quantified by real-time reverse transcription polymerase chain reaction combined with laser-captured microdissection. The Dihydropyrimidine dehydrogenase mRNA level in patients with grade B liver damage was significantly lower than that in patients with grade A liver damage (p=0.009). The Dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferase mRNA level in al samples was higher than that in a2 and a3 samples (p= 0.01 and 0.013, respectively). Statistically significant correlations were found between the hyaluronic acid and the thymidylate phosphorylase mRNA level (p= 0.0001), and the T-BIL and the dihydropyrimidine dehydrogenase mRNA level (p=0.01). The level of Dihydropyrimidine dehydrogenase mRNA may be affected by the clinicopathological status of patients with cirrhosis.

Nuclear Matrix Proteins in Disparity of Prostate Cancer

DTIC Science & Technology

2013-09-01

nuclear coactivator-3 (NCOA3). 5 Methods Patients and Prostate Cancer Specimens Fresh, flash -frozen specimens were obtained from age- (50 to...for reliable data interpretation. Gene Array Analysis Total RNA isolated from LCM-procured normal epithelium and tumor cells from flash -frozen...PCR Briefly, RNA was extracted from matched LCM procured normal epithelium and tumor cells of age-, tumor grade-matched flash -frozen sections (n=24

Influence of WIN 55,212-2 on the anticonvulsant and acute neurotoxic potential of clobazam and lacosamide in the maximal electroshock-induced seizure model and chimney test in mice.

PubMed

Florek-Luszczki, Magdalena; Zagaja, Miroslaw; Luszczki, Jarogniew J

2014-12-01

The influence of WIN 55,212-2 mesylate (WIN) on the anticonvulsant activity and acute neurotoxic potential of clobazam (CLB) and lacosamide (LCM) was studied in the maximal electroshock-induced seizure (MES) model and chimney test in mice. indicate that WIN administered intraperitoneally, at doses of 2.5 and 5 mg/kg significantly enhanced the anticonvulsant action of CLB in the MES test by reducing its median effective dose (ED50) from 20.80 mg/kg to 12.05 mg/kg (P<0.05), and 8.22 mg/kg (P<0.001), respectively. In contrast, WIN (1.25 mg/kg) did not significantly potentiate the anticonvulsant activity of CLB against MES-induced seizures. Similarly, WIN at doses of 1.25, 2.5 and 5 mg/kg had no significant impact on the anticonvulsant action of LCM in the MES test. On the other hand, WIN (5 mg/kg) had no impact on the acute neurotoxic effects of CLB and LCM in the chimney test and the median toxic doses (TD50) for CLB and LCM were almost unchanged. Thus, WIN (5 mg/kg) elevated the protective index values for CLB (from 1.41 to 3.07) and LCM (from 3.60 to 4.91). In conclusion, WIN potentiates suppression of tonic-clonic seizures produced by CLB in the mouse MES model, without affecting acute neurotoxic adverse effects of CLB in the chimney test in mice, which is favorable from a preclinical point of view. Copyright © 2014 Elsevier B.V. All rights reserved.

Randomized double-blind comparison of cognitive and EEG effects of lacosamide and carbamazepine.

PubMed

Meador, Kimford J; Loring, David W; Boyd, Alan; Echauz, Javier; LaRoche, Suzette; Velez-Ruiz, Naymee; Korb, Pearce; Byrnes, William; Dilley, Deanne; Borghs, Simon; De Backer, Marc; Story, Tyler; Dedeken, Peter; Webster, Elizabeth

2016-09-01

Differential effectiveness of antiepileptic drugs (AEDs) is more commonly determined by tolerability than efficacy. Cognitive effects of AEDs can adversely affect tolerability and quality of life. This study evaluated cognitive and EEG effects of lacosamide (LCM) compared with carbamazepine immediate-release (CBZ-IR). A randomized, double-blind, double-dummy, two-period crossover, fixed-dose study in healthy subjects compared neuropsychological and EEG effects of LCM (150mg, b.i.d.) and CBZ-IR (200mg, t.i.d.). Testing was conducted at screening, predrug baseline, the end of each treatment period (3-week titration; 3-week maintenance), and the end of each washout period (4weeks after treatment). A composite Z-score was derived for the primary outcome variable (computerized cognitive tests and traditional neuropsychological measures) and separately for the EEG measures. Other variables included individual computer, neuropsychological, and EEG scores and adverse events (AEs). Subjects included 60 healthy adults (57% female; mean age: 34.4years [SD: 10.5]); 44 completed both treatments; 41 were per protocol subjects. Carbamazepine immediate-release had worse scores compared with LCM for the primary composite neuropsychological outcome (mean difference=0.33 [SD: 1.36], p=0.011) and for the composite EEG score (mean difference=0.92 [SD: 1.77], p=0.003). Secondary analyses across the individual variables revealed that CBZ-IR was statistically worse than LCM on 36% (4/11) of the neuropsychological tests (computerized and noncomputerized) and 0% of the four EEG measures; none favored CBZ-IR. Drug-related AEs occurred more with CBZ-IR (49%) than LCM (22%). Lacosamide had fewer untoward neuropsychological and EEG effects and fewer AEs and AE-related discontinuations than CBZ-IR in healthy subjects. Lacosamide exhibits a favorable cognitive profile. Copyright © 2016 Elsevier Inc. All rights reserved.

Laser micro-dissection and qPCR for identifying specific HPV types responsible for malignancy in penile lesions.

PubMed

Lebelo, Ramokone L; Thys, Sofie; Benoy, Ina; Depuydt, Christophe E; Bogers, John-Paul; Bida, Meshack N; Mphahlele, M Jeffrey

2015-10-01

The aim of the study was to identify specific human papillomavirus (HPV) type responsible for malignancy in penile tissue samples using laser micro-dissection and TaqMan quantitative real-time PCR (qPCR). The study was based on two pre-malignant and seven malignant penile tissue samples and laser micro-dissection was performed on all. Genotyping was performed on whole tissue sections and laser micro-dissection samples using qPCR. Two whole tissue section samples were HPV negative while seven were HPV positive. In four samples that were single HPV infections with whole tissue section PCR, identical HPV types were confirmed with laser micro-dissection PCR. Clearly confirming that the single HPV type detected is responsible for malignancy. In two samples that had multiple HPV infections with whole tissue section PCR, only one HPV type with the highest viral load was detected with laser micro-dissection PCR, suggesting that the HPV type with the highest viral load is most likely the cause of that particular lesion. HPV 11 and/or HPV 16 were the only types detected with laser micro-dissection PCR in these cases, compared to multiple HPV types (HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, and HPV 39) initially detected with whole tissue section PCR. HPV 11 was associated with verrucous lesions while HPV 16 was associated with squamous cell carcinoma and PIN 3 lesions. This study confirms that laser micro-dissection and qPCR are essential tools in identifying the HPV types responsible for malignancy in penile lesions, particularly in samples with multiple infections. © 2015 Wiley Periodicals, Inc.

Noteworthy performance of La(1-x)Ca(x)MnO3 perovskites in generating H2 and CO by the thermochemical splitting of H2O and CO2.

PubMed

Dey, Sunita; Naidu, B S; Govindaraj, A; Rao, C N R

2015-01-07

Perovskite oxides of the composition La1-xCaxMnO3 (LCM) have been investigated for the thermochemical splitting of H2O and CO2 to produce H2 and CO, respectively. The study was carried out in comparison with La1-xSrxMnO3, CeO2 and other oxides. The LCM system exhibits superior characteristics in high-temperature evolution of oxygen, and in reducing CO2 to CO and H2O to H2. The best results were obtained with La0.5Ca0.5MnO3 whose performance is noteworthy compared to that of other oxides including ceria. The orthorhombic structure of LCM seems to be a crucial factor.

Laser confocal microscope for analysis of 3013 inner container closure weld region

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

Martinez-Rodriguez, M. J.

As part of the protocol to investigate the corrosion in the inner container closure weld region (ICCWR) a laser confocal microscope (LCM) was used to perform close visual examination of the surface and measurements of corrosion features on the surface. However, initial analysis of selected destructively evaluated (DE) containers using the LCM revealed several challenges for acquiring, processing and interpreting the data. These challenges include topography of the ICCWR sample, surface features, and the amount of surface area for collecting data at high magnification conditions. In FY17, the LCM parameters were investigated to identify the appropriate parameter values for datamore » acquisition and identification of regions of interest. Using these parameter values, selected DE containers were analyzed to determine the extent of the ICCWR to be examined.« less

The Balance-Scale Task Revisited: A Comparison of Statistical Models for Rule-Based and Information-Integration Theories of Proportional Reasoning

PubMed Central

Hofman, Abe D.; Visser, Ingmar; Jansen, Brenda R. J.; van der Maas, Han L. J.

2015-01-01

We propose and test three statistical models for the analysis of children’s responses to the balance scale task, a seminal task to study proportional reasoning. We use a latent class modelling approach to formulate a rule-based latent class model (RB LCM) following from a rule-based perspective on proportional reasoning and a new statistical model, the Weighted Sum Model, following from an information-integration approach. Moreover, a hybrid LCM using item covariates is proposed, combining aspects of both a rule-based and information-integration perspective. These models are applied to two different datasets, a standard paper-and-pencil test dataset (N = 779), and a dataset collected within an online learning environment that included direct feedback, time-pressure, and a reward system (N = 808). For the paper-and-pencil dataset the RB LCM resulted in the best fit, whereas for the online dataset the hybrid LCM provided the best fit. The standard paper-and-pencil dataset yielded more evidence for distinct solution rules than the online data set in which quantitative item characteristics are more prominent in determining responses. These results shed new light on the discussion on sequential rule-based and information-integration perspectives of cognitive development. PMID:26505905

Altered anti-inflammatory response of mononuclear cells to neuropeptide PACAP is associated with deregulation of NF-{kappa}B in chronic pancreatitis.

PubMed

Michalski, Christoph W; Selvaggi, Federico; Bartel, Michael; Mitkus, Tomas; Gorbachevski, Andrej; Giese, Thomas; Sebastiano, Pierluigi Di; Giese, Nathalia A; Friess, Helmut

2008-01-01

Although it is recognized that neurogenic influences contribute to progression of chronic inflammatory diseases, the molecular basis of neuroimmune interactions in the pathogenesis of chronic pancreatitis (CP) is not well defined. Here we report that responsiveness of peripheral blood mononuclear cells (PBMC) to the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is altered in CP. Expression of PACAP and its receptors in human CP was analyzed with quantitative RT-PCR, laser-capture microdissection, and immunohistochemistry. Regulation of PACAP expression was studied in coculture systems using macrophages and acinar cells. Responsiveness of donor and CP PBMC to PACAP was determined based on cytokine profiles and NF-kappaB activation of LPS- or LPS+PACAP-exposed cells. Although donor and CP PBMC responded equally to LPS, PACAP-mediated counteraction of LPS-induced cytokine response was switched from inhibiting TNF-alpha to decreasing IL-1beta and increasing IL-10 secretion. The change of PACAP-mediated anti-inflammatory pattern was associated with altered activation of NF-kappaB: compared with LPS alone, a combination of LPS and PACAP had no effect on NF-kappaB p65 nuclear translocation in CP PBMC, whereas NF-kappaB was significantly decreased in donor PBMC. According to laser-capture microdissection and coculture experiments, PBMC also contributed to generation of a PACAP-rich intrapancreatic environment by upregulating PACAP expression in macrophages encountering apoptotic pancreatic acini. The nociceptive status of CP patients correlated with pancreatic PACAP levels and with IL-10 bias of PACAP-exposed CP PBMC. Thus the ability of PBMC to produce and to respond to PACAP might influence neuroimmune interactions that regulate pain and inflammation in CP.

Myosin content of individual human muscle fibers isolated by laser capture microdissection.

PubMed

Stuart, Charles A; Stone, William L; Howell, Mary E A; Brannon, Marianne F; Hall, H Kenton; Gibson, Andrew L; Stone, Michael H

2016-03-01

Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle. Copyright © 2016 the American Physiological Society.

Myosin content of individual human muscle fibers isolated by laser capture microdissection

PubMed Central

Stone, William L.; Howell, Mary E. A.; Brannon, Marianne F.; Hall, H. Kenton; Gibson, Andrew L.; Stone, Michael H.

2015-01-01

Muscle fiber composition correlates with insulin resistance, and exercise training can increase slow-twitch (type I) fibers and, thereby, mitigate diabetes risk. Human skeletal muscle is made up of three distinct fiber types, but muscle contains many more isoforms of myosin heavy and light chains, which are coded by 15 and 11 different genes, respectively. Laser capture microdissection techniques allow assessment of mRNA and protein content in individual fibers. We found that specific human fiber types contain different mixtures of myosin heavy and light chains. Fast-twitch (type IIx) fibers consistently contained myosin heavy chains 1, 2, and 4 and myosin light chain 1. Type I fibers always contained myosin heavy chains 6 and 7 (MYH6 and MYH7) and myosin light chain 3 (MYL3), whereas MYH6, MYH7, and MYL3 were nearly absent from type IIx fibers. In contrast to cardiomyocytes, where MYH6 (also known as α-myosin heavy chain) is seen solely in fast-twitch cells, only slow-twitch fibers of skeletal muscle contained MYH6. Classical fast myosin heavy chains (MHC1, MHC2, and MHC4) were present in variable proportions in all fiber types, but significant MYH6 and MYH7 expression indicated slow-twitch phenotype, and the absence of these two isoforms determined a fast-twitch phenotype. The mixed myosin heavy and light chain content of type IIa fibers was consistent with its role as a transition between fast and slow phenotypes. These new observations suggest that the presence or absence of MYH6 and MYH7 proteins dictates the slow- or fast-twitch phenotype in skeletal muscle. PMID:26676053

Laser Capture Microdissection Assessment of Virus Compartmentalization in the Central Nervous Systems of Macaques Infected with Neurovirulent Simian Immunodeficiency Virus

PubMed Central

Matsuda, Kenta; Brown, Charles R.; Foley, Brian; Goeken, Robert; Whitted, Sonya; Dang, Que; Wu, Fan; Plishka, Ronald; Buckler-White, Alicia

2013-01-01

Nonhuman primate-simian immunodeficiency virus (SIV) models are powerful tools for studying the pathogenesis of human immunodeficiency virus type 1 (HIV-1) in the brain. Our laboratory recently isolated a neuropathogenic viral swarm, SIVsmH804E, a derivative of SIVsmE543-3, which was the result of sequential intravenous passages of viruses isolated from the brains of rhesus macaques with SIV encephalitis. Animals infected with SIVsmH804E or its precursor (SIVsmH783Br) developed SIV meningitis and/or encephalitis at high frequencies. Since we observed macaques with a combination of meningitis and encephalitis, as well as animals in which meningitis or encephalitis was the dominant component, we hypothesized that distinct mechanisms could be driving the two pathological states. Therefore, we assessed viral populations in the meninges and the brain parenchyma by laser capture microdissection. Viral RNAs were isolated from representative areas of the meninges, brain parenchyma, terminal plasma, and cerebrospinal fluid (CSF) and from the inoculum, and the SIV envelope fragment was amplified by PCR. Phylogenetic analysis of envelope sequences from the conventional progressors revealed compartmentalization of viral populations between the meninges and the parenchyma. In one of these animals, viral populations in meninges were closely related to those from CSF and shared signature truncations in the cytoplasmic domain of gp41, consistent with a common origin. Apart from magnetic resonance imaging (MRI) and positron-emission tomography (PET) imaging, CSF is the most accessible assess to the central nervous system for HIV-1-infected patients. However, our results suggest that the virus in the CSF may not always be representative of viral populations in the brain and that caution should be applied in extrapolating between the properties of viruses in these two compartments. PMID:23720733

Isolating RNA from precursor and mature melanocytes from human vitiligo and normal skin using laser capture microdissection.

PubMed

Goldstein, Nathaniel B; Koster, Maranke I; Hoaglin, Laura G; Wright, Michael J; Robinson, Steven E; Robinson, William A; Roop, Dennis R; Norris, David A; Birlea, Stanca A

2016-10-01

To characterize the gene expression profile of regenerated melanocytes in the narrow band UVB (NBUVB)-treated vitiligo epidermis and their precursors in the hair follicle, we present here a strategy of RNA isolation from in situ melanocytes using human frozen skin. We developed a rapid immunostaining protocol using the NKI-beteb antibody, which labels differentiated and precursor melanocytes, followed by fluorescent laser capture microdissection. This technique enabled the direct isolation, from melanocyte and adjacent keratinocyte populations, of satisfactory quality RNA that was successfully amplified and analysed by qRT-PCR. The melanocyte-specific gene transcripts TYR, DCT, TYRP1 and PMEL were significantly upregulated in our NBUVB-treated melanocyte samples as compared with the keratinocyte samples, while keratinocyte-specific genes (KRT5 and KRT14) were expressed significantly higher in the keratinocyte samples as compared with the melanocyte samples. Furthermore, in both NBUVB-treated vitiligo skin and normal skin, when bulge melanocytes were compared with epidermal melanocytes, we found significantly lower expression of melanocyte-specific genes and significantly higher expression of three melanocytic stem cell genes (SOX9, WIF1 and SFRP1), while ALCAM and ALDH1A1 transcripts did not show significant variation. We found significantly higher expression of melanocyte-specific genes in the epidermis of NBUVB-treated vitiligo, as compared to the normal skin. When comparing bulge melanocyte samples from untreated vitiligo, NBUVB-treated vitiligo and normal skin, we did not find significant differences in the expression of melanocyte-specific genes or melanocytic stem cell genes. These techniques offer valuable opportunities to study melanocytes and their precursors in vitiligo and other pigmentation disorders. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The Chromosome Microdissection and Microcloning Technique.

PubMed

Zhang, Ying-Xin; Deng, Chuan-Liang; Hu, Zan-Min

2016-01-01

Chromosome microdissection followed by microcloning is an efficient tool combining cytogenetics and molecular genetics that can be used for the construction of the high density molecular marker linkage map and fine physical map, the generation of probes for chromosome painting, and the localization and cloning of important genes. Here, we describe a modified technique to microdissect a single chromosome, paint individual chromosomes, and construct single-chromosome DNA libraries.

Analysis of Particulates in the Exhaust Plume of a TF30 Engine at Military Power.

DTIC Science & Technology

1984-05-01

DIADAH-LCA (2) flHDAR LCAI1’ 2, D)AfI-L(:E (21 DRtDAR-LCE-C. Cieistry Branch (1 DRtDAB-LCE-D (2) D1IDAR-LCNI (2) DRDAR-LCM-SA. R. Westerdahl (1) DRDAR...DRDAR-LCM-SA, R. Westerdahl (1) DBRDAR-LCN-C, Concepts and Analysis Branch (4) DRDAR-LCU (2) DFIDAB-LCU-E (1) 4 Army Ballistic Research Laboratory

Quality of life, mood and seizure control in patients with brain tumor related epilepsy treated with lacosamide as add-on therapy: A prospective explorative study with a historical control group.

PubMed

Maschio, Marta; Zarabla, Alessia; Maialetti, Andrea; Fabi, Alessandra; Vidiri, Antonello; Villani, Veronica; Giannarelli, Diana

2017-08-01

Brain tumor-related epilepsy (BTRE) is often drug resistant and patients can be forced to take polytherapy that can adversely affect their quality of life (QoL). Lacosamide (LCM) is a new antiepileptic drug (AED) used as adjunctive therapy in patients with partial seizures with or without secondary generalization, with a favorable pharmacokinetic profile that seems to be effective and well tolerated. Therefore it represents a possible therapeutic choice for patients with BTRE. We propose a prospective study with a historical control group to evaluate the effect of LCM as add-on therapy on seizure control and quality of life in patients with BTRE. This study has been designed to test the superiority of Lacosamide over Levetiracetam as an add-on. We compared a prospective cohort of 25 patients treated with Lacosamide with a historical control group (n=19) treated with Levetiracetam as an add-on. We recruited 25 adult patients (M 18, F 7; mean age 41.9) affected by BTRE with uncontrolled partial-onset seizures treated with AED polytherapy. We added LCM as an add-on. Patients were evaluated at baseline, after 3months and at 6months. This population has been compared with a historical control group of 19 BTRE adult patients (M 13, F 6; median age 48.0, range: 28-70) with uncontrolled partial-onset seizures treated with LEV as add-on. The patients underwent QoL, mood and adverse events tests (Adverse Event Profile-AEP) and evaluation of seizure frequency. Twelve patients had high grade gliomas, and thirteen had low grade gliomas. During follow-up, thirteen patients underwent chemotherapy, three radiotherapy and five patients had disease progression. Nine patients had simple partial seizures, eight had complex partial seizures, and eight had secondary generalized seizures. Fifteen patients were in monotherapy and ten in polytherapy with AEDs. LCM was added up to reach the maximum dosage of 400mg/die (mean final dose 300mg/die). Four patients dropped out due to poor compliance and 1 for inefficacy. In the historical control group treated with LEV (mean final dose 2000mg/die) 12 patients had high-grade gliomas, and 7 had low grade gliomas. Thirteen patients were in monotherapy and 6 in polytherapy with AEDs. In the 22 patients evaluable of 25 patients treated with LCM, we observed at final follow-up 7 patients seizure free, 12 with a significant reduction of seizures≥50%, 2 stable and 1 patient with number of seizures increased. Mean seizure frequency at baseline compared with baseline period: the mean number of seizures significantly decreased from baseline (9.4) to final follow-up (1.2) (P=0.005). The Responder Rate was 86.4%. Comparing responder rate of 22 evaluable patients with LCM with responder rate of 19 patients with LEV we didn't observe significant differences (p=0.31). In our patients treated with LCM we didn't observe significant difference at 3 and 6months in QoL tests results; we observe a significant reduction in the mean score of Karnofsky Performance Status (KPS) and Barthel Index (BI) between baseline and 6months of follow-up (KPS p=0.003; BI p=0.007). No clinical side effects were observed. Comparing the LCM with the historical group treated with LEV in add-on, we observed that LCM seems to have a higher clinical efficacy than LEV. In our patients, we did not observe any significant changes in QoL tests, indicating stability in all quality of life domains explored, despite the objective worsening in their functional status. Although this is a small series with a relatively short follow-up, our data indicates that LCM in add-on in patients with BTRE appears to be as effective as LEV in add-on, without impact on mood and quality of life. Copyright © 2017 Elsevier Inc. All rights reserved.

Oil Palm Empty Fruit Bunch (OPEFB) Fiber as Lost Circulation Material (LCM) in Water Based Mud (WBM)

NASA Astrophysics Data System (ADS)

Ghazali, N. A.; Sauki, A.; Abu Bakar, N. F.; Mohamed, S.

2018-05-01

Lost Circulation Material (LCM) is an additive used to prevent lost of mud to the formation as a results from natural or induced fractured during drilling operation. Losses of mud could give great impact to the oil industry as it increases mud cost and rig time. The objective of this research was to investigate the effect of size and concentration of Oil Palm Empty Fruit Bunch (OPEFB) as LCM in water based mud (WBM). Several important properties of WBM rheology after adding the OPEFB namely plastic viscosity, apparent viscosity, yield point and gel strength were characterized. The sizes of OPEFB added into the WBM were 150μm, 250μm, 500μm and 1000μm while the concentration of OPEFB used were 5g, 10g, 15g and 20g in 350 mL of WBM. Results indicated that the plastic viscosity and apparent viscosity increased with increasing of the OPEFB concentrations. On the other hand, the plastic viscosity and apparent viscosity decreased with increasing sizes of OPEFB. Yield point increased as the concentration and size of OPEFB increases. This study indicated that OPEFB was effective to be used as LCM for size of 150μm and concentration of 15g whereby it produced least amount of filtrate volume as well as good control in mud rheology.

Economic contribution of draught animals to Mazahua smallholder Campesino farming systems in the highlands of Central Mexico.

PubMed

Arriaga-Jordán, C M; Pedraza-Fuentes, A M; Velázquez-Beltrán, L G; Nava-Bernal, E G; Chávez-Mejía, M C

2005-10-01

The economic contribution of draught animals to smallholder Mazahua campesino systems in two mountain villages of San Felipe del Progreso, in the central highlands of Mexico, was assessed. Campesinos rely on draught animals for cultivation tasks, as pack animals, and as transport for agricultural and domestic activities. The villages were San Pablo Tlalchichilpa (SPT) and La Concepción Mayorazgo (LCM). Twelve households that possessed draught animals were monitored from July 1999 to June 2000, nine in SPT and three in LCM, in terms of animal inventories and income from their draught animals, in cash and opportunity values. Equines in SPT have substituted bulls, and are recognized for their multipurpose contribution, while in LCM bulls are still used for ploughing the land. Overall total mean gross income was US dollar 490.78 per farm per year, plus US dollar 56 as opportunity value of the fertilizer value of manure for both villages. Deducting estimated costs, owning draught animals leaves a mean net margin of US dollar 412.50/year in SPT and of US dollar 285.64/year in LCM. There is a significant correlation (p < 0.05) between ownership of draught animals and incomes, with a regression coefficient of US dollar 279.16 per year per draught animal. Besides positive economic returns, having work animals alleviates drudgery for the campesino families.

In Vivo Antiviral Activity of 1,3-Bis(2-Chloroethyl)-1-Nitrosourea

PubMed Central

Sidwell, Robert W.; Dixon, Glen J.; Sellers, Sara M.; Schabel, Frank M.

1965-01-01

A prolongation in the lives of Swiss mice inoculated intracerebrally with lymphocytic choriomeningitis virus (LCM) was observed after treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). A variety of treatment schedules, including therapy once or twice daily up to 17 days and single treatments at various times after virus inoculation, were employed. Virus titers ranging to greater than 104 were detected in the blood and brains of surviving drug-treated animals. In three comparative studies in which different treatment schedules were used, BCNU was shown to exert a protective effect approximately equal to that of methotrexate in LCM virus-infected mice. Tests were also carried out to investigate the activity of BCNU in mice experimentally infected with eastern equine encephalomyelitis (EEE) virus, western equine encephalomyelitis virus, Semliki Forest (SF) virus, herpes simplex virus, influenza virus strain PR8, vaccinia virus strain WR, Rous sarcoma virus, Friend leukemia virus (FLV), and poliovirus. Slight increases in life span were observed in the treated EEE, SF, and influenza PR8 virus-infected animals. Significant reduction in splenomegaly in FLV-infected animals treated with BCNU was demonstrated. The possible mechanisms of LCM virus inhibition by BCNU, on the basis of these and other studies, were postulated to be either specific antiviral activity or inhibition of “lethal” immune response to the LCM virus. Each of these postulates is discussed. PMID:14339266

Like will to like: abundances of closely related species can predict susceptibility to intestinal colonization by pathogenic and commensal bacteria.

PubMed

Stecher, Bärbel; Chaffron, Samuel; Käppeli, Rina; Hapfelmeier, Siegfried; Freedrich, Susanne; Weber, Thomas C; Kirundi, Jorum; Suar, Mrutyunjay; McCoy, Kathy D; von Mering, Christian; Macpherson, Andrew J; Hardt, Wolf-Dietrich

2010-01-01

The intestinal ecosystem is formed by a complex, yet highly characteristic microbial community. The parameters defining whether this community permits invasion of a new bacterial species are unclear. In particular, inhibition of enteropathogen infection by the gut microbiota ( = colonization resistance) is poorly understood. To analyze the mechanisms of microbiota-mediated protection from Salmonella enterica induced enterocolitis, we used a mouse infection model and large scale high-throughput pyrosequencing. In contrast to conventional mice (CON), mice with a gut microbiota of low complexity (LCM) were highly susceptible to S. enterica induced colonization and enterocolitis. Colonization resistance was partially restored in LCM-animals by co-housing with conventional mice for 21 days (LCM(con21)). 16S rRNA sequence analysis comparing LCM, LCM(con21) and CON gut microbiota revealed that gut microbiota complexity increased upon conventionalization and correlated with increased resistance to S. enterica infection. Comparative microbiota analysis of mice with varying degrees of colonization resistance allowed us to identify intestinal ecosystem characteristics associated with susceptibility to S. enterica infection. Moreover, this system enabled us to gain further insights into the general principles of gut ecosystem invasion by non-pathogenic, commensal bacteria. Mice harboring high commensal E. coli densities were more susceptible to S. enterica induced gut inflammation. Similarly, mice with high titers of Lactobacilli were more efficiently colonized by a commensal Lactobacillus reuteri(RR) strain after oral inoculation. Upon examination of 16S rRNA sequence data from 9 CON mice we found that closely related phylotypes generally display significantly correlated abundances (co-occurrence), more so than distantly related phylotypes. Thus, in essence, the presence of closely related species can increase the chance of invasion of newly incoming species into the gut ecosystem. We provide evidence that this principle might be of general validity for invasion of bacteria in preformed gut ecosystems. This might be of relevance for human enteropathogen infections as well as therapeutic use of probiotic commensal bacteria.

Layered composites made from bimetallic strips produced by plasma spraying of TiAl on niobium

NASA Astrophysics Data System (ADS)

Burmistrov, V. I.; Antonova, A. V.; Povarova, K. B.; Bannykh, I. O.

2007-12-01

The production and structure of a multilayer TiAl/Nb composite material made from bimetallic TiAl/Nb strips fabricated by plasma spraying of TiAl granules onto niobium plates are studied. Here, 3-mm-and 2-mm-thick plates of a layered composite material (LCM) are produced by hot isostatic pressing of a stack of 35 bimetallic plates followed by hot rolling (the total degree of reduction is 78.6 and 85.7%, respectively). The LCM consists of discontinuous TiAl layers separated by niobium layers, and the adhesion between the layers is good. Diffusional intermediate layers form at the TiAl/Nb interfaces in the 3-mm-thick LCM and consist of the following two solid solutions: an α2-Ti3Al-based solid solution contains up to 28 at % Nb, and a niobiumbased solid solution contains up to 27 at % Ti and 32 at % Al. The diffusional intermediate layers in the 2-mmthick LCM plates consist of an α2-Ti3Al-based solid solution with up to 16.0 at % Nb; a τ-Ti3Al2Nb-or Ti4Al3Nb-based solid solution with 51.5 at % Ti, 32 at % Al, and 16.5 at % Nb; and a niobium-based solid solution with up to 22 at % Ti and 30.5 at % Al. When a bimetallic TiAl/Nb strip is fabricated by plasma spraying of granules of the Ti-48 at % Al alloy, this alloy is depleted of aluminum to 42 45 at %, and the fraction of the α2-Ti3Al phase in the sprayed layer increases. When the LCM is produced by hot isostatic pressing followed by hot rolling, the layer of plain niobium (Nb1) dissolves up to 5 at % Ti and 7 at % Al.

Establishment of a protocol for the gene expression analysis of laser microdissected rat kidney samples with affymetrix genechips

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

Stemmer, Kerstin; Ellinger-Ziegelbauer, Heidrun; Lotz, Kerstin

2006-11-15

Laser microdissection in conjunction with microarray technology allows selective isolation and analysis of specific cell populations, e.g., preneoplastic renal lesions. To date, only limited information is available on sample preparation and preservation techniques that result in both optimal histomorphological preservation of sections and high-quality RNA for microarray analysis. Furthermore, amplification of minute amounts of RNA from microdissected renal samples allowing analysis with genechips has only scantily been addressed to date. The objective of this study was therefore to establish a reliable and reproducible protocol for laser microdissection in conjunction with microarray technology using kidney tissue from Eker rats p.o. treatedmore » for 7 days and 6 months with 10 and 1 mg Aristolochic acid/kg bw, respectively. Kidney tissues were preserved in RNAlater or snap frozen. Cryosections were cut and stained with either H and E or cresyl violet for subsequent morphological and RNA quality assessment and laser microdissection. RNA quality was comparable in snap frozen and RNAlater-preserved samples, however, the histomorphological preservation of renal sections was much better following cryopreservation. Moreover, the different staining techniques in combination with sample processing time at room temperature can have an influence on RNA quality. Different RNA amplification protocols were shown to have an impact on gene expression profiles as demonstrated with Affymetrix Rat Genome 230{sub 2}.0 arrays. Considering all the parameters analyzed in this study, a protocol for RNA isolation from laser microdissected samples with subsequent Affymetrix chip hybridization was established that was also successfully applied to preneoplastic lesions laser microdissected from Aristolochic acid-treated rats.« less

Army Training and Testing Area Carrying Capacity (ATTACC) LS Factor Calculator User Manual, Version 1.0

DTIC Science & Technology

2002-08-01

of these elevation files depends on the contour interval and map scale of the original contour data. The choice of data source for determining the...the ATTACC LCM Installation CD. Not all versions of the ATTACC LCM Installation CD have the LS Factor Calculator software included. The process for ...PAGE Form Approved OMB No. 0704-0188 Public reporting burder for this collection of information is estibated to average 1 hour per response

Latent Class Models in action: bridging social capital & Internet usage.

PubMed

Neves, Barbara Barbosa; Fonseca, Jaime R S

2015-03-01

This paper explores how Latent Class Models (LCM) can be applied in social research, when the basic assumptions of regression models cannot be validated. We examine the usefulness of this method with data collected from a study on the relationship between bridging social capital and the Internet. Social capital is defined here as the resources that are potentially available in one's social ties. Bridging is a dimension of social capital, usually related to weak ties (acquaintances), and a source of instrumental resources such as information. The study surveyed a stratified random sample of 417 inhabitants of Lisbon, Portugal. We used LCM to create the variable bridging social capital, but also to estimate the relationship between bridging social capital and Internet usage when we encountered convergence problems with the logistic regression analysis. We conclude by showing a positive relationship between bridging and Internet usage, and by discussing the potential of LCM for social science research. Copyright © 2014 Elsevier Inc. All rights reserved.

Functional genomics of a generalist parasitic plant: Laser microdissection of host-parasite interface reveals host-specific patterns of parasite gene expression

PubMed Central

2013-01-01

Background Orobanchaceae is the only plant family with members representing the full range of parasitic lifestyles plus a free-living lineage sister to all parasitic lineages, Lindenbergia. A generalist member of this family, and an important parasitic plant model, Triphysaria versicolor regularly feeds upon a wide range of host plants. Here, we compare de novo assembled transcriptomes generated from laser micro-dissected tissues at the host-parasite interface to uncover details of the largely uncharacterized interaction between parasitic plants and their hosts. Results The interaction of Triphysaria with the distantly related hosts Zea mays and Medicago truncatula reveals dramatic host-specific gene expression patterns. Relative to above ground tissues, gene families are disproportionally represented at the interface including enrichment for transcription factors and genes of unknown function. Quantitative Real-Time PCR of a T. versicolor β-expansin shows strong differential (120x) upregulation in response to the monocot host Z. mays; a result that is concordant with our read count estimates. Pathogenesis-related proteins, other cell wall modifying enzymes, and orthologs of genes with unknown function (annotated as such in sequenced plant genomes) are among the parasite genes highly expressed by T. versicolor at the parasite-host interface. Conclusions Laser capture microdissection makes it possible to sample the small region of cells at the epicenter of parasite host interactions. The results of our analysis suggest that T. versicolor’s generalist strategy involves a reliance on overlapping but distinct gene sets, depending upon the host plant it is parasitizing. The massive upregulation of a T. versicolor β-expansin is suggestive of a mechanism for parasite success on grass hosts. In this preliminary study of the interface transcriptomes, we have shown that T. versicolor, and the Orobanchaceae in general, provide excellent opportunities for the characterization of plant genes with unknown functions. PMID:23302495

Solid Cattle Manure Less Prone to Phosphorus Loss in Tile Drainage Water.

PubMed

Wang, Y T; Zhang, T Q; Tan, C S; Qi, Z M; Welacky, T

2018-03-01

Forms (e.g., liquid and solid) of manure influence the risk of P loss after land application. The objective of this study was to investigate the effects of P-based application of various forms of cattle manure (liquid, LCM; or solid, SCM) or inorganic P as triple superphosphate (IP) on soil P losses in tile drainage water. A 4-yr field experiment was conducted in a clay loam soil with a corn ( L.)-soybean [ (L.) Merr.] rotation in the Lake Erie basin. Over the 4 yr, the dissolved reactive P (DRP) flow-weighted mean concentration (FWMC) in tile drainage water was greater under SCM fertilization than under either IP or LCM fertilization. Despite its lower value on an annual basis, DRP FWMC rose dramatically immediately after LCM application. However, the differences in DRP FWMC did not result in detectable differences in DRP loads. Regarding particulate P and total P losses during the 4 yr, they were 68 and 47%, respectively, lower in the soils amended with SCM than in those with IP, whereas both values were similar between IP and LCM treatments. Overall, the P contained in solid cattle manure was less prone to P loss after land application. Accordingly, the present results can provide a basis for manure storage and application of best management practices designed to reduce P losses and improve crop growth. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

Development of a system emulating the global carbon cycle in Earth system models

NASA Astrophysics Data System (ADS)

Tachiiri, K.; Hargreaves, J. C.; Annan, J. D.; Oka, A.; Abe-Ouchi, A.; Kawamiya, M.

2010-08-01

Recent studies have indicated that the uncertainty in the global carbon cycle may have a significant impact on the climate. Since state of the art models are too computationally expensive for it to be possible to explore their parametric uncertainty in anything approaching a comprehensive fashion, we have developed a simplified system for investigating this problem. By combining the strong points of general circulation models (GCMs), which contain detailed and complex processes, and Earth system models of intermediate complexity (EMICs), which are quick and capable of large ensembles, we have developed a loosely coupled model (LCM) which can represent the outputs of a GCM-based Earth system model, using much smaller computational resources. We address the problem of relatively poor representation of precipitation within our EMIC, which prevents us from directly coupling it to a vegetation model, by coupling it to a precomputed transient simulation using a full GCM. The LCM consists of three components: an EMIC (MIROC-lite) which consists of a 2-D energy balance atmosphere coupled to a low resolution 3-D GCM ocean (COCO) including an ocean carbon cycle (an NPZD-type marine ecosystem model); a state of the art vegetation model (Sim-CYCLE); and a database of daily temperature, precipitation, and other necessary climatic fields to drive Sim-CYCLE from a precomputed transient simulation from a state of the art AOGCM. The transient warming of the climate system is calculated from MIROC-lite, with the global temperature anomaly used to select the most appropriate annual climatic field from the pre-computed AOGCM simulation which, in this case, is a 1% pa increasing CO2 concentration scenario. By adjusting the effective climate sensitivity (equivalent to the equilibrium climate sensitivity for an energy balance model) of MIROC-lite, the transient warming of the LCM could be adjusted to closely follow the low sensitivity (with an equilibrium climate sensitivity of 4.0 K) version of MIROC3.2. By tuning of the physical and biogeochemical parameters it was possible to reasonably reproduce the bulk physical and biogeochemical properties of previously published CO2 stabilisation scenarios for that model. As an example of an application of the LCM, the behavior of the high sensitivity version of MIROC3.2 (with a 6.3 K equilibrium climate sensitivity) is also demonstrated. Given the highly adjustable nature of the model, we believe that the LCM should be a very useful tool for studying uncertainty in global climate change, and we have named the model, JUMP-LCM, after the name of our research group (Japan Uncertainty Modelling Project).

Characterizing the heterogeneity of triple-negative breast cancers using microdissected normal ductal epithelium and RNA-sequencing

PubMed Central

Radovich, Milan; Clare, Susan E.; Atale, Rutuja; Pardo, Ivanesa; Hancock, Bradley A.; Solzak, Jeffrey P.; Kassem, Nawal; Mathieson, Theresa; Storniolo, Anna Maria V.; Rufenbarger, Connie; Lillemoe, Heather A.; Blosser, Rachel J.; Choi, Mi Ran; Sauder, Candice A.; Doxey, Diane; Henry, Jill E.; Hilligoss, Eric E.; Sakarya, Onur; Hyland, Fiona C.; Hickenbotham, Matthew; Zhu, Jin; Glasscock, Jarret; Badve, Sunil; Ivan, Mircea; Liu, Yunlong; Sledge, George W.; Schneider, Bryan P.

2014-01-01

Triple-negative breast cancers (TNBCs) are a heterogeneous set of tumors defined by an absence of actionable therapeutic targets (ER−,PR−,HER2−). Microdissected normal ductal epithelium from healthy volunteers represents a novel comparator to reveal insights into TNBC heterogeneity and to inform drug development. Using RNA-sequencing data from our institution and The Cancer Genome Atlas (TCGA) we compared the transcriptomes of 94 TNBCs, 20 microdissected normal breast tissues from healthy volunteers from the Susan G. Komen for the Cure Tissue Bank, and 10 histologically normal tissues adjacent to tumor. Pathway analysis comparing TNBCs to optimized normal controls of microdissected normal epithelium versus classic controls composed of adjacent normal tissue revealed distinct molecular signatures. Differential gene expression of TNBC compared with normal comparators demonstrated important findings for TNBC-specific clinical trials testing targeted agents; lack of over-expression for negative studies and over-expression in studies with drug activity. Next, by comparing each individual TNBC to the set of microdissected normals, we demonstrate that TNBC heterogeneity is attributable to transcriptional chaos, is associated with non-silent DNA mutational load, and explains transcriptional heterogeneity in addition to known molecular subtypes. Finally, chaos analysis identified 146 core genes dysregulated in >90% of TNBCs revealing an over-expressed central network. In conclusion, Use of microdissected normal ductal epithelium from healthy volunteers enables an optimized approach for studying TNBC and uncovers biological heterogeneity mediated by transcriptional chaos. PMID:24292813

Characterizing the heterogeneity of triple-negative breast cancers using microdissected normal ductal epithelium and RNA-sequencing.

PubMed

Radovich, Milan; Clare, Susan E; Atale, Rutuja; Pardo, Ivanesa; Hancock, Bradley A; Solzak, Jeffrey P; Kassem, Nawal; Mathieson, Theresa; Storniolo, Anna Maria V; Rufenbarger, Connie; Lillemoe, Heather A; Blosser, Rachel J; Choi, Mi Ran; Sauder, Candice A; Doxey, Diane; Henry, Jill E; Hilligoss, Eric E; Sakarya, Onur; Hyland, Fiona C; Hickenbotham, Matthew; Zhu, Jin; Glasscock, Jarret; Badve, Sunil; Ivan, Mircea; Liu, Yunlong; Sledge, George W; Schneider, Bryan P

2014-01-01

Triple-negative breast cancers (TNBCs) are a heterogeneous set of tumors defined by an absence of actionable therapeutic targets (ER, PR, and HER-2). Microdissected normal ductal epithelium from healthy volunteers represents a novel comparator to reveal insights into TNBC heterogeneity and to inform drug development. Using RNA-sequencing data from our institution and The Cancer Genome Atlas (TCGA) we compared the transcriptomes of 94 TNBCs, 20 microdissected normal breast tissues from healthy volunteers from the Susan G. Komen for the Cure Tissue Bank, and 10 histologically normal tissues adjacent to tumor. Pathway analysis comparing TNBCs to optimized normal controls of microdissected normal epithelium versus classic controls composed of adjacent normal tissue revealed distinct molecular signatures. Differential gene expression of TNBC compared with normal comparators demonstrated important findings for TNBC-specific clinical trials testing targeted agents; lack of over-expression for negative studies and over-expression in studies with drug activity. Next, by comparing each individual TNBC to the set of microdissected normals, we demonstrate that TNBC heterogeneity is attributable to transcriptional chaos, is associated with non-silent DNA mutational load, and explains transcriptional heterogeneity in addition to known molecular subtypes. Finally, chaos analysis identified 146 core genes dysregulated in >90 % of TNBCs revealing an over-expressed central network. In conclusion, use of microdissected normal ductal epithelium from healthy volunteers enables an optimized approach for studying TNBC and uncovers biological heterogeneity mediated by transcriptional chaos.

Expression microdissection adapted to commercial laser dissection instruments

PubMed Central

Hanson, Jeffrey C; Tangrea, Michael A; Kim, Skye; Armani, Michael D; Pohida, Thomas J; Bonner, Robert F; Rodriguez-Canales, Jaime; Emmert-Buck, Michael R

2016-01-01

Laser-based microdissection facilitates the isolation of specific cell populations from clinical or animal model tissue specimens for molecular analysis. Expression microdissection (xMD) is a second-generation technology that offers considerable advantages in dissection capabilities; however, until recently the method has not been accessible to investigators. This protocol describes the adaptation of xMD to commonly used laser microdissection instruments and to a commercially available handheld laser device in order to make the technique widely available to the biomedical research community. The method improves dissection speed for many applications by using a targeting probe for cell procurement in place of an operator-based, cell-by-cell selection process. Moreover, xMD can provide improved dissection precision because of the unique characteristics of film activation. The time to complete the protocol is highly dependent on the target cell population and the number of cells needed for subsequent molecular analysis. PMID:21412274

Investigation of the shear thinning behavior of epoxy resins for utilization in vibration assisted liquid composite molding processes

NASA Astrophysics Data System (ADS)

Meier, R.; Kirdar, C.; Rudolph, N.; Zaremba, S.; Drechsler, K.

2014-05-01

Efficient production and consumption of energy are of greatest importance for contemporary industries and their products. This has led to an increasing application of lightweight materials in general and of Carbon Fiber Reinforced Plastics (CFRP) in particular. However, broader application of CFRP is often limited by high costs and manual labor production processes. These constraints are addressed by Liquid Composite Molding (LCM) processes. In LCM a dry fibrous preform is placed into a cavity and infiltrated mostly by thermoset resins; epoxy resins are wide spread in CFRP applications. One crucial parameter for a fast mold filling is the viscosity of the resin, which is affected by the applied shear rates as well as temperature and curing time. The work presented focuses on the characterization of the shear thinning behavior of epoxy resins. Furthermore, the correlation with the conditions in vibration assisted LCM processes, where additional shear rates are created during manufacture, is discussed. Higher shear rates result from high frequencies and/or high amplitudes of the vibration motions which are created by a vibration engine mounted on the mold. In rheological investigations the shear thinning behavior of a representative epoxy resin is studied by means of rotational and oscillatory experiments. Moreover, possible effects of shear rates on the chemical curing reaction are studied. Here, the time for gelation is measured for different levels of shear rates in a pre-shearing phase. Based on the rheological studies, the beneficial effect of vibration assistance in LCM processes with respect to mold filling can further be predicted and utilized.

The Implementation of APIQ Creative Mathematics Game Method in the Subject Matter of Greatest Common Factor and Least Common Multiple in Elementary School

NASA Astrophysics Data System (ADS)

Rahman, Abdul; Saleh Ahmar, Ansari; Arifin, A. Nurani M.; Upu, Hamzah; Mulbar, Usman; Alimuddin; Arsyad, Nurdin; Ruslan; Rusli; Djadir; Sutamrin; Hamda; Minggi, Ilham; Awi; Zaki, Ahmad; Ahmad, Asdar; Ihsan, Hisyam

2018-01-01

One of causal factors for uninterested feeling of the students in learning mathematics is a monotonous learning method, like in traditional learning method. One of the ways for motivating students to learn mathematics is by implementing APIQ (Aritmetika Plus Intelegensi Quantum) creative mathematics game method. The purposes of this research are (1) to describe students’ responses toward the implementation of APIQ creative mathematics game method on the subject matter of Greatest Common Factor (GCF) and Least Common Multiple (LCM) and (2) to find out whether by implementing this method, the student’s learning completeness will improve or not. Based on the results of this research, it is shown that the responses of the students toward the implementation of APIQ creative mathematics game method in the subject matters of GCF and LCM were good. It is seen in the percentage of the responses were between 76-100%. (2) The implementation of APIQ creative mathematics game method on the subject matters of GCF and LCM improved the students’ learning.

A new procedure to produce lignocellulosic anion exchangers from agricultural waste materials.

PubMed

Orlando, U S; Baes, A U; Nishijima, W; Okada, M

2002-07-01

Two lignocellulosic agricultural waste materials (LCM), sugarcane bagasse (BG) and rice hull (RH), were converted into weak-base anion exchanger and evaluated for their exchanger capacity for nitrate. Pure cellulose (PC) and pure alkaline lignin (PL) were also used as reference materials to elucidate possible reactivity in LCM. Epoxy and amino groups were introduced into BG, RH, PC and PL substrates after the reaction with epichlorohydrin and dimethylamine in the presence of pyridine and an organic solvent N,N-dimethylformamide (DMF). Amino group incorporation into cellulose decreased with the presence of water in the reaction mixture and increased with the reaction time and presence of a catalyst (pyridine). The highest maximum nitrate exchange capacity (Qmax) and yields of the prepared exchangers was obtained from PL (1.8 mmol g(-1) and 412.5%), followed by BG (1.41 mmol g(-1) and 300%), PC (1.34 mmol g(-1) and 166%) and RH (1.32 mmol g(-1) and 180%). The proposed synthetic procedure was effective in modifying PL, PC and LCM chemically resulting in a higher yield and nitrate removal capacity.

Cloud Microphysics Parameterization in a Shallow Cumulus Cloud Simulated by a Largrangian Cloud Model

NASA Astrophysics Data System (ADS)

Oh, D.; Noh, Y.; Hoffmann, F.; Raasch, S.

2017-12-01

Lagrangian cloud model (LCM) is a fundamentally new approach of cloud simulation, in which the flow field is simulated by large eddy simulation and droplets are treated as Lagrangian particles undergoing cloud microphysics. LCM enables us to investigate raindrop formation and examine the parameterization of cloud microphysics directly by tracking the history of individual Lagrangian droplets simulated by LCM. Analysis of the magnitude of raindrop formation and the background physical conditions at the moment at which every Lagrangian droplet grows from cloud droplets to raindrops in a shallow cumulus cloud reveals how and under which condition raindrops are formed. It also provides information how autoconversion and accretion appear and evolve within a cloud, and how they are affected by various factors such as cloud water mixing ratio, rain water mixing ratio, aerosol concentration, drop size distribution, and dissipation rate. Based on these results, the parameterizations of autoconversion and accretion, such as Kessler (1969), Tripoli and Cotton (1980), Beheng (1994), and Kharioutdonov and Kogan (2000), are examined, and the modifications to improve the parameterizations are proposed.

Utilization of Ionic Liquids in Lignocellulose Biorefineries as Agents for Separation, Derivatization, Fractionation, or Pretreatment.

PubMed

Peleteiro, Susana; Rivas, Sandra; Alonso, José L; Santos, Valentín; Parajó, Juan C

2015-09-23

Ionic liquids (ILs) can play multiple roles in lignocellulose biorefineries, including utilization as agents for the separation of selected compounds or as reaction media for processing lignocellulosic materials (LCM). Imidazolium-based ILs have been proposed for separating target components from LCM biorefinery streams, for example, the dehydration of ethanol-water mixtures or the extractive separation of biofuels (ethanol, butanol) or lactic acid from the respective fermentation broths. As in other industries, ILs are potentially suitable for removing volatile organic compounds or carbon dioxide from gaseous biorefinery effluents. On the other hand, cellulose dissolution in ILs allows homogeneous derivatization reactions to be carried out, opening new ways for product design or for improving the quality of the products. Imidazolium-based ILs are also suitable for processing native LCM, allowing the integral benefit of the feedstocks via separation of polysaccharides and lignin. Even strongly lignified materials can yield cellulose-enriched substrates highly susceptible to enzymatic hydrolysis upon ILs processing. Recent developments in enzymatic hydrolysis include the identification of ILs causing limited enzyme inhibition and the utilization of enzymes with improved performance in the presence of ILs.

Beyond laser microdissection technology: follow the yellow brick road for cancer research

PubMed Central

Legres, Luc G; Janin, Anne; Masselon, Christophe; Bertheau, Philippe

2014-01-01

Normal biological tissues harbour different populations of cells with intricate spacial distribution patterns resulting in heterogeneity of their overall cellular composition. Laser microdissection involving direct viewing and expertise by a pathologist, enables access to defined cell populations or specific region on any type of tissue sample, thus selecting near-pure populations of targeted cells. It opens the way for molecular methods directed towards well-defined populations, and provides also a powerful tool in studies focused on a limited number of cells. Laser microdissection has wide applications in oncology (diagnosis and research), cellular and molecular biology, biochemistry and forensics for tissue selection, but other areas have been gradually opened up to these new methodological approaches, such as cell cultures and cytogenetics. In clinical oncology trials, molecular profiling of microdissected samples can yield global “omics” information which, together, with the morphological analysis of cells, can provide the basis for diagnosis, prognosis and patient-tailored treatments. This remarkable technology has brought new insights in the understanding of DNA, RNA, and the biological functions and regulation of proteins to identify molecular disease signatures. We review herein the different applications of laser microdissection in a variety of fields, and we particularly focus attention on the pre-analytical steps that are crucial to successfully perform molecular-level investigations. PMID:24482735

The occasional role of low-risk human papillomaviruses 6, 11, 42, 44, and 70 in anogenital carcinoma defined by laser capture microdissection/PCR methodology: results from a global study.

PubMed

Guimerà, Núria; Lloveras, Belén; Lindeman, Jan; Alemany, Laia; van de Sandt, Miekel; Alejo, Maria; Hernandez-Suarez, Gustavo; Bravo, Ignacio G; Molijn, Anco; Jenkins, David; Cubilla, Antonio; Muñoz, Nubia; de Sanjose, Silvia; Bosch, Francesc Xavier; Quint, Wim

2013-09-01

Low-risk human papillomaviruses (LR-HPVs) have been associated occasionally with clinically and pathologically unusual anogenital malignancies. The relation between clinicopathologic features and any pathogenetic role of LR-HPV remains unclear. From a global study of 13,328 anogenital carcinomas, we identified 57 cases in which whole-tissue polymerase chain reaction using SPF10-LiPA25 showed single LR-HPV infection. In 43/46 (93.5%) available carcinomas, multiple polymerase chain reaction assays confirmed single detection of HPV6, 11, 42, 44, or 70 DNA. In 75% (n=32) of these, LR-HPV DNA was confirmed in tumor cells by laser capture microdissection. In 2 cases, including 1 adenocarcinoma, viral DNA was only found outside the tumor. All anogenital tumors with confirmed HPV6/11 showed a distinctive range of papillary, warty or warty-basaloid, squamous, or transitional histology with patchy or negative p16 expression. HPV6-associated cervical tumors occurred at a low median age. HPV42/70 was associated with typical squamous cell carcinoma showing diffuse p16 staining like high-risk HPV-related malignancies. HPV44 was found in malignant cells in 1 case. Viral taxonomy and theoretical analysis show that HPV6/11 belong to a different genus from HPV42/70 with E6/E7 gene products that would not bind pRb or p53, whereas HPV42/70 could bind pRb. Our data support the causal involvement of LR-HPVs in the carcinogenesis of <2% of anogenital malignancies of 2 distinct clinicopathologic patterns related to the genetic structure of the HPV types 6/11 and 70/42. HPV42/70 was associated with typical squamous carcinomas. Importantly all carcinomas associated with HPV6/11 globally showed verruco-papillary, well-differentiated, squamous, or transitional histology without p16 expression.

Laser capture microdissection as a tool to evaluate human papillomavirus genotyping and methylation as biomarkers of persistence and progression of anal lesions

PubMed Central

Cornall, Alyssa M; Roberts, Jennifer M; Molano, Monica; Machalek, Dorothy A; Phillips, Samuel; Hillman, Richard J; Grulich, Andrew E; Jin, Fengyi; Poynten, I Mary; Templeton, David J; Garland, Suzanne M; Tabrizi, Sepehr N

2015-01-01

Introduction Anal squamous cell carcinoma is preceded by persistent infection with high-risk human papillomavirus (HPV) and the cancer precursor, high-grade squamous intraepithelial lesion (HSIL). Detection of specific HPV genotypes and HPV-related biomarkers may be an option for primary anal screening. However, more data on the natural history of HPV-related anal lesions are required. The outcomes from this study will enhance our understanding of the clinical and biological behaviour of HPV-related anal lesions and inform the development of future HPV genotype and/or biomarker screening tests. Methods and analysis HIV-negative and HIV-positive men who have sex with men, aged 35 years and over, recruited from community-based settings in Sydney, Australia, attend 6 clinic visits over 3 years. At the first 5 visits, participants undergo a digital anorectal examination, an anal swab for HPV genotyping and anal cytology, and high-resolution anoscopy with directed biopsy of any visible abnormalities that are suggestive of any abnormality suspicious of SIL. Tissue sections from participants diagnosed with histologically confirmed HSIL at the baseline clinic visit will undergo laser capture microdissection, HPV detection and genotyping, and quantitation of CpG methylation in baseline and follow-up biopsies. Histological and cytological findings in combination with HPV genotyping data will be used to identify persistent HSIL. HSIL will be stratified as non-persistent and persistent based on their status at 12 months. The performance of HPV genotype and methylation status in predicting disease persistence at 12 months will be assessed, along with associations with HIV status and other covariates such as age. Ethics and dissemination The St Vincent's Hospital Ethics Committee granted ethics approval for the study. Written informed consent is obtained from all individuals before any study-specific procedures are performed. Findings from this study will be disseminated to participants and the community through study newsletters, and through peer-reviewed publications and international conferences. PMID:26310402

Comparative quantitative proteomic analysis of disease stratified laser captured microdissected human islets identifies proteins and pathways potentially related to type 1 diabetes.

PubMed

Nyalwidhe, Julius O; Grzesik, Wojciech J; Burch, Tanya C; Semeraro, Michele L; Waseem, Tayab; Gerling, Ivan C; Mirmira, Raghavendra G; Morris, Margaret A; Nadler, Jerry L

2017-01-01

Type 1 diabetes (T1D) is a chronic inflammatory disease that is characterized by autoimmune destruction of insulin-producing pancreatic beta cells. The goal of this study was to identify novel protein signatures that distinguish Islets from patients with T1D, patients who are autoantibody positive without symptoms of diabetes, and from individuals with no evidence of disease. High resolution high mass accuracy label free quantitative mass spectrometry analysis was applied to islets isolated by laser capture microdissection from disease stratified human pancreata from the Network for Pancreatic Organ Donors with Diabetes (nPOD), these included donors without diabetes, donors with T1D-associated autoantibodies in the absence of diabetes, and donors with T1D. Thirty-nine proteins were found to be differentially regulated in autoantibody positive cases compared to the no-disease group, with 25 upregulated and 14 downregulated proteins. For the T1D cases, 63 proteins were differentially expressed, with 24 upregulated and 39 downregulated, compared to the no disease controls. We have identified functional annotated enriched gene families and multiple protein-protein interaction clusters of proteins are involved in biological and molecular processes that may have a role in T1D. The proteins that are upregulated in T1D cases include S100A9, S100A8, REG1B, REG3A and C9 amongst others. These proteins have important biological functions, such as inflammation, metabolic regulation, and autoimmunity, all of which are pathways linked to the pathogenesis of T1D. The identified proteins may be involved in T1D development and pathogenesis. Our findings of novel proteins uniquely upregulated in T1D pancreas provides impetus for further investigations focusing on their expression profiles in beta cells/ islets to evaluate their role in the disease pathogenesis. Some of these molecules may be novel therapeutic targets T1D.

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

Park, Kwangwook; Kang, Seokjin; Ravindran, Sooraj

Here, we report changes at the interface between Ga-rich/In-rich GaInP vertical slabs in laterally composition modulated (LCM) GaInP as a function of the V/III ratio. The photoluminescence exhibits satellite peaks, indicating that the parasitic potential between the GaInP vertical slabs disappears as the V/III ratio decreases. However, a high V/III ratio leads to an abrupt interface, increasing the parasitic potential because of the phosphorus-amount-dependent diffusion of group-III atoms during growth. These results suggest that the V/III ratio is an important parameter that must be wisely chosen in designing optoelectronic devices incorporating LCM structure.

Modeling and control of flow during impregnation of heterogeneous porous media, with application to composite mold-filling processes

NASA Astrophysics Data System (ADS)

Bickerton, Simon

Liquid Composite Molding (LCM) encompasses a growing list of composite material manufacturing techniques. These processes have provided the promise for complex fiber reinforced plastics parts, manufactured from a single molding step. In recent years a significant research effort has been invested in development of process simulations, providing tools that have advanced current LCM technology and broadened the range of applications. The requirement for manufacture of larger, more complex parts has motivated investigation of active control of LCM processes. Due to the unlimited variety of part geometries that can be produced, finite element based process simulations will be used to some extent in design of actively controlled processes. Ongoing efforts are being made to improve material parameter specification for process simulations, increasing their value as design tools. Several phenomena occurring during mold filling have been addressed through flow visualization experimentation and analysis of manufactured composite parts. The influence of well defined air channels within a mold cavity is investigated, incorporating their effects within existing filling simulations. Three different flow configurations have been addressed, testing the application of 'equivalent permeabilities', effectively approximating air channels as representative porous media. LCM parts having doubly curved regions require preform fabrics to undergo significant, and varying deformation throughout a mold cavity. Existing methods for predicting preform deformation, and the resulting permeability distribution have been applied to a conical mold geometry. Comparisons between experiment and simulation are promising, while the geometry studied has required large deformation over much of the part, shearing the preform fabric beyond the scope of the models applied. An investigational study was performed to determine the magnitude of effect, if any, on mold filling caused by corners within LCM mold cavities. The molds applied in this study have required careful consideration of cavity thickness variations. Any effects on mold filling due to corner radii have been overshadowed by those due to preform compression. While numerical tools are available to study actively controlled mold filling in a virtual environment, some development is required for the physical equipment to implement this in practice. A versatile, multiple line fluid injection system is developed here. The equipment and control algorithms employed have provided servo control of flow rate, or injection pressure, and have been tested under very challenging conditions. The single injection line developed is expanded to a multiple line system, and shows great potential for application to actual resin systems. A case study is presented, demonstrating design and implementation of a simple actively controlled injection scheme. The experimental facility developed provides an excellent testbed for application of actively controlled mold filling concepts, an area that is providing great promise for the advancement of LCM processes.

The preferred magnetic resonance imaging planes in quantifying visceral adipose tissue and evaluating cardiovascular risk.

PubMed

Liu, K H; Chan, Y L; Chan, J C N; Chan, W B; Kong, M O; Poon, M Y

2005-09-01

Magnetic Resonance Imaging (MRI) is a well-accepted non-invasive method in the quantification of visceral adipose tissue. However, a standard method of measurement has not yet been universally agreed. The objectives of the present study were 2-fold, firstly, to identify the imaging plane in the Chinese population which gives the best correlation with total visceral adipose tissue volume and cardiovascular risk factors; and secondly to compare the correlations between single-slice and multiple-slice approach with cardiovascular risk factors. Thirty-seven Chinese subjects with no known medical history underwent MRI examination for quantifying total visceral adipose tissue volume. The visceral adipose tissue area at five axial imaging levels within abdomen and pelvis were determined. All subjects had blood pressure measured and fasting blood taken for analysis of cardiovascular risk factors. Framingham risk score for each subject was calculated. The imaging plane at the level of 'lower costal margin' (LCM) in both men and women had the highest correlation with total visceral adipose tissue volume (r = 0.97 and 0.99 respectively). The visceral adipose tissue area at specific imaging levels showed higher correlations with various cardiovascular risk factors and Framingham risk score than total visceral adipose tissue volume. The visceral adipose tissue area at 'umbilicus' (UMB) level in men (r = 0.88) and LCM level in women (r = 0.70) showed the best correlation with Framingham risk score. The imaging plane at the level of LCM is preferred for reflecting total visceral adipose tissue volume in Chinese subjects. For investigating the association of cardiovascular risk with visceral adipose tissue in MRI-obesity research, the single-slice approach is superior to the multiple-slice approach, with the level of UMB in men and LCM in women as the preferred imaging planes.

Adsorption Device Based on a Langatate Crystal Microbalance for High Temperature High Pressure Gas Adsorption in Zeolite H-ZSM-5.

PubMed

Ding, Wenjin; Baracchini, Giulia; Klumpp, Michael; Schwieger, Wilhelm; Dittmeyer, Roland

2016-08-25

We present a high-temperature and high-pressure gas adsorption measurement device based on a high-frequency oscillating microbalance (5 MHz langatate crystal microbalance, LCM) and its use for gas adsorption measurements in zeolite H-ZSM-5. Prior to the adsorption measurements, zeolite H-ZSM-5 crystals were synthesized on the gold electrode in the center of the LCM, without covering the connection points of the gold electrodes to the oscillator, by the steam-assisted crystallization (SAC) method, so that the zeolite crystals remain attached to the oscillating microbalance while keeping good electroconductivity of the LCM during the adsorption measurements. Compared to a conventional quartz crystal microbalance (QCM) which is limited to temperatures below 80 °C, the LCM can realize the adsorption measurements in principle at temperatures as high as 200-300 °C (i.e., at or close to the reaction temperature of the target application of one-stage DME synthesis from the synthesis gas), owing to the absence of crystalline-phase transitions up to its melting point (1,470 °C). The system was applied to investigate the adsorption of CO2, H2O, methanol and dimethyl ether (DME), each in the gas phase, on zeolite H-ZSM-5 in the temperature and pressure range of 50-150 °C and 0-18 bar, respectively. The results showed that the adsorption isotherms of these gases in H-ZSM-5 can be well fitted by Langmuir-type adsorption isotherms. Furthermore, the determined adsorption parameters, i.e., adsorption capacities, adsorption enthalpies, and adsorption entropies, compare well to literature data. In this work, the results for CO2 are shown as an example.

Adsorption Device Based on a Langatate Crystal Microbalance for High Temperature High Pressure Gas Adsorption in Zeolite H-ZSM-5

PubMed Central

Ding, Wenjin; Baracchini, Giulia; Klumpp, Michael; Schwieger, Wilhelm; Dittmeyer, Roland

2016-01-01

We present a high-temperature and high-pressure gas adsorption measurement device based on a high-frequency oscillating microbalance (5 MHz langatate crystal microbalance, LCM) and its use for gas adsorption measurements in zeolite H-ZSM-5. Prior to the adsorption measurements, zeolite H-ZSM-5 crystals were synthesized on the gold electrode in the center of the LCM, without covering the connection points of the gold electrodes to the oscillator, by the steam-assisted crystallization (SAC) method, so that the zeolite crystals remain attached to the oscillating microbalance while keeping good electroconductivity of the LCM during the adsorption measurements. Compared to a conventional quartz crystal microbalance (QCM) which is limited to temperatures below 80 °C, the LCM can realize the adsorption measurements in principle at temperatures as high as 200-300 °C (i.e., at or close to the reaction temperature of the target application of one-stage DME synthesis from the synthesis gas), owing to the absence of crystalline-phase transitions up to its melting point (1,470 °C). The system was applied to investigate the adsorption of CO2, H2O, methanol and dimethyl ether (DME), each in the gas phase, on zeolite H-ZSM-5 in the temperature and pressure range of 50-150 °C and 0-18 bar, respectively. The results showed that the adsorption isotherms of these gases in H-ZSM-5 can be well fitted by Langmuir-type adsorption isotherms. Furthermore, the determined adsorption parameters, i.e., adsorption capacities, adsorption enthalpies, and adsorption entropies, compare well to literature data. In this work, the results for CO2 are shown as an example. PMID:27585356

Improved resolution by mounting of tissue sections for laser microdissection.

PubMed

van Dijk, M C R F; Rombout, P D M; Dijkman, H B P M; Ruiter, D J; Bernsen, M R

2003-08-01

Laser microbeam microdissection has greatly facilitated the procurement of specific cell populations from tissue sections. However, the fact that a coverslip is not used means that the morphology of the tissue sections is often poor. To develop a mounting method that greatly improves the morphological quality of tissue sections for laser microbeam microdissection purposes so that the identification of target cells can be facilitated. Fresh frozen tissue and formalin fixed, paraffin wax embedded tissue specimens were used to test the morphological quality of mounted and unmounted tissue. The mounting solution consisted of an adhesive gum and blue ink diluted in water. Interference of the mounting solution with DNA quality was analysed by the polymerase chain reaction using 10-2000 cells isolated by microdissection from mounted and unmounted tissue. The mounting solution greatly improved the morphology of tissue sections for laser microdissection purposes and had no detrimental effects on the isolation and efficiency of amplification of DNA. One disadvantage was that the mounting solution reduced the cutting efficiency of the ultraviolet laser. To minimise this effect, the mounting solution should be diluted as much as possible. Furthermore, the addition of blue ink to the mounting medium restores the cutting efficiency of the laser. The mounting solution is easy to prepare and apply and can be combined with various staining methods without compromising the quality of the DNA extracted.

Improved resolution by mounting of tissue sections for laser microdissection

PubMed Central

van Dijk, M C R F; Rombout, P D M; Dijkman, H B P M; Ruiter, D J; Bernsen, M R

2003-01-01

Background: Laser microbeam microdissection has greatly facilitated the procurement of specific cell populations from tissue sections. However, the fact that a coverslip is not used means that the morphology of the tissue sections is often poor. Aims: To develop a mounting method that greatly improves the morphological quality of tissue sections for laser microbeam microdissection purposes so that the identification of target cells can be facilitated. Methods: Fresh frozen tissue and formalin fixed, paraffin wax embedded tissue specimens were used to test the morphological quality of mounted and unmounted tissue. The mounting solution consisted of an adhesive gum and blue ink diluted in water. Interference of the mounting solution with DNA quality was analysed by the polymerase chain reaction using 10–2000 cells isolated by microdissection from mounted and unmounted tissue. Results: The mounting solution greatly improved the morphology of tissue sections for laser microdissection purposes and had no detrimental effects on the isolation and efficiency of amplification of DNA. One disadvantage was that the mounting solution reduced the cutting efficiency of the ultraviolet laser. To minimise this effect, the mounting solution should be diluted as much as possible. Furthermore, the addition of blue ink to the mounting medium restores the cutting efficiency of the laser. Conclusions: The mounting solution is easy to prepare and apply and can be combined with various staining methods without compromising the quality of the DNA extracted. PMID:12890747

An Exploratory Analysis of Personality, Attitudes, and Study Skills on the Learning Curve within a Team-based Learning Environment

PubMed Central

Henry, Teague; Campbell, Ashley

2015-01-01

Objective. To examine factors that determine the interindividual variability of learning within a team-based learning environment. Methods. Students in a pharmacokinetics course were given 4 interim, low-stakes cumulative assessments throughout the semester and a cumulative final examination. Students’ Myers-Briggs personality type was assessed, as well as their study skills, motivations, and attitudes towards team-learning. A latent curve model (LCM) was applied and various covariates were assessed to improve the regression model. Results. A quadratic LCM was applied for the first 4 assessments to predict final examination performance. None of the covariates examined significantly impacted the regression model fit except metacognitive self-regulation, which explained some of the variability in the rate of learning. There were some correlations between personality type and attitudes towards team learning, with introverts having a lower opinion of team-learning than extroverts. Conclusion. The LCM could readily describe the learning curve. Extroverted and introverted personality types had the same learning performance even though preference for team-learning was lower in introverts. Other personality traits, study skills, or practice did not significantly contribute to the learning variability in this course. PMID:25861101

An exploratory analysis of personality, attitudes, and study skills on the learning curve within a team-based learning environment.

PubMed

Persky, Adam M; Henry, Teague; Campbell, Ashley

2015-03-25

To examine factors that determine the interindividual variability of learning within a team-based learning environment. Students in a pharmacokinetics course were given 4 interim, low-stakes cumulative assessments throughout the semester and a cumulative final examination. Students' Myers-Briggs personality type was assessed, as well as their study skills, motivations, and attitudes towards team-learning. A latent curve model (LCM) was applied and various covariates were assessed to improve the regression model. A quadratic LCM was applied for the first 4 assessments to predict final examination performance. None of the covariates examined significantly impacted the regression model fit except metacognitive self-regulation, which explained some of the variability in the rate of learning. There were some correlations between personality type and attitudes towards team learning, with introverts having a lower opinion of team-learning than extroverts. The LCM could readily describe the learning curve. Extroverted and introverted personality types had the same learning performance even though preference for team-learning was lower in introverts. Other personality traits, study skills, or practice did not significantly contribute to the learning variability in this course.

A single CD4 test with 250 cells/mm3 threshold predicts viral suppression in HIV-infected adults failing first-line therapy by clinical criteria.

PubMed

Gilks, Charles F; Walker, A Sarah; Munderi, Paula; Kityo, Cissy; Reid, Andrew; Katabira, Elly; Goodall, Ruth L; Grosskurth, Heiner; Mugyenyi, Peter; Hakim, James; Gibb, Diana M

2013-01-01

In low-income countries, viral load (VL) monitoring of antiretroviral therapy (ART) is rarely available in the public sector for HIV-infected adults or children. Using clinical failure alone to identify first-line ART failure and trigger regimen switch may result in unnecessary use of costly second-line therapy. Our objective was to identify CD4 threshold values to confirm clinically-determined ART failure when VL is unavailable. 3316 HIV-infected Ugandan/Zimbabwean adults were randomised to first-line ART with Clinically-Driven (CDM, CD4s measured but blinded) or routine Laboratory and Clinical Monitoring (LCM, 12-weekly CD4s) in the DART trial. CD4 at switch and ART failure criteria (new/recurrent WHO 4, single/multiple WHO 3 event; LCM: CD4<100 cells/mm(3)) were reviewed in 361 LCM, 314 CDM participants who switched over median 5 years follow-up. Retrospective VLs were available in 368 (55%) participants. Overall, 265/361 (73%) LCM participants failed with CD4<100 cells/mm(3); only 7 (2%) switched with CD4≥250 cells/mm(3), four switches triggered by WHO events. Without CD4 monitoring, 207/314 (66%) CDM participants failed with WHO 4 events, and 77(25%)/30(10%) with single/multiple WHO 3 events. Failure/switching with single WHO 3 events was more likely with CD4≥250 cells/mm(3) (28/77; 36%) (p = 0.0002). CD4 monitoring reduced switching with viral suppression: 23/187 (12%) LCM versus 49/181 (27%) CDM had VL<400 copies/ml at failure/switch (p<0.0001). Amongst CDM participants with CD4<250 cells/mm(3) only 11/133 (8%) had VL<400 copies/ml, compared with 38/48 (79%) with CD4≥250 cells/mm(3) (p<0.0001). Multiple, but not single, WHO 3 events predicted first-line ART failure. A CD4 threshold 'tiebreaker' of ≥250 cells/mm(3) for clinically-monitored patients failing first-line could identify ∼80% with VL<400 copies/ml, who are unlikely to benefit from second-line. Targeting CD4s to single WHO stage 3 'clinical failures' would particularly avoid premature, costly switch to second-line ART.

A Single CD4 Test with 250 Cells/Mm3 Threshold Predicts Viral Suppression in HIV-Infected Adults Failing First-Line Therapy by Clinical Criteria

PubMed Central

Munderi, Paula; Kityo, Cissy; Reid, Andrew; Katabira, Elly; Goodall, Ruth L.; Grosskurth, Heiner; Mugyenyi, Peter; Hakim, James; Gibb, Diana M.

2013-01-01

Background In low-income countries, viral load (VL) monitoring of antiretroviral therapy (ART) is rarely available in the public sector for HIV-infected adults or children. Using clinical failure alone to identify first-line ART failure and trigger regimen switch may result in unnecessary use of costly second-line therapy. Our objective was to identify CD4 threshold values to confirm clinically-determined ART failure when VL is unavailable. Methods 3316 HIV-infected Ugandan/Zimbabwean adults were randomised to first-line ART with Clinically-Driven (CDM, CD4s measured but blinded) or routine Laboratory and Clinical Monitoring (LCM, 12-weekly CD4s) in the DART trial. CD4 at switch and ART failure criteria (new/recurrent WHO 4, single/multiple WHO 3 event; LCM: CD4<100 cells/mm3) were reviewed in 361 LCM, 314 CDM participants who switched over median 5 years follow-up. Retrospective VLs were available in 368 (55%) participants. Results Overall, 265/361 (73%) LCM participants failed with CD4<100 cells/mm3; only 7 (2%) switched with CD4≥250 cells/mm3, four switches triggered by WHO events. Without CD4 monitoring, 207/314 (66%) CDM participants failed with WHO 4 events, and 77(25%)/30(10%) with single/multiple WHO 3 events. Failure/switching with single WHO 3 events was more likely with CD4≥250 cells/mm3 (28/77; 36%) (p = 0.0002). CD4 monitoring reduced switching with viral suppression: 23/187 (12%) LCM versus 49/181 (27%) CDM had VL<400 copies/ml at failure/switch (p<0.0001). Amongst CDM participants with CD4<250 cells/mm3 only 11/133 (8%) had VL<400copies/ml, compared with 38/48 (79%) with CD4≥250 cells/mm3 (p<0.0001). Conclusion Multiple, but not single, WHO 3 events predicted first-line ART failure. A CD4 threshold ‘tiebreaker’ of ≥250 cells/mm3 for clinically-monitored patients failing first-line could identify ∼80% with VL<400 copies/ml, who are unlikely to benefit from second-line. Targeting CD4s to single WHO stage 3 ‘clinical failures’ would particularly avoid premature, costly switch to second-line ART. PMID:23437399

Combined histochemical staining, RNA amplification, regional, and single cell cDNA analysis within the hippocampus.

PubMed

Ginsberg, Stephen D; Che, Shaoli

2004-08-01

The use of five histochemical stains (cresyl violet, thionin, hematoxylin & eosin, silver stain, and acridine orange) was evaluated in combination with an expression profiling paradigm that included regional and single cell analyses within the hippocampus of post-mortem human brains and adult mice. Adjacent serial sections of human and mouse hippocampus were labeled by histochemistry or neurofilament immunocytochemistry. These tissue sections were used as starting material for regional and single cell microdissection followed by a newly developed RNA amplification procedure (terminal continuation (TC) RNA amplification) and subsequent hybridization to custom-designed cDNA arrays. Results indicated equivalent levels of global hybridization signal intensity and relative expression levels for individual genes for hippocampi stained by cresyl violet, thionin, and hematoxylin & eosin, and neurofilament immunocytochemistry. Moreover, no significant differences existed between the Nissl stains and neurofilament immunocytochemistry for individual CA1 neurons obtained via laser capture microdissection. In contrast, a marked decrement was observed in adjacent hippocampal sections stained for silver stain and acridine orange, both at the level of the regional dissection and at the CA1 neuron population level. Observations made on the cDNA array platform were validated by real-time qPCR using primers directed against beta-actin and glyceraldehyde-3 phosphate dehydrogenase. Thus, this report demonstrated the utility of using specific Nissl stains, but not stains that bind RNA species directly, in both human and mouse brain tissues at the regional and cellular level for state-of-the-art molecular fingerprinting studies.

Characterization of a microdissection library from human chromosome region 3p14

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

Bardenheuer, W.; Szymanski, S.; Lux, A.

1994-01-15

Structural alterations in human chromosome region 3p14-p23 resulting in the inactivation of one or more tumor suppressor genes are thought to play a pathogenic role in small cell lung cancer, renal cell carcinoma, and other human neoplasms. To identify putative tumor suppressor genes, 428 recombinant clones from a microdissection library specific for human chromosome region 3p14 were isolated and characterized. Ninety-six of these (22.5%) were human single-copy DNA sequences, 57 of which were unique sequence clones. Forty-four of these were mapped to the microdissected region using a cell hybrid mapping panel. Within this mapping panel, four probes detected two newmore » chromosome breakpoints that were previously indistinguishable from the translocation breakpoint t(3;8) in 3p14.2 in hereditary renal cell carcinoma. One probe maps to the homozygously deleted region of the small cell lung cancer cell line U2020. In addition, microdissection clones have been shown to be suitable for isolation of yeast artificial chromosomes. 52 refs., 3 figs., 2 tabs.« less

Sequence analysis of cultivated strawberry (Fragaria × ananassa Duch.) using microdissected single somatic chromosomes.

PubMed

Yanagi, Tomohiro; Shirasawa, Kenta; Terachi, Mayuko; Isobe, Sachiko

2017-01-01

Cultivated strawberry ( Fragaria  ×  ananassa Duch.) has homoeologous chromosomes because of allo-octoploidy. For example, two homoeologous chromosomes that belong to different sub-genome of allopolyploids have similar base sequences. Thus, when conducting de novo assembly of DNA sequences, it is difficult to determine whether these sequences are derived from the same chromosome. To avoid the difficulties associated with homoeologous chromosomes and demonstrate the possibility of sequencing allopolyploids using single chromosomes, we conducted sequence analysis using microdissected single somatic chromosomes of cultivated strawberry. Three hundred and ten somatic chromosomes of the Japanese octoploid strawberry 'Reiko' were individually selected under a light microscope using a microdissection system. DNA from 288 of the dissected chromosomes was successfully amplified using a DNA amplification kit. Using next-generation sequencing, we decoded the base sequences of the amplified DNA segments, and on the basis of mapping, we identified DNA sequences from 144 samples that were best matched to the reference genomes of the octoploid strawberry, F.  ×  ananassa , and the diploid strawberry, F. vesca . The 144 samples were classified into seven pseudo-molecules of F. vesca . The coverage rates of the DNA sequences from the single chromosome onto all pseudo-molecular sequences varied from 3 to 29.9%. We demonstrated an efficient method for sequence analysis of allopolyploid plants using microdissected single chromosomes. On the basis of our results, we believe that whole-genome analysis of allopolyploid plants can be enhanced using methodology that employs microdissected single chromosomes.

Microdissection and molecular manipulation of single chromosomes in woody fruit trees with small chromosomes using pomelo (Citrus grandis) as a model. I. Construction of single chromosomal DNA libraries.

PubMed

Huang, D; Wu, W; Zhou, Y; Hu, Z; Lu, L

2004-05-01

Construction of single chromosomal DNA libraries by means of chromosome microdissection and microcloning will be useful for genomic research, especially for those species that have not been extensively studied genetically. Application of the technology of microdissection and microcloning to woody fruit plants has not been reported hitherto, largely due to the generally small sizes of metaphase chromosomes and the difficulty of chromosome preparation. The present study was performed to establish a method for single chromosome microdissection and microcloning in woody fruit species using pomelo as a model. The standard karyotype of a pomelo cultivar ( Citrus grandis cv. Guanxi) was established based on 20 prometaphase photomicrographs. According to the standard karyotype, chromosome 1 was identified and isolated with fine glass microneedles controlled by a micromanipulator. DNA fragments ranging from 0.3 kb to 2 kb were acquired from the isolated single chromosome 1 via two rounds of PCR mediated by Sau3A linker adaptors and then cloned into T-easy vectors to generate a DNA library of chromosome 1. Approximately 30,000 recombinant clones were obtained. Evaluation based on 108 randomly selected clones showed that the sizes of the cloned inserts varied from 0.5 kb to 1.5 kb with an average of 860 bp. Our research suggests that microdissection and microcloning of single small chromosomes in woody plants is feasible.

Unveiling interfaces between In-rich and Ga-rich GaInP vertical slabs of laterally composition modulated structures

DOE PAGES

Park, Kwangwook; Kang, Seokjin; Ravindran, Sooraj; ...

2017-01-16

Here, we report changes at the interface between Ga-rich/In-rich GaInP vertical slabs in laterally composition modulated (LCM) GaInP as a function of the V/III ratio. The photoluminescence exhibits satellite peaks, indicating that the parasitic potential between the GaInP vertical slabs disappears as the V/III ratio decreases. However, a high V/III ratio leads to an abrupt interface, increasing the parasitic potential because of the phosphorus-amount-dependent diffusion of group-III atoms during growth. These results suggest that the V/III ratio is an important parameter that must be wisely chosen in designing optoelectronic devices incorporating LCM structure.

Land use change and conversion effects on ground water quality trends: An integration of land change modeler in GIS and a new Ground Water Quality Index developed by fuzzy multi-criteria group decision-making models.

PubMed

Shooshtarian, Mohammad Reza; Dehghani, Mansooreh; Margherita, Ferrante; Gea, Oliveri Conti; Mortezazadeh, Shima

2018-04-01

This study aggregated Land Change Modeller (LCM) as a useful model in GIS with an extended Groundwater Quality Index (GWQI) developed by fuzzy Multi-Criteria Group Decision-Making models to investigate the effect of land use change and conversion on groundwater quality being supplied for drinking. The model's performance was examined through an applied study in Shiraz, Iran, in a five year period (2011 to 2015). Four land use maps including urban, industrial, garden, and bare were employed in LCM model and the impact of change in area and their conversion to each other on GWQI changes was analysed. The correlation analysis indicated that increase in the urban land use area and conversion of bare to the residential/industrial land uses, had a relation with water quality decrease. Integration of LCM and GWQI can accurately and logically provide a numerical analysis of the possible impact of land use change and conversion, as one of the influencing factors, on the groundwater quality. Hence, the methodology could be used in urban development planning and management in macro level. Copyright © 2018. Published by Elsevier Ltd.

Gene recovery microdissection (GRM) a process for producing chromosome region-specific libraries of expressed genes

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

Christian, A T; Coleman, M A; Tucker, J D

2001-02-08

Gene Recovery Microdissection (GRM) is a unique and cost-effective process for producing chromosome region-specific libraries of expressed genes. It accelerates the pace, reduces the cost, and extends the capabilities of functional genomic research, the means by which scientists will put to life-saving, life-enhancing use their knowledge of any plant or animal genome.

The Development of Chromosome Microdissection and Microcloning Technique and its Applications in Genomic Research

PubMed Central

Zhou, Ruo-Nan; Hu, Zan-Min

2007-01-01

The technique of chromosome microdissection and microcloning has been developed for more than 20 years. As a bridge between cytogenetics and molecular genetics, it leads to a number of applications: chromosome painting probe isolation, genetic linkage map and physical map construction, and expressed sequence tags generation. During those 20 years, this technique has not only been benefited from other technological advances but also cross-fertilized with other techniques. Today, it becomes a practicality with extensive uses. The purpose of this article is to review the development of this technique and its application in the field of genomic research. Moreover, a new method of generating ESTs of specific chromosomes developed by our lab is introduced. By using this method, the technique of chromosome microdissection and microcloning would be more valuable in the advancement of genomic research. PMID:18645627

IL2RG, identified as overexpressed by RNA-seq profiling of pancreatic intraepithelial neoplasia, mediates pancreatic cancer growth

PubMed Central

Ayars, Michael; O’Sullivan, Eileen; Macgregor-Das, Anne; Shindo, Koji; Kim, Haeryoung; Borges, Michael; Yu, Jun; Hruban, Ralph H.; Goggins, Michael

2017-01-01

Pancreatic ductal adenocarcinoma evolves from precursor lesions, the most common of which is pancreatic intraepithelial neoplasia (PanIN). We performed RNA-sequencing analysis of laser capture microdissected PanINs and normal pancreatic duct cells to identify differentially expressed genes between PanINs and normal pancreatic duct, and between low-grade and high-grade PanINs. One of the most highly overexpressed transcripts identified in PanIN is interleukin-2 receptor subunit gamma (IL2RG) encoding the common gamma chain, IL2Rγ. CRISPR-mediated knockout of IL2RG in orthotopically implanted pancreatic cancer cells resulted in attenuated tumor growth in mice and reduced JAK3 expression in orthotopic tumors. These results indicate that IL2Rγ/JAK3 signaling contributes to pancreatic cancer cell growth in vivo. PMID:29137350

Comparing Habitat Suitability and Connectivity Modeling Methods for Conserving Pronghorn Migrations

PubMed Central

Poor, Erin E.; Loucks, Colby; Jakes, Andrew; Urban, Dean L.

2012-01-01

Terrestrial long-distance migrations are declining globally: in North America, nearly 75% have been lost. Yet there has been limited research comparing habitat suitability and connectivity models to identify migration corridors across increasingly fragmented landscapes. Here we use pronghorn (Antilocapra americana) migrations in prairie habitat to compare two types of models that identify habitat suitability: maximum entropy (Maxent) and expert-based (Analytic Hierarchy Process). We used distance to wells, distance to water, NDVI, land cover, distance to roads, terrain shape and fence presence to parameterize the models. We then used the output of these models as cost surfaces to compare two common connectivity models, least-cost modeling (LCM) and circuit theory. Using pronghorn movement data from spring and fall migrations, we identified potential migration corridors by combining each habitat suitability model with each connectivity model. The best performing model combination was Maxent with LCM corridors across both seasons. Maxent out-performed expert-based habitat suitability models for both spring and fall migrations. However, expert-based corridors can perform relatively well and are a cost-effective alternative if species location data are unavailable. Corridors created using LCM out-performed circuit theory, as measured by the number of pronghorn GPS locations present within the corridors. We suggest the use of a tiered approach using different corridor widths for prioritizing conservation and mitigation actions, such as fence removal or conservation easements. PMID:23166656

Comparing habitat suitability and connectivity modeling methods for conserving pronghorn migrations.

PubMed

Poor, Erin E; Loucks, Colby; Jakes, Andrew; Urban, Dean L

2012-01-01

Terrestrial long-distance migrations are declining globally: in North America, nearly 75% have been lost. Yet there has been limited research comparing habitat suitability and connectivity models to identify migration corridors across increasingly fragmented landscapes. Here we use pronghorn (Antilocapra americana) migrations in prairie habitat to compare two types of models that identify habitat suitability: maximum entropy (Maxent) and expert-based (Analytic Hierarchy Process). We used distance to wells, distance to water, NDVI, land cover, distance to roads, terrain shape and fence presence to parameterize the models. We then used the output of these models as cost surfaces to compare two common connectivity models, least-cost modeling (LCM) and circuit theory. Using pronghorn movement data from spring and fall migrations, we identified potential migration corridors by combining each habitat suitability model with each connectivity model. The best performing model combination was Maxent with LCM corridors across both seasons. Maxent out-performed expert-based habitat suitability models for both spring and fall migrations. However, expert-based corridors can perform relatively well and are a cost-effective alternative if species location data are unavailable. Corridors created using LCM out-performed circuit theory, as measured by the number of pronghorn GPS locations present within the corridors. We suggest the use of a tiered approach using different corridor widths for prioritizing conservation and mitigation actions, such as fence removal or conservation easements.

Isolation of single Chlamydia-infected cells using laser microdissection.

PubMed

Podgorny, Oleg V; Polina, Nadezhda F; Babenko, Vladislav V; Karpova, Irina Y; Kostryukova, Elena S; Govorun, Vadim M; Lazarev, Vassili N

2015-02-01

Chlamydia are obligate intracellular parasites of humans and animals that cause a wide range of acute and chronic infections. To elucidate the genetic basis of chlamydial parasitism, several approaches for making genetic modifications to Chlamydia have recently been reported. However, the lack of the available methods for the fast and effective selection of genetically modified bacteria restricts the application of genetic tools. We suggest the use of laser microdissection to isolate of single live Chlamydia-infected cells for the re-cultivation and whole-genome sequencing of single inclusion-derived Chlamydia. To visualise individual infected cells, we made use of the vital labelling of inclusions with the fluorescent Golgi-specific dye BODIPY® FL C5-ceramide. We demonstrated that single Chlamydia-infected cells isolated by laser microdissection and placed onto a host cell monolayer resulted in new cycles of infection. We also demonstrated the successful use of whole-genome sequencing to study the genomic variability of Chlamydia derived from a single inclusion. Our work provides the first evidence of the successful use of laser microdissection for the isolation of single live Chlamydia-infected cells, thus demonstrating that this method can help overcome the barriers to the fast and effective selection of Chlamydia. Copyright © 2014 Elsevier B.V. All rights reserved.

A Methodological Approach for Conducting a Business Case Analysis for the Joint Distance Support and Response (JDSR) Advanced Concept Technology Demonstration (ACTD)

DTIC Science & Technology

2006-12-01

24 $8,018.84 63 $104,722.97 AOE 0006 USS SUPPLY 4 $6,319.85 21 $26,655.18 AOE 0007 USS RAINIER 0 $0.00 4 $2,547.89 AOE 0008 USS ARCTIC 8 $1,778.17...0.00 4 $9,987.61 LCC 0020 USS MOUNT WHITNEY 5 $709.19 90 $99,210.49 LCM 0008 USS LCM-8 0 $0.00 2 $590.00 38 LCU 1634 LCU-1634 0 $0.00 2 $2,281.91...636.88 38 $38,446.13 LSD 0036 USS ANCHORAGE 1 $320.22 1 $582.12 LSD 0037 USS PORTLAND 1 $0.00 46 $53,501.29 LSD 0039 USS MOUNT VERNON 1 $339.04 0

Protein analysis through Western blot of cells excised individually from human brain and muscle tissue

PubMed Central

Koob, A.O.; Bruns, L.; Prassler, C.; Masliah, E.; Klopstock, T.; Bender, A.

2016-01-01

Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. PMID:22402104

Protein analysis through Western blot of cells excised individually from human brain and muscle tissue.

PubMed

Koob, A O; Bruns, L; Prassler, C; Masliah, E; Klopstock, T; Bender, A

2012-06-15

Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. Copyright © 2012 Elsevier Inc. All rights reserved.

Percutaneous Dilational Tracheotomy in Solid-Organ Transplant Recipients.

PubMed

Ozdemirkan, Aycan; Ersoy, Zeynep; Zeyneloglu, Pinar; Gedik, Ender; Pirat, Arash; Haberal, Mehmet

2015-11-01

Solid-organ transplant recipients may require percutaneous dilational tracheotomy because of prolonged mechanical ventilation or airway issues, but data regarding its safety and effectiveness in solid-organ transplant recipients are scarce. Here, we evaluated the safety, effectiveness, and benefits in terms of lung mechanics, complications, and patient comfort of percutaneous dilational tracheotomy in solid-organ transplant recipients. Medical records from 31 solid-organ transplant recipients (median age of 41.0 years [interquartile range, 18.0-53.0 y]) who underwent percutaneous dilational tracheotomy at our hospital between January 2010 and March 2015 were analyzed, including primary diagnosis, comorbidities, duration of orotracheal intubation and mechanical ventilation, length of intensive care unit and hospital stays, the time interval between transplant to percutaneous dilational tracheotomy, Acute Physiology and Chronic Health Evaluation II score, tracheotomy-related complications, and pulmonary compliance and ratio of partial pressure of arterial oxygen to fraction of inspired oxygen. The median Acute Physiology and Chronic Health Evaluation II score on admission was 24.0 (interquartile range, 18.0-29.0). The median interval from transplant to percutaneous dilational tracheotomy was 105.5 days (interquartile range, 13.0-2165.0 d). The only major complication noted was left-sided pneumothorax in 1 patient. There were no significant differences in ratio of partial pressure of arterial oxygen to fraction of inspired oxygen before and after procedure (170.0 [interquartile range, 102.2-302.0] vs 210.0 [interquartile range, 178.5-345.5]; P = .052). However, pulmonary compliance results preprocedure and postprocedure were significantly different (0.020 L/cm H2O [interquartile range, 0.015-0.030 L/cm H2O] vs 0.030 L/cm H2O [interquartile range, 0.020-0.041 L/cm H2O); P = .001]). Need for sedation significantly decreased after tracheotomy (from 17 patients [54.8%] to 8 patients [25.8%]; P = .004]). Percutaneous dilational tracheotomy with bronchoscopic guidance is an efficacious and safe technique for maintaining airways in solidorgan transplant recipients who require prolonged mechanical ventilation, resulting in possible improvements in ventilatory mechanics and patient comfort.

A Novel Combination of Homeobox Genes Is Expressed in Mesenchymal Chorionic Stem/Stromal Cells in First Trimester and Term Pregnancies

PubMed Central

Liu, Haiying; Murthi, Padma; Qin, Sharon; Kusuma, Gina D.; Borg, Anthony J.; Knöfler, Martin; Haslinger, Peter; Manuelpillai, Ursula; Pertile, Mark D.; Abumaree, Mohamed

2014-01-01

Human chorionic mesenchymal stem/stromal cells (CMSCs) derived from the placenta are similar to adult tissue-derived MSCs. The aim of this study was to investigate the role of these cells in normal placental development. Transcription factors, particularly members of the homeobox gene family, play crucial roles in maintaining stem cell proliferation and lineage specification in embryonic tissues. In adult tissues and organs, stem cells proliferate at low levels in their niche until they receive cues from the microenvironment to differentiate. The homeobox genes that are expressed in the CMSC niche in placental tissues have not been identified. We used the novel strategy of laser capture microdissection to isolate the stromal component of first trimester villi and excluded the cytotrophoblast and syncytiotrophoblast layers that comprise the outer layer of the chorionic villi. Microarray analysis was then used to screen for homeobox genes in the microdissected tissue. Candidate homeobox genes were selected for further RNA analysis. Immunohistochemistry of candidate genes in first trimester placental villous stromal tissue revealed homeobox genes Meis1, myeloid ectropic viral integration site 1 homolog 2 (MEIS2), H2.0-like Drosophila (HLX), transforming growth factor β-induced factor (TGIF), and distal-less homeobox 5 (DLX5) were expressed in the vascular niche where CMSCs have been shown to reside. Expression of MEIS2, HLX, TGIF, and DLX5 was also detected in scattered stromal cells. Real-time polymerase chain reaction and immunocytochemistry verified expression of MEIS2, HLX, TGIF, and DLX5 homeobox genes in first trimester and term CMSCs. These data suggest a combination of regulatory homeobox genes is expressed in CMSCs from early placental development to term, which may be required for stem cell proliferation and differentiation. PMID:24692208

Differential Gene Expression in Benign Prostate Epithelium of Men with and without Prostate Cancer: Evidence for a Prostate Cancer Field Effect

PubMed Central

Risk, Michael C; Knudsen, Beatrice S; Coleman, Ilsa; Dumpit, Ruth F; Kristal, Alan R; LeMeur, Nolwenn; Gentleman, Robert C; True, Lawrence D; Nelson, Peter S; Lin, Daniel W

2010-01-01

Background Several malignancies are known to exhibit a “field-effect” whereby regions beyond tumor boundaries harbor histological or molecular changes that are associated with cancer. We sought to determine if histologically benign prostate epithelium collected from men with prostate cancer exhibits features indicative of pre-malignancy or field effect. Methods Prostate needle biopsies from 15 men with high grade(Gleason 8–10) prostate cancer and 15 age- and BMI-matched controls were identified from a biospecimen repository. Benign epithelia from each patient were isolated by laser capture microdissection. RNA was isolated, amplified, and used for microarray hybridization. Quantitative PCR(qPCR) was used to determine the expression of specific genes of interest. Alterations in protein expression were analyzed through immunohistochemistry. Results Overall patterns of gene expression in microdissected benign-associated benign epithelium (BABE) and cancer-associated benign epithelium (CABE) were similar. Two genes previously associated with prostate cancer, PSMA and SSTR1, were significantly upregulated in the CABE group(FDR <1%). Expression of other prostate cancer-associated genes, including ERG, HOXC4, HOXC5 and MME, were also increased in CABE by qRT-PCR, although other genes commonly altered in prostate cancer were not different between the BABE and CABE samples. The expression of MME and PSMA proteins on IHC coincided with their mRNA alterations. Conclusion Gene expression profiles between benign epithelia of patients with and without prostate cancer are very similar. However, these tissues exhibit differences in the expression levels of several genes previously associated with prostate cancer development or progression. These differences may comprise a field effect and represent early events in carcinogenesis. PMID:20935156

Laser microdissection and capture of pure cardiomyocytes and fibroblasts from infarcted heart regions: perceived hyperoxia induces p21 in peri-infarct myocytes.

PubMed

Kuhn, Donald E; Roy, Sashwati; Radtke, Jared; Khanna, Savita; Sen, Chandan K

2007-03-01

Myocardial infarction caused by ischemia-reperfusion in the coronary vasculature is a focal event characterized by an infarct-core, bordering peri-infarct zone and remote noninfarct zone. Recently, we have reported the first technique, based on laser microdissection pressure catapulting (LMPC), enabling the dissection of infarction-induced biological responses in multicellular regions of the heart. Molecular mechanisms in play at the peri-infarct zone are central to myocardial healing. At the infarct site, myocytes are more sensitive to insult than robust fibroblasts. Understanding of cell-specific responses in the said zones is therefore critical. In this work, we describe the first technique to collect the myocardial tissue with a single-cell resolution. The infarcted myocardium was identified by using a truncated hematoxylin-eosin stain. Cell elements from the infarct, peri-infarct, and noninfarct zones were collected in a chaotropic RNA lysis solution with micron-level surgical precision. Isolated RNA was analyzed for quality by employing microfluidics technology and reverse transcribed to generate cDNA. Purity of the collected specimen was established by real-time PCR analyses of cell-specific genes. Previously, we have reported that the oxygen-sensitive induction of p21/Cip1/Waf1/Sdi1 in cardiac fibroblasts in the peri-infarct zone plays a vital role in myocardial remodeling. Using the novel LMPC technique developed herein, we confirmed that finding and report for the first time that the induction of p21 in the peri-infarct zone is not limited to fibroblasts but is also evident in myocytes. This work presents the first account of an analytical technique that applies the LMPC technology to study myocardial remodeling with a cell-type specific resolution.

Different Sarcocystis spp. are present in bovine eosinophilic myositis.

PubMed

Vangeel, Lieve; Houf, Kurt; Geldhof, Peter; De Preter, Katleen; Vercruysse, Jozef; Ducatelle, Richard; Chiers, Koen

2013-11-08

It has been suggested that Sarcocystis species are associated with bovine eosinophilic myositis (BEM). To date, parasite identification in this myopathy has been based on morphological techniques. The aim of the present study was to use molecular techniques to identify Sarcocystis species inside lesions of BEM. Histologically, BEM lesions of 97 condemned carcasses were examined for the presence of Sarcocystis species. Intralesional and extralesional cysts were collected using laser capture microdissection and the species was determined with a PCR-based technique based on 18S rDNA. Intralesional sarcocysts or remnants were found in BEM lesions in 28% of the carcasses. The majority (82%) of intralesional Sarcocystis species were found to be S. hominis. However S. cruzi and S. hirsuta were also found, as well as an unidentified species. It can be concluded that Sarcocystis species present in lesions of BEM are not restricted to one species. Copyright © 2013 Elsevier B.V. All rights reserved.

Spatially Distinct Neutrophil Responses within the Inflammatory Lesions of Pneumonic Plague

PubMed Central

Stasulli, Nikolas M.; Eichelberger, Kara R.; Price, Paul A.; Pechous, Roger D.; Montgomery, Stephanie A.; Parker, Joel S.

2015-01-01

ABSTRACT During pneumonic plague, the bacterium Yersinia pestis elicits the development of inflammatory lung lesions that continue to expand throughout infection. This lesion development and persistence are poorly understood. Here, we examine spatially distinct regions of lung lesions using laser capture microdissection and transcriptome sequencing (RNA-seq) analysis to identify transcriptional differences between lesion microenvironments. We show that cellular pathways involved in leukocyte migration and apoptosis are downregulated in the center of lung lesions compared to the periphery. Probing for the bacterial factor(s) important for the alteration in neutrophil survival, we show both in vitro and in vivo that Y. pestis increases neutrophil survival in a manner that is dependent on the type III secretion system effector YopM. This research explores the complexity of spatially distinct host-microbe interactions and emphasizes the importance of cell relevance in assays in order to fully understand Y. pestis virulence. PMID:26463167

Precision toxicology based on single cell sequencing: an evolving trend in toxicological evaluations and mechanism exploration.

PubMed

Zhang, Boyang; Huang, Kunlun; Zhu, Liye; Luo, Yunbo; Xu, Wentao

2017-07-01

In this review, we introduce a new concept, precision toxicology: the mode of action of chemical- or drug-induced toxicity can be sensitively and specifically investigated by isolating a small group of cells or even a single cell with typical phenotype of interest followed by a single cell sequencing-based analysis. Precision toxicology can contribute to the better detection of subtle intracellular changes in response to exogenous substrates, and thus help researchers find solutions to control or relieve the toxicological effects that are serious threats to human health. We give examples for single cell isolation and recommend laser capture microdissection for in vivo studies and flow cytometric sorting for in vitro studies. In addition, we introduce the procedures for single cell sequencing and describe the expected application of these techniques to toxicological evaluations and mechanism exploration, which we believe will become a trend in toxicology.

Gene expression profiles in anatomically and functionally distinct regions of the normal aged human brain

PubMed Central

Liang, Winnie S.; Dunckley, Travis; Beach, Thomas G.; Grover, Andrew; Mastroeni, Diego; Walker, Douglas G.; Caselli, Richard J.; Kukull, Walter A.; McKeel, Daniel; Morris, John C.; Hulette, Christine; Schmechel, Donald; Alexander, Gene E.; Reiman, Eric M.; Rogers, Joseph; Stephan, Dietrich A.

2008-01-01

In this article, we have characterized and compared gene expression profiles from laser capture microdissected neurons in six functionally and anatomically distinct regions from clinically and histopathologically normal aged human brains. These regions, which are also known to be differentially vulnerable to the histopathological and metabolic features of Alzheimer’s disease (AD), include the entorhinal cortex and hippocampus (limbic and paralimbic areas vulnerable to early neurofibrillary tangle pathology in AD), posterior cingulate cortex (a paralimbic area vulnerable to early metabolic abnormalities in AD), temporal and prefrontal cortex (unimodal and heteromodal sensory association areas vulnerable to early neuritic plaque pathology in AD), and primary visual cortex (a primary sensory area relatively spared in early AD). These neuronal profiles will provide valuable reference information for future studies of the brain, in normal aging, AD and other neurological and psychiatric disorders. PMID:17077275

HANFORD RIVER PROTECTION PROJECT ENHANCED MISSION PLANNING THROUGH INNOVATIVE TOOLS LIFECYCLE COST MODELING AND AQUEOUS THERMODYNAMIC MODELING - 12134

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

PIERSON KL; MEINERT FL

2012-01-26

Two notable modeling efforts within the Hanford Tank Waste Operations Simulator (HTWOS) are currently underway to (1) increase the robustness of the underlying chemistry approximations through the development and implementation of an aqueous thermodynamic model, and (2) add enhanced planning capabilities to the HTWOS model through development and incorporation of the lifecycle cost model (LCM). Since even seemingly small changes in apparent waste composition or treatment parameters can result in large changes in quantities of high-level waste (HLW) and low-activity waste (LAW) glass, mission duration or lifecycle cost, a solubility model that more accurately depicts the phases and concentrations ofmore » constituents in tank waste is required. The LCM enables evaluation of the interactions of proposed changes on lifecycle mission costs, which is critical for decision makers.« less

Ultrasonography of longus colli muscle: A reliability study on healthy subjects and patients with chronic neck pain.

PubMed

Javanshir, Khodabakhsh; Mohseni-Bandpei, Mohammad Ali; Rezasoltani, Asghar; Amiri, Mohsen; Rahgozar, Mehdi

2011-01-01

In this study, the reliability of the longus colli muscle (LCM) size was assessed in a relaxed state by a real time ultrasonography (US) device in a group of healthy subjects and a group of patients with chronic neck pain. Fifteen healthy subjects (19-41 years old) and 10 patients with chronic neck pain (27-44 years old) were recruited for the purpose of this study. LCM size was measured at the level of thyroid cartilage. Two images were taken on the same day with an hour interval to assess the within day reliability and the third image was taken 1 week later to determine between days reliability. Cross sectional area (CSA), anterior posterior dimension (APD), and lateral dimension (LD) were measured each time. The shape ratio was calculated as LD/APD. Intraclass correlation coefficients (ICC) and standard error of measurement (SEM) were computed for data analysis. The ICC of left and right CSA for within day and between days reliability in healthy subjects were (0.90, 0.93) and (0.85, 0.82), respectively. The ICC of left and right CSA for within day and between days reliability in patients with neck pain were (0.86, 0.82) and (0.76, 0.81), respectively. The results indicated that US could be used as a reliable tool to measure the LCM dimensions in healthy subjects and patients with chronic neck pain. Copyright © 2009 Elsevier Ltd. All rights reserved.

Accuracy of p53 Codon 72 Polymorphism Status Determined by Multiple Laboratory Methods: A Latent Class Model Analysis

PubMed Central

Walter, Stephen D.; Riddell, Corinne A.; Rabachini, Tatiana; Villa, Luisa L.; Franco, Eduardo L.

2013-01-01

Introduction Studies on the association of a polymorphism in codon 72 of the p53 tumour suppressor gene (rs1042522) with cervical neoplasia have inconsistent results. While several methods for genotyping p53 exist, they vary in accuracy and are often discrepant. Methods We used latent class models (LCM) to examine the accuracy of six methods for p53 determination, all conducted by the same laboratory. We also examined the association of p53 with cytological cervical abnormalities, recognising potential test inaccuracy. Results Pairwise disagreement between laboratory methods occurred approximately 10% of the time. Given the estimated true p53 status of each woman, we found that each laboratory method is most likely to classify a woman to her correct status. Arg/Arg women had the highest risk of squamous intraepithelial lesions (SIL). Test accuracy was independent of cytology. There was no strong evidence for correlations of test errors. Discussion Empirical analyses ignore possible laboratory errors, and so are inherently biased, but test accuracy estimated by the LCM approach is unbiased when model assumptions are met. LCM analysis avoids ambiguities arising from empirical test discrepancies, obviating the need to regard any of the methods as a “gold” standard measurement. The methods we presented here to analyse the p53 data can be applied in many other situations where multiple tests exist, but where none of them is a gold standard. PMID:23441193

Synthesis and structure-activity relationships of novel lincomycin derivatives. Part 4: synthesis of novel lincomycin analogs modified at the 6- and 7-positions and their potent antibacterial activities.

PubMed

Wakiyama, Yoshinari; Kumura, Ko; Umemura, Eijiro; Ueda, Kazutaka; Watanabe, Takashi; Yamada, Keiko; Okutomi, Takafumi; Ajito, Keiichi

2017-07-01

To modify lincomycin (LCM) at the C-6 and the C-7 positions, we firstly prepared various substituted proline intermediates (7, 11-15 and 17). These proline intermediates were coupled with methyl 1-thio-α-lincosamide and tetrakis-O-trimethylsilylation followed by selective deprotection of the TMS group at the 7-position gave a wide variety of key intermediates (23-27, 47 and 50). Then, we synthesized a variety of novel LCM analogs modified at the 7-position in application of the Mitsunobu reaction, an S N 2 reaction, and a Pd-catalyzed cross-coupling reaction. Compounds 34 and 35 (1'-NH derivatives) exhibited enhanced antibacterial activities against resistant pathogens with erm gene compared with the corresponding 1'-N-methyl derivatives (3 and 37). On the basis of reported SAR, we modified the 4'-position of LCM derivatives possessing a 5-(2-nitrophenyl)-1,3,4-thiadiazol-2-yl group at the C-7 position. Compound 56 showed significantly potent antibacterial activities against S. pneumoniae and S. pyogenes with erm gene, and its activities against S. pneumoniae with erm gene were improved compared with those of 34 and 57. Although we synthesized novel analogs by transformation of a C-7 substituent focusing on the 1'-demethyl framework to prepare very potent analogs 73 and 75, it was impossible to generate novel derivatives exhibiting stronger antibacterial activities against S. pneumoniae with erm gene compared with 56.

Can the rapid identification of mature spermatozoa during microdissection testicular sperm extraction guide operative planning?

PubMed

Alrabeeah, K; Doucet, R; Boulet, E; Phillips, S; Al-Hathal, N; Bissonnette, F; Kadoch, I J; Zini, A

2015-05-01

The minimum sperm count and quality that must be identified during microdissection testicular sperm extraction (micro-TESE) to deem the procedure successful remains to be established. We conducted a retrospective study of 81 consecutive men with non-obstructive azoospermia who underwent a primary (first) micro-TESE between March 2007 and October 2013. Final assessment of sperm recovery [reported on the day of (intracytoplasmic sperm injection) ICSI] was recorded as (i) successful (available spermatozoa for ICSI) or (ii) unsuccessful (no spermatozoa for ICSI). The decision to perform a unilateral (with limited or complete microdissection) or bilateral micro-TESE was guided by the intra-operative identification of sperm recovery (≥5 motile or non-motile sperm) from the first testicle. Overall, sperm recovery was successful in 56% (45/81) of the men. A unilateral micro-TESE was performed in 47% (38/81) of the men (based on intra-operative identification of sperm) and in 100% (38/38) of these men, spermatozoa was found on final assessment. In 42% (16/38) of the unilateral cases, a limited microdissection was performed (owing to the rapid intra-operative identification of sperm). The remaining 43 men underwent a bilateral micro-TESE and 16% (7/43) of these men had sperm identified on final assessment. The cumulative ICSI pregnancy rates (per cycle started and per embryo transfer) were 47% (21/45) and 60% (21/35), respectively, with a mean (±SD) of 1.9 ± 1.0 embryos transferred. The data demonstrate that intra-operative assessment of sperm recovery can correctly identify those men that require a unilateral micro-TESE. Moreover, the rapid identification of sperm recovery can allow some men to undergo a limited unilateral micro-TESE and avoid the need for complete testicular microdissection. © 2015 American Society of Andrology and European Academy of Andrology.

Intratumoral heterogeneity in breast carcinoma revealed by laser-microdissection and comparative genomic hybridization.

PubMed

Aubele, M; Mattis, A; Zitzelsberger, H; Walch, A; Kremer, M; Hutzler, P; Höfler, H; Werner, M

1999-04-15

To evaluate the potential cytogenetic heterogeneity in breast carcinoma, several small cell groups (each consisting of 20 to 50 cells) were investigated within paraffin sections. By laser-microdissection, three to seven cell groups were taken per case. The DNA was amplified by degenerate oligonucleotide primed PCR (DOP-PCR), and the samples were analyzed by CGH for chromosomal gains and losses. Two ductal invasive breast carcinomas, one of them with two lymphnode metastases, were investigated. To compare the results from the small samples, CGH was also performed on DNA isolated from the tumorous regions of three to five serial sections (10(7) to 10(6) cells). The aberrations observed in the microdissected tumor samples were multiple and involved up to 14 different chromosomal or subchromosomal regions. The most frequent changes were gains on chromosomes 12q (14/20) and 20q (16/20), and loss on 13q (12/20). Some aberrations have rarely been detected (e.g., loss on 2p, gain on 8q). Comparing chromosomal imbalances in primary tumors and lymph node metastases, more consistent changes were found between the primary tumor and its corresponding metastases than between both primary tumors. The laser-microdissected samples in general showed more chromosomal aberrations than DNA isolated from several tumor sections. Our CGH results were confirmed by fluorescence in situ hybridization (FISH) for the chromosomal regions of centromere 1 and 20, and 20q13. In addition, microsatellite analyses on 31 samples confirmed our CGH findings for selected chromosome regions 2p and 11q. It can be concluded that there is a distinct intratumoral heterogeneity in primary breast tumors as well as in the corresponding lymph node metastases. The combination of microdissection and CGH enabled us to detect cytogenetic aberrations from important clones which are missed when analyzing DNA extracted from large cell numbers.

The utility of endoscopic radical resection with microdissection electrodes for lingual thyroglossal duct cysts.

PubMed

Cho, Jung-Hae; Jung, Won-Sang; Sun, Dong-Il

2014-03-01

Lingual thyroglossal duct cysts (LTGDCs) are very rare and liable to be misdiagnosed as simple vallecular or mucus retention cysts. We recognized the importance of complete resection by means of the Sistrunk operation and applied the revised surgical technique to the treatment of LTGDCs. The aim of this study was to evaluate the results of surgical management of LTGDCs from the author's series and analyze its utility. Twelve patients, 10 male and 2 female, who were diagnosed with LTGDCs between January 2007 and December 2012, underwent endoscopic radical resection with microdissection electrodes. All cases were evaluated by enhanced CT and flexible laryngoscope before surgery. We reviewed the collected data including presentation, CT findings, surgical techniques, postoperative complication, and recurrence. Most adult LTGDCs presented with foreign body sensation, while one infant presented acute upper airway obstruction. All cysts abutted on the hyoid bone and were located at the midline of the posterior tongue. Endoscopic radical resection with microdissection electrodes was possible by dissecting hyoid periosteum without significant morbidity. All patients excluding 1 infant were not intubated electively overnight and went home the following morning. All patients showed no evidence of recurrence during follow-up. We found that the diagnosis of LTGDCs must be based on the anatomic relationship with the hyoid bone by enhanced sagittal neck CT. Endoscopic radical resection with microdissection electrodes can be recommended for reducing recurrence and morbidity by dissecting the hyoid perichondrium in the treatment of LTGDCs.

Long-term exposure of mice to nucleoside analogues disrupts mitochondrial DNA maintenance in cortical neurons.

PubMed

Zhang, Yulin; Song, Fengli; Gao, Ziyun; Ding, Wei; Qiao, Luxin; Yang, Sufang; Chen, Xi; Jin, Ronghua; Chen, Dexi

2014-01-01

Nucleoside analogue reverse transcriptase inhibitor (NRTI), an integral component of highly active antiretroviral therapy (HAART), was widely used to inhibit HIV replication. Long-term exposure to NRTIs can result in mitochondrial toxicity which manifests as lipoatrophy, lactic acidosis, cardiomyopathy and myopathy, as well as polyneuropathy. But the cerebral neurotoxicity of NRTIs is still not well known partly due to the restriction of blood-brain barrier (BBB) and the complex microenvironment of the central nervous system (CNS). In this study, the Balb/c mice were administered 50 mg/kg stavudine (D4T), 100 mg/kg zidovudine (AZT), 50 mg/kg lamivudine (3TC) or 50 mg/kg didanosine (DDI) per day by intraperitoneal injection, five days per week for one or four months, and primary cortical neurons were cultured and exposed to 25 µM D4T, 50 µM AZT, 25 µM 3TC or 25 µM DDI for seven days. Then, single neuron was captured from mouse cerebral cortical tissues by laser capture microdissection. Mitochondrial DNA (mtDNA) levels of the primary cultured cortical neurons, and captured neurons or glial cells, and the tissues of brains and livers and muscles were analyzed by relative quantitative real-time PCR. The data showed that mtDNA did not lose in both NRTIs exposed cultured neurons and one month NRTIs treated mouse brains. In four months NRTIs treated mice, brain mtDNA levels remained unchanged even if the mtDNA levels of liver (except for 3TC) and muscle significantly decreased. However, mtDNA deletion was significantly higher in the captured neurons from mtDNA unchanged brains. These results suggest that long-term exposure to NRTIs can result in mtDNA deletion in mouse cortical neurons.

Apostle Islands National Lakeshore: alternatives analysis for LCM replacement

DOT National Transportation Integrated Search

2014-10-01

This report documents a study which analyzed various transportation options for Apostle Islands National Lakeshore (APIS) to pump toilet systems and perform construction support activities. The report presents the background of the study; describes t...

Localized Chemical Remodeling for Live Cell Imaging of Protein-Specific Glycoform.

PubMed

Hui, Jingjing; Bao, Lei; Li, Siqiao; Zhang, Yi; Feng, Yimei; Ding, Lin; Ju, Huangxian

2017-07-03

Live cell imaging of protein-specific glycoforms is important for the elucidation of glycosylation mechanisms and identification of disease states. The currently used metabolic oligosaccharide engineering (MOE) technology permits routinely global chemical remodeling (GCM) for carbohydrate site of interest, but can exert unnecessary whole-cell scale perturbation and generate unpredictable metabolic efficiency issue. A localized chemical remodeling (LCM) strategy for efficient and reliable access to protein-specific glycoform information is reported. The proof-of-concept protocol developed for MUC1-specific terminal galactose/N-acetylgalactosamine (Gal/GalNAc) combines affinity binding, off-on switchable catalytic activity, and proximity catalysis to create a reactive handle for bioorthogonal labeling and imaging. Noteworthy assay features associated with LCM as compared with MOE include minimum target cell perturbation, short reaction timeframe, effectiveness as a molecular ruler, and quantitative analysis capability. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

CCR7 directs the recruitment of T cells into inflamed pancreatic islets of nonobese diabetic (NOD) mice.

PubMed

Shan, Zhongyan; Xu, Baohui; Mikulowska-Mennis, Anna; Michie, Sara A

2014-05-01

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease characterized by the destruction of insulin-producing β cells in the pancreatic islets. The migration of T cells from blood vessels into pancreas is critical for the development of islet inflammation and β cell destruction in T1D. To define the roles of C-C chemokine receptor type 7 (CCR7) in recruitment of T cells into islets, we used laser capture microdissection to isolate tissue from inflamed islets of nonobese diabetic (NOD) mice and uninflamed islets of BALB/c and young NOD mice. RT-PCR analyses detected mRNAs for CCR7 and its chemokine ligands CCL19 (ELC; MIP-3β) and CCL21 (SLC) in captures from inflamed, but not from uninflamed, islets. Immunohistology studies revealed that high endothelial venules in inflamed islets co-express CCL21 protein and MAdCAM-1 (an adhesion molecule that recruits lymphocytes into islets). Desensitization of lymphocyte CCR7 blocked about 75 % of T cell migration from the bloodstream into inflamed islets, but had no effect on B cell migration into islets. These results indicate that CCR7 and its ligands are important in the recruitment of T cells into inflamed islets and thus in the pathogenesis of T1D.

Evaluating Intervention Programs with a Pretest-Posttest Design: A Structural Equation Modeling Approach

PubMed Central

Alessandri, Guido; Zuffianò, Antonio; Perinelli, Enrico

2017-01-01

A common situation in the evaluation of intervention programs is the researcher's possibility to rely on two waves of data only (i.e., pretest and posttest), which profoundly impacts on his/her choice about the possible statistical analyses to be conducted. Indeed, the evaluation of intervention programs based on a pretest-posttest design has been usually carried out by using classic statistical tests, such as family-wise ANOVA analyses, which are strongly limited by exclusively analyzing the intervention effects at the group level. In this article, we showed how second order multiple group latent curve modeling (SO-MG-LCM) could represent a useful methodological tool to have a more realistic and informative assessment of intervention programs with two waves of data. We offered a practical step-by-step guide to properly implement this methodology, and we outlined the advantages of the LCM approach over classic ANOVA analyses. Furthermore, we also provided a real-data example by re-analyzing the implementation of the Young Prosocial Animation, a universal intervention program aimed at promoting prosociality among youth. In conclusion, albeit there are previous studies that pointed to the usefulness of MG-LCM to evaluate intervention programs (Muthén and Curran, 1997; Curran and Muthén, 1999), no previous study showed that it is possible to use this approach even in pretest-posttest (i.e., with only two time points) designs. Given the advantages of latent variable analyses in examining differences in interindividual and intraindividual changes (McArdle, 2009), the methodological and substantive implications of our proposed approach are discussed. PMID:28303110

Evaluating Intervention Programs with a Pretest-Posttest Design: A Structural Equation Modeling Approach.

PubMed

Alessandri, Guido; Zuffianò, Antonio; Perinelli, Enrico

2017-01-01

A common situation in the evaluation of intervention programs is the researcher's possibility to rely on two waves of data only (i.e., pretest and posttest), which profoundly impacts on his/her choice about the possible statistical analyses to be conducted. Indeed, the evaluation of intervention programs based on a pretest-posttest design has been usually carried out by using classic statistical tests, such as family-wise ANOVA analyses, which are strongly limited by exclusively analyzing the intervention effects at the group level. In this article, we showed how second order multiple group latent curve modeling (SO-MG-LCM) could represent a useful methodological tool to have a more realistic and informative assessment of intervention programs with two waves of data. We offered a practical step-by-step guide to properly implement this methodology, and we outlined the advantages of the LCM approach over classic ANOVA analyses. Furthermore, we also provided a real-data example by re-analyzing the implementation of the Young Prosocial Animation, a universal intervention program aimed at promoting prosociality among youth. In conclusion, albeit there are previous studies that pointed to the usefulness of MG-LCM to evaluate intervention programs (Muthén and Curran, 1997; Curran and Muthén, 1999), no previous study showed that it is possible to use this approach even in pretest-posttest (i.e., with only two time points) designs. Given the advantages of latent variable analyses in examining differences in interindividual and intraindividual changes (McArdle, 2009), the methodological and substantive implications of our proposed approach are discussed.

Hepatoprotective potential of Lavandula coronopifolia extracts against ethanol induced oxidative stress-mediated cytotoxicity in HepG2 cells.

PubMed

Farshori, Nida Nayyar; Al-Sheddi, Ebtsam S; Al-Oqail, Mai M; Hassan, Wafaa H B; Al-Khedhairy, Abdulaziz A; Musarrat, Javed; Siddiqui, Maqsood A

2015-08-01

The present investigations were carried out to study the protective potential of four extracts (namely petroleum ether extract (LCR), chloroform extract (LCM), ethyl acetate extract (LCE), and alcoholic extract (LCL)) of Lavandula coronopifolia on oxidative stress-mediated cell death induced by ethanol, a known hepatotoxin in human hapatocellular carcinoma (HepG2) cells. Cells were pretreated with LCR, LCM, LCE, and LCL extracts (10-50 μg/ml) of L. coronopifolia for 24 h and then ethanol was added and incubated further for 24 h. After the exposure, cell viability using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and neutral red uptake assays and morphological changes in HepG2 cells were studied. Pretreatment with various extracts of L. coronpifolia was found to be significantly effective in countering the cytotoxic responses of ethanol. Antioxidant properties of these L. coronopifolia extracts against reactive oxygen species (ROS) generation, lipid peroxidation (LPO), and glutathione (GSH) levels induced by ethanol were investigated. Results show that pretreatment with these extracts for 24 h significantly inhibited ROS generation and LPO induced and increased the GSH levels reduced by ethanol. The data from the study suggests that LCR, LCM, LCE, and LCL extracts of L. coronopifolia showed hepatoprotective activity against ethanol-induced damage in HepG2 cells. However, a comparative study revealed that the LCE extract was found to be the most effective and LCL the least effective. The hepatoprotective effects observed in the study could be associated with the antioxidant properties of these extracts of L. coronopifolia. © The Author(s) 2013.

RNA analysis of inner ear cells from formalin fixed paraffin embedded (FFPE) archival human temporal bone section using laser microdissection--a technical report.

PubMed

Kimura, Yurika; Kubo, Sachiho; Koda, Hiroko; Shigemoto, Kazuhiro; Sawabe, Motoji; Kitamura, Ken

2013-08-01

Molecular analysis using archival human inner ear specimens is challenging because of the anatomical complexity, long-term fixation, and decalcification. However, this method may provide great benefit for elucidation of otological diseases. Here, we extracted mRNA for RT-PCR from tissues dissected from archival FFPE human inner ears by laser microdissection. Three human temporal bones obtained at autopsy were fixed in formalin, decalcified by EDTA, and embedded in paraffin. The samples were isolated into spiral ligaments, outer hair cells, spiral ganglion cells, and stria vascularis by laser microdissection. RNA was extracted and heat-treated in 10 mM citrate buffer to remove the formalin-derived modification. To identify the sites where COCH and SLC26A5 mRNA were expressed, semi-nested RT-PCR was performed. We also examined how long COCH mRNA could be amplified by semi-nested RT-PCR in archival temporal bone. COCH was expressed in the spiral ligament and stria vascularis. However, SLC26A5 was expressed only in outer hair cells. The maximum base length of COCH mRNA amplified by RT-PCR was 98 bp in 1 case and 123 bp in 2 cases. We detected COCH and SLC26A5 mRNA in specific structures and cells of the inner ear from archival human temporal bone. Our innovative method using laser microdissection and semi-nested RT-PCR should advance future RNA study of human inner ear diseases. Copyright © 2013 Elsevier B.V. All rights reserved.

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

PubMed

Bova, G Steven; Eltoum, Isam A; Kiernan, John A; Siegal, Gene P; Frost, Andra R; Best, Carolyn J M; Gillespie, John W; Emmert-Buck, Michael R

2005-01-01

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of pancreatic malignancy and other biological phenomena. This chapter reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed-over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification. High-quality tissue microdissection does not necessarily mean high-quality samples to analyze. The quality of biomaterials obtained for analysis is highly dependent on steps upstream and downstream from tissue microdissection. We provide protocols for each of these steps, and encourage you to improve upon these. It is worth the effort of every laboratory to optimize and document its technique at each stage of the process, and we provide a starting point for those willing to spend the time to optimize. In our view, poor documentation of tissue and cell type of origin and the use of nonoptimized protocols is a source of inefficiency in current life science research. Even incremental improvement in this area will increase productivity significantly.

Combined maceration procedure permits advanced microsurgical dissection of Thiel-embalmed specimens.

PubMed

Bangerter, Hannes; Boemke, Susanne; Röthlisberger, Raphael; Schwartz, Valerie; Bergmann, Mathias; Müller, Michael D; Djonov, Valentin

2017-03-01

Due to the realistic colour, texture conservation and preservation of biomechanical properties, Thiel-embalming is becoming the main embalming procedure for clinical courses and research based on human cadaver material. The aim of this study is to establish a new procedure that allows advanced microdissection of small vessels and intraorganic nerves in Thiel-embalmed material. After a classical gross anatomical dissection, human hemipelves underwent repetitive application of 3 consecutive steps: (i) maceration with alloy of nitric acid and MiliQ water 1:10 for 24-48h. (ii) Immersion: the hemipelves were rinsed under tap water for 20-30min. and placed in a water bath for 1h. The nerves become more prominent due to the swelling and increased water content. (iii) microdissection under surgical microscope. To facilitate the organ visualization perfusion with polyurethane (Pu4ii, VasQtec ® , Switzerland) in red/blue for arteries/veins respectively has been performed. By using the proposed procedure, we performed satisfactory microdissection on Thiel-embalmed samples. The combination with polyurethane vascular casting permits visualization of small arterioles and venules in a range of 20-25μm. The method is very suitable for demonstration of somatic and vegetative nerves. Branches of the sacral plexuses and autonomic nerves from the superior and inferior hypogastric plexus have been tracked up to the smallest intraorganic branches in a range of 12.5-15μm. In conclusion, the established combined procedure offers a new possibility for advanced microdissection, which will allow acquisition of clinically relevant information about organ specific micro- vascularization and innervation. Copyright © 2016 Elsevier GmbH. All rights reserved.

From life cycle talking to taking action

EPA Science Inventory

The series of Life Cycle Management (LCM) conferences has aimed to create a platform for users and developers of life cycle assessment tools to share their experiences as they challenge traditional environmental management practices, which are narrowly confined (“gate-to-gate”) a...

No difference in acute effects of supplemental v. dietary calcium on blood pressure and microvascular function in obese women challenged with a high-fat meal: a cross-over randomised study.

PubMed

Ferreira, Thaís da Silva; Leal, Priscila Mansur; Antunes, Vanessa Parada; Sanjuliani, Antonio Felipe; Klein, Márcia Regina Simas Torres

2016-11-01

Recent studies suggest that supplemental Ca (SC) increases the risk of cardiovascular events, whereas dietary Ca (DC) decreases the risk of cardiovascular events. Although frequently consumed with meals, it remains unclear whether Ca can mitigate or aggravate the deleterious effects of a high-fat meal on cardiovascular risk factors. This study aimed to evaluate the effects of SC or DC on blood pressure (BP) and microvascular function (MVF) in the postprandial period in obese women challenged with a high-fat meal. In this cross-over controlled trial, sixteen obese women aged 20-50 years were randomly assigned to receive three test meals (2908 kJ (695 kcal); 48 % fat): high DC (HDCM; 547 mg DC), high SC (HSCM; 500 mg SC-calcium carbonate) and low Ca (LCM; 42 mg DC). BP was continuously evaluated from 15 min before to 120 min after meals by digital photoplethysmography. Before and 120 min after meals, participants underwent evaluation of serum Ca and microvascular flow after postocclusive reactive hyperaemia (PORH) by laser speckle contrast imaging. Ionised serum Ca rose significantly only after HSCM. Systolic BP increased after the three meals, whereas diastolic BP increased after LCM and HDCM. Hyperaemia peak, hyperaemia amplitude and AUC evaluated after PORH decreased with LCM. After HDCM, there was a reduction in hyperaemia peak and hyperaemia amplitude, whereas HSCM decreased only hyperaemia peak. However, comparative analyses of the effects of three test meals on serum Ca, BP and MVF revealed no significant meal×time interaction. This study suggests that in obese women SC and DC do not interfere with the effects of a high-fat meal on BP and MVF.

Ab Initio Calculations of Water Line Strengths

NASA Technical Reports Server (NTRS)

Schwenke, David W.; Partridge, Harry

1998-01-01

We report on the determination of a high quality ab initiu potential energy surface (PES) and dipole moment function for water. This PES is empirically adjusted to improve the agreement between the computed line positions and those from the HITRAN 92 data base with J less than 6 for H2O. The changes in the PES are small, nonetheless including an estimate of core (oxygen 1s) electron correlation greatly improves the agreement with experiment. Using this adjusted PES, we can match 30,092 of the 30,117 transitions in the HITRAN 96 data base for H2O with theoretical lines. The 10,25,50,75, and 90 percentiles of the difference between the calculated and tabulated line positions are -0.11, -0.04, -0.01, 0.02, and 0.07 l/cm. Non-adiabatic effects are not explicitly included. About 3% of the tabulated line positions appear to be incorrect. Similar agreement using this adjusted PES is obtained for the oxygen 17 and oxygen 18 isotopes. For HDO, the agreement is not as good, with root-mean-square error of 0.25 l/cm for lines with J less than 6. This error is reduced to 0.02 l/cm by including a small asymmetric correction to the PES, which is parameterized by simultaneously fitting to HDO md D2O data. Scaling this correction by mass factors yields good results for T2O and HTO. The intensities summed over vibrational bands are usually in good agreement between the calculations and the tabulated results, but individual lines strengths can differ greatly. A high temperature list consisting of 307,721,352 lines is generated for H2O using our PES and dipole moment function.

Biomimicry of quorum sensing using bacterial lifecycle model.

PubMed

Niu, Ben; Wang, Hong; Duan, Qiqi; Li, Li

2013-01-01

Recent microbiologic studies have shown that quorum sensing mechanisms, which serve as one of the fundamental requirements for bacterial survival, exist widely in bacterial intra- and inter-species cell-cell communication. Many simulation models, inspired by the social behavior of natural organisms, are presented to provide new approaches for solving realistic optimization problems. Most of these simulation models follow population-based modelling approaches, where all the individuals are updated according to the same rules. Therefore, it is difficult to maintain the diversity of the population. In this paper, we present a computational model termed LCM-QS, which simulates the bacterial quorum-sensing (QS) mechanism using an individual-based modelling approach under the framework of Agent-Environment-Rule (AER) scheme, i.e. bacterial lifecycle model (LCM). LCM-QS model can be classified into three main sub-models: chemotaxis with QS sub-model, reproduction and elimination sub-model and migration sub-model. The proposed model is used to not only imitate the bacterial evolution process at the single-cell level, but also concentrate on the study of bacterial macroscopic behaviour. Comparative experiments under four different scenarios have been conducted in an artificial 3-D environment with nutrients and noxious distribution. Detailed study on bacterial chemotatic processes with quorum sensing and without quorum sensing are compared. By using quorum sensing mechanisms, artificial bacteria working together can find the nutrient concentration (or global optimum) quickly in the artificial environment. Biomimicry of quorum sensing mechanisms using the lifecycle model allows the artificial bacteria endowed with the communication abilities, which are essential to obtain more valuable information to guide their search cooperatively towards the preferred nutrient concentrations. It can also provide an inspiration for designing new swarm intelligence optimization algorithms, which can be used for solving the real-world problems.

Biomimicry of quorum sensing using bacterial lifecycle model

PubMed Central

2013-01-01

Background Recent microbiologic studies have shown that quorum sensing mechanisms, which serve as one of the fundamental requirements for bacterial survival, exist widely in bacterial intra- and inter-species cell-cell communication. Many simulation models, inspired by the social behavior of natural organisms, are presented to provide new approaches for solving realistic optimization problems. Most of these simulation models follow population-based modelling approaches, where all the individuals are updated according to the same rules. Therefore, it is difficult to maintain the diversity of the population. Results In this paper, we present a computational model termed LCM-QS, which simulates the bacterial quorum-sensing (QS) mechanism using an individual-based modelling approach under the framework of Agent-Environment-Rule (AER) scheme, i.e. bacterial lifecycle model (LCM). LCM-QS model can be classified into three main sub-models: chemotaxis with QS sub-model, reproduction and elimination sub-model and migration sub-model. The proposed model is used to not only imitate the bacterial evolution process at the single-cell level, but also concentrate on the study of bacterial macroscopic behaviour. Comparative experiments under four different scenarios have been conducted in an artificial 3-D environment with nutrients and noxious distribution. Detailed study on bacterial chemotatic processes with quorum sensing and without quorum sensing are compared. By using quorum sensing mechanisms, artificial bacteria working together can find the nutrient concentration (or global optimum) quickly in the artificial environment. Conclusions Biomimicry of quorum sensing mechanisms using the lifecycle model allows the artificial bacteria endowed with the communication abilities, which are essential to obtain more valuable information to guide their search cooperatively towards the preferred nutrient concentrations. It can also provide an inspiration for designing new swarm intelligence optimization algorithms, which can be used for solving the real-world problems. PMID:23815296

Flow monitoring of microwave pre-heated resin in LCM processes

NASA Astrophysics Data System (ADS)

Rubino, F.; Paradiso, V.; Carlone, P.

2017-10-01

Liquid composite molding is manufacturing techniques that involve the injection or infusion of catalyzed liquid resin into a mold to impregnate a dry fiber preform. The challenges of LCM processes are related to the obtaining of a complete wetting of the reinforcement as well as a reduction of the void to obtain a final product with high mechanical properties. The heating of the resin prior the injection into the mold cavity has proven to be useful to improve the LCM processes. The increasing of temperature results in a reduction of resin viscosity and allows the resin to flow more easily through the reinforcement; the cure stage is also improved resulting in a reduction of global process time required. Besides the conventional solutions to heat up the resin based on the thermal conduction, in-line microwave heating is a suitable method to heat dielectric materials providing an even temperature distribution through the resin, thereby avoiding a thermal gradient between the surface and the core of liquid resin, which could result in a premature and uncontrolled cure. In the present work, an in-line microwave system, manually controlled, have been coupled with a VARTM apparatus to heat the resin before the infusion. In addition, parallel-plate dielectric sensors and pressure sensors, embedded into the mold, were employed to track the flow front through the fiber reinforcement in two distinct cases: unheated resin and pre-heated resin. The aim of work was to assess the effectiveness of microwave pre-heating to improve the macro and micro-impregnation of dry preform. The obtained results showed capability of in-line microwave heating to shorten the impregnation of dry fabric and provide a homogeneous wetting of fibers.

Allelic loss studies do not provide evidence for the "endometriosis-as-tumor" theory.

PubMed

Prowse, Amanda H; Fakis, Giannoulis; Manek, Sanjiv; Churchman, Michael; Edwards, Sarah; Rowan, Andrew; Koninckx, Philippe; Kennedy, Stephen; Tomlinson, Ian P M

2005-04-01

To identify consistent genetic changes in endometriosis samples to determine whether endometriosis lesions are true neoplasms. We analyzed ovarian endometriosis lesions for loss of heterozygosity (LOH) at 12 loci of potential importance (D9S1870, D9S265, D9S270, D9S161, D11S29, D1S199, D8S261, APOA2, PTCH, TP53, D10S541, and D10S1765), including some at which genetic changes were previously reported in endometriosis. Molecular biology laboratory in a university hospital department. Seventeen women with ovarian endometriosis. Laser capture microdissection to separate the endometriotic epithelium, the adjacent endometriotic stroma, and surrounding normal ovarian stromal tissue, followed by DNA extraction and polymerase chain reaction amplification of polymorphic microsatellite markers. Fluorescence-based quantitation for the LOH analysis. We identified LOH in only one lesion at one locus (D8S261). Our data do not support the hypothesis that ovarian endometriosis is a true neoplasm.

Thymidylate synthase (TS) protein expression as a prognostic factor in advanced colorectal cancer: a comparison with TS mRNA expression.

PubMed

Nakagawa, Tateo; Shimada, Mitsuo; Kurita, Nobuhiro; Iwata, Takashi; Nishioka, Masanori; Yoshikawa, Kozo; Higashijima, Jun; Utsunomiya, Tohru

2012-06-01

The role of intratumoral thymidylate synthase (TS) mRNA or protein expression is still controversial and little has been reported regarding relation of them in colorectal cancer. Forty-six patients with advanced colorectal cancer who underwent surgical resection were included. TS mRNA expression was determined by the Danenberg tumor profile method based on laser-captured micro-dissection of the tumor cells. TS protein expression was evaluated using immunohistochemical staining. TS mRNA expression tended to relate TS protein expression. Statistical significance was not found in overall survival between the TS mRNA high group and low group regardless of performing adjuvant chemotherapy. The overall survival in the TS protein negative group was significantly higher than that in positive group in all and the patients without adjuvant chemotherapy. Multivariate analysis showed TS protein expression was as an independent prognostic factor. TS protein expression tends to be related TS mRNA expression and is an independent prognostic factor in advanced colorectal cancer.

Negative Enrichment and Isolation of Circulating Tumor Cells for Whole Genome Amplification.

PubMed

Kanwar, Nisha; Done, Susan J

2017-01-01

Circulating tumor cells (CTCs) are a rare population of cells found in the peripheral blood of patients with many types of cancer such as breast, prostate, colon, and lung cancers. Higher numbers of these cells in blood are associated with a poorer prognosis of patients. Genomic profiling of CTCs would help characterize markers specific for the identification of these cells in blood, and also define genomic alterations that give these cells a metastatic advantage over other cells in the primary tumor. Here, we describe an immunomagnetic method to enrich CTCs from the blood of patients with breast cancer, followed by single-cell laser capture microdissection to isolate single CTCs. Whole genome amplification of isolated CTCs allows for many downstream applications to be performed to aide in their characterization, such as whole genome or exome sequencing, Single Nucleotide Polymorphism (SNP) and copy number analysis, and targeted sequencing or quantitative Polymerase Chain Reaction (qPCR) for genomic analyses.

Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis.

PubMed

Kang, Yun; McMillan, Ian; Norris, Michael H; Hoang, Tung T

2015-07-01

Until recently, transcriptome analyses of single cells have been confined to eukaryotes. The information obtained from single-cell transcripts can provide detailed insight into spatiotemporal gene expression, and it could be even more valuable if expanded to prokaryotic cells. Transcriptome analysis of single prokaryotic cells is a recently developed and powerful tool. Here we describe a procedure that allows amplification of the total transcript of a single prokaryotic cell for in-depth analysis. This is performed by using a laser-capture microdissection instrument for single-cell isolation, followed by reverse transcription via Moloney murine leukemia virus, degradation of chromosomal DNA with McrBC and DpnI restriction enzymes, single-stranded cDNA (ss-cDNA) ligation using T4 polynucleotide kinase and CircLigase, and polymerization of ss-cDNA to double-stranded cDNA (ds-cDNA) by Φ29 polymerase. This procedure takes ∼5 d, and sufficient amounts of ds-cDNA can be obtained from single-cell RNA template for further microarray analysis.

Inflammatory signaling in human Tuberculosis granulomas is spatially organized

PubMed Central

Marakalala, Mohlopheni J.; Raju, Ravikiran M.; Sharma, Kirti; Zhang, Yanjia J.; Eugenin, Eliseo A.; Prideaux, Brendan; Daudelin, Isaac B.; Chen, Pei-Yu; Booty, Matthew G.; Kim, Jin Hee; Eum, Seok Yong; Via, Laura E.; Behar, Samuel M.; Barry, Clifton E.; Mann, Matthias; Dartois, Véronique; Rubin, Eric J.

2016-01-01

Granulomas are the pathological hallmark of tuberculosis (TB). However, their function and mechanisms of formation remain poorly understood. To understand the role of granulomas in TB, we analyzed the proteomes of granulomas from subjects with tuberculosis in an unbiased fashion. Using laser capture microdissection, mass spectrometry and confocal microscopy, we generated detailed molecular maps of human granulomas. We found that the centers of granulomas possess a pro-inflammatory environment characterized by anti-microbial peptides, ROS and pro-inflammatory eicosanoids. Conversely, the tissue surrounding the caseum possesses a comparatively anti-inflammatory signature. These findings are consistent across a set of six subjects and in rabbits. While the balance between systemic pro- and anti-inflammatory signals is crucial to TB disease outcome, here we find that these signals are physically segregated within each granuloma. The protein and lipid snapshots of human and rabbit lesions analysed here suggest that the pathologic response to TB is shaped by the precise anatomical localization of these inflammatory pathways during the development of the granuloma. PMID:27043495

Downregulation of miR-125b in metastatic cutaneous malignant melanoma.

PubMed

Glud, Martin; Rossing, Maria; Hother, Christoffer; Holst, Line; Hastrup, Nina; Nielsen, Finn C; Gniadecki, Robert; Drzewiecki, Krzysztof T

2010-12-01

This study aimed to identify microRNA species involved in the earliest metastatic event in cutaneous malignant melanoma (MM). Samples from 28 patients with MM [stage T2 (tumor), M0 (distant metastasis)] were grouped by the presence of micrometastasis in the sentinel lymph nodes (N0/N1). Melanoma cells were harvested from primary, cutaneous MM tumors by laser-capture microdissection, and microRNA expression profiles were obtained by the microarray technique. Results were validated by quantitative reverse transcription PCR. We found that miR-125b was downregulated in the primary cutaneous melanomas that produced early metastases (T2, N1, M0) compared with the sentinel lymph node-negative (T2, N0, M0) melanomas. MiR-125b has earlier been found to be downregulated in other tumor types and in atypic naevi compared with the common acquired naevi. In conclusion, miR-125b may be involved in an early progression of cutaneous MM.

9 CFR 113.42 - Detection of lymphocytic choriomeningitis contamination.

Code of Federal Regulations, 2010 CFR

2010-01-01

... of at least 10 mice obtained from a source free of LCM shall be injected in the footpad of a hindfoot with 0.02 ml of the material being tested and observed each day for 21 days. (b) If any of the mice...

9 CFR 113.42 - Detection of lymphocytic choriomeningitis contamination.

Code of Federal Regulations, 2011 CFR

2011-01-01

... of at least 10 mice obtained from a source free of LCM shall be injected in the footpad of a hindfoot with 0.02 ml of the material being tested and observed each day for 21 days. (b) If any of the mice...

9 CFR 113.42 - Detection of lymphocytic choriomeningitis contamination.

Code of Federal Regulations, 2012 CFR

2012-01-01

... of at least 10 mice obtained from a source free of LCM shall be injected in the footpad of a hindfoot with 0.02 ml of the material being tested and observed each day for 21 days. (b) If any of the mice...

9 CFR 113.42 - Detection of lymphocytic choriomeningitis contamination.

Code of Federal Regulations, 2013 CFR

2013-01-01

... of at least 10 mice obtained from a source free of LCM shall be injected in the footpad of a hindfoot with 0.02 ml of the material being tested and observed each day for 21 days. (b) If any of the mice...

9 CFR 113.42 - Detection of lymphocytic choriomeningitis contamination.

Code of Federal Regulations, 2014 CFR

2014-01-01

... of at least 10 mice obtained from a source free of LCM shall be injected in the footpad of a hindfoot with 0.02 ml of the material being tested and observed each day for 21 days. (b) If any of the mice...

Habituation in acoustic startle reflex: individual differences in personality.

PubMed

Blanch, Angel; Balada, Ferran; Aluja, Anton

2014-03-01

This study analyzed the relationship of individual differences in personality with habituation in the acoustic startle response (ASR). Data from nine trials in ASR to white noise bursts and a personality questionnaire based on the alternative big five personality approach were modelled with a latent growth curve (LCM) including intercept and slope habituation growth factors. There was a negative correlation between the intercept and slope, indicating that individuals with higher initial ASR levels had also a more pronounced and faster decrease in the ASR. Contrary to expectations, Extraversion and Sensation Seeking did not relate with habituation in ASR. Neuroticism and Aggressiveness related asymmetrically with the habituation rate in ASR. Higher levels of Neuroticism were related with faster habituation, whereas higher levels of Aggressiveness were related with slower habituation. Further studies with the LCM should be undertaken to clarify in a greater extent the association of personality with habituation in ASR. Copyright © 2014 Elsevier B.V. All rights reserved.

Induction technique in manufacturing preforms

NASA Astrophysics Data System (ADS)

Frauenhofer, M.; Ströhlein, T.; Fabig, S.; Böhm, S.; Herbeck, L.; Dilger, K.

2008-09-01

The prepreg technology is a state-of-the-art method to produce high-performance CFRP parts. Due to the high material prices, the restricted process rate, and limitations to the component complexity, in future, more and more parts will be assembled by using liquid composite moulding. Especially in the case of series larger than 100 parts per year, the LCM technology offers the best cost-effectiveness. This technology is based on resin injection into dry multilayer fibre textiles (preforms). The Institute of Joining and Welding (TU, Braunschweig), together with the Institute of Composite Structures and Adaptive Systems (DLR), has elaborated a new technology to speed up the preform process, which is the most labour-intensive step within the LCM process chain. A novel concept to consolidate binder-coated fabrics is under development. By applying the high energy transfer rate of induction technology, it is possible to heat up a preform with rates up to 50 K/s to melt the binder and consolidate the preform.

Factors that affect the postnatal increase in superior mesenteric artery blood flow velocity in very low birth weight preterm infants.

PubMed

Havranek, Thomas; Miladinovic, Branko; Wadhawan, Rajan; Carver, Jane D

2012-04-15

To identify factors related to the postnatal increase in superior mesenteric artery blood flow velocity (SMA BFV). SMA BFV was measured in 35 infants (birth weight 1047±246 g) on day of life (DOL) 1, 3, 5, 7 10 and 14. Latent curve modeling (LCM) was used to measure the longitudinal change in BFV for each subject, and the correlation between changes in BFV and baseline values. Non-parametric correlations were calculated between BFV and variables previously reported to be related to SMA BFV. There was significant variability in SMA BFV on DOL 1, a significant increase from DOL 1-14, and significant variability in the postnatal increase. Infants with higher enteral feeding volumes had greater increases, while infants receiving positive pressure ventilation or hyperalimentation had lower increases. Several clinical factors affect the postnatal increase in SMA BFV. The use of LCM is useful in longitudinal studies of very low birth weight (VLBW) infants, who are clinically and demographically heterogeneous.

Operations and Modeling Analysis

NASA Technical Reports Server (NTRS)

Ebeling, Charles

2005-01-01

The Reliability and Maintainability Analysis Tool (RMAT) provides NASA the capability to estimate reliability and maintainability (R&M) parameters and operational support requirements for proposed space vehicles based upon relationships established from both aircraft and Shuttle R&M data. RMAT has matured both in its underlying database and in its level of sophistication in extrapolating this historical data to satisfy proposed mission requirements, maintenance concepts and policies, and type of vehicle (i.e. ranging from aircraft like to shuttle like). However, a companion analyses tool, the Logistics Cost Model (LCM) has not reached the same level of maturity as RMAT due, in large part, to nonexistent or outdated cost estimating relationships and underlying cost databases, and it's almost exclusive dependence on Shuttle operations and logistics cost input parameters. As a result, the full capability of the RMAT/LCM suite of analysis tools to take a conceptual vehicle and derive its operations and support requirements along with the resulting operating and support costs has not been realized.

Precise, motion-free polarization control in Second Harmonic Generation microscopy using a liquid crystal modulator in the infinity space.

PubMed

Lien, Chi-Hsiang; Tilbury, Karissa; Chen, Shean-Jen; Campagnola, Paul J

2013-01-01

Second Harmonic Generation (SHG) microscopy coupled with polarization analysis has great potential for use in tissue characterization, as molecular and supramolecular structural details can be extracted. Such measurements are difficult to perform quickly and accurately. Here we present a new method that uses a liquid crystal modulator (LCM) located in the infinity space of a SHG laser scanning microscope that allows the generation of any desired linear or circular polarization state. As the device contains no moving parts, polarization can be rotated accurately and faster than by manual or motorized control. The performance in terms of polarization purity was validated using Stokes vector polarimetry, and found to have minimal residual polarization ellipticity. SHG polarization imaging characteristics were validated against well-characterized specimens having cylindrical and/or linear symmetries. The LCM has a small footprint and can be implemented easily in any standard microscope and is cost effective relative to other technologies.

Teaching strategies in the learning of highest common factor and lowest common multiple

NASA Astrophysics Data System (ADS)

Halim, N. L. A.; Li, H.-C.; Shahrill, M.; Prahmana, R. C. I.

2017-12-01

The main purpose of this study is to improve students’ understanding of Highest Common Factor (HCF) and Lowest Common Multiple (LCM). Several alternative teaching strategies were integrated in the Year 7 lessons involving 20 students from one secondary school in Brunei Darussalam. The categories identified in the teaching strategy were the application of group work, embedding real-life problems, using presentations and the traditional drilling practice method. An open-ended survey was disseminated to collate the students’ feedback, and among the questions posed was in relation to the different types of lesson activities utilised within each of the teaching strategies. Although 55% of the students preferred the traditional drilling practice on the calculation of HCF and LCM, mainly due to the ease of the task as opposed to activities involving real-life problems, the findings also indicated that most students favour sharing their ideas in a healthy competition learning style between each other or between the groups.

Extracting the Data From the LCM vk4 Formatted Output File

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

Wendelberger, James G.

These are slides about extracting the data from the LCM vk4 formatted output file. The following is covered: vk4 file produced by Keyence VK Software, custom analysis, no off the shelf way to read the file, reading the binary data in a vk4 file, various offsets in decimal lines, finding the height image data, directly in MATLAB, binary output beginning of height image data, color image information, color image binary data, color image decimal and binary data, MATLAB code to read vk4 file (choose a file, read the file, compute offsets, read optical image, laser optical image, read and computemore » laser intensity image, read height image, timing, display height image, display laser intensity image, display RGB laser optical images, display RGB optical images, display beginning data and save images to workspace, gamma correction subroutine), reading intensity form the vk4 file, linear in the low range, linear in the high range, gamma correction for vk4 files, computing the gamma intensity correction, observations.« less

Precise, motion-free polarization control in Second Harmonic Generation microscopy using a liquid crystal modulator in the infinity space

PubMed Central

Lien, Chi-Hsiang; Tilbury, Karissa; Chen, Shean-Jen; Campagnola, Paul J.

2013-01-01

Second Harmonic Generation (SHG) microscopy coupled with polarization analysis has great potential for use in tissue characterization, as molecular and supramolecular structural details can be extracted. Such measurements are difficult to perform quickly and accurately. Here we present a new method that uses a liquid crystal modulator (LCM) located in the infinity space of a SHG laser scanning microscope that allows the generation of any desired linear or circular polarization state. As the device contains no moving parts, polarization can be rotated accurately and faster than by manual or motorized control. The performance in terms of polarization purity was validated using Stokes vector polarimetry, and found to have minimal residual polarization ellipticity. SHG polarization imaging characteristics were validated against well-characterized specimens having cylindrical and/or linear symmetries. The LCM has a small footprint and can be implemented easily in any standard microscope and is cost effective relative to other technologies. PMID:24156059

Transmission Geometry Laser Ablation into a Non-Contact Liquid Vortex Capture Probe for Mass Spectrometry Imaging

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

Ovchinnikova, Olga S; Bhandari, Deepak; Lorenz, Matthias

2014-01-01

RATIONALE: Capture of material from a laser ablation plume into a continuous flow stream of solvent provides the means for uninterrupted sampling, transport and ionization of collected material for coupling with mass spectral analysis. Reported here is the use of vertically aligned transmission geometry laser ablation in combination with a new non-contact liquid vortex capture probe coupled with electrospray ionization for spot sampling and chemical imaging with mass spectrometry. Methods: A vertically aligned continuous flow liquid vortex capture probe was positioned directly underneath a sample surface in a transmission geometry laser ablation (355 nm, 10 Hz, 7 ns pulse width)more » setup to capture into solution the ablated material. The outlet of the vortex probe was coupled to the Turbo V ion source of an AB SCIEX TripleTOF 5600+ mass spectrometer. System operation and performance metrics were tested using inked patterns and thin tissue sections. Glass slides and slides designed especially for laser capture microdissection, viz., DIRECTOR slides and PEN 1.0 (polyethylene naphthalate) membrane slides, were used as sample substrates. Results: The estimated capture efficiency of laser ablated material was 24%, which was enabled by the use of a probe with large liquid surface area (~ 2.8 mm2) and with gravity to help direct ablated material vertically down towards the probe. The swirling vortex action of the liquid surface potentially enhanced capture and dissolution of not only particulates, but also gaseous products of the laser ablation. The use of DIRECTOR slides and PEN 1.0 (polyethylene naphthalate) membrane slides as sample substrates enabled effective ablation of a wide range of sample types (basic blue 7, polypropylene glycol, insulin and cyctochrome c) without photodamage using a UV laser. Imaging resolution of about 6 m was demonstrated for stamped ink on DIRECTOR slides based on the ability to distinguish features present both in the optical and in the chemical image. This imaging resolution was 20 times better than the previous best reported results with laser ablation/liquid sample capture mass spectrometry imaging. Using thin sections of brain tissue the chemical image of a selected lipid was obtained with an estimated imaging resolution of about 50 um. Conclusions: A vertically aligned, transmission geometry laser ablation liquid vortex capture probe, electrospray ionization mass spectrometry system provides an effective means for spatially resolved spot sampling and imaging with mass spectrometry.« less

Transmission geometry laser ablation into a non-contact liquid vortex capture probe for mass spectrometry imaging.

PubMed

Ovchinnikova, Olga S; Bhandari, Deepak; Lorenz, Matthias; Van Berkel, Gary J

2014-08-15

Capture of material from a laser ablation plume into a continuous flow stream of solvent provides the means for uninterrupted sampling, transport and ionization of collected material for coupling with mass spectral analysis. Reported here is the use of vertically aligned transmission geometry laser ablation in combination with a new non-contact liquid vortex capture probe coupled with electrospray ionization for spot sampling and chemical imaging with mass spectrometry. A vertically aligned continuous flow liquid vortex capture probe was positioned directly underneath a sample surface in a transmission geometry laser ablation (355 nm, 10 Hz, 7 ns pulse width) set up to capture into solution the ablated material. The outlet of the vortex probe was coupled to the Turbo V™ ion source of an AB SCIEX TripleTOF 5600+ mass spectrometer. System operation and performance metrics were tested using inked patterns and thin tissue sections. Glass slides and slides designed especially for laser capture microdissection, viz., DIRECTOR(®) slides and PEN 1.0 (polyethylene naphthalate) membrane slides, were used as sample substrates. The estimated capture efficiency of laser-ablated material was 24%, which was enabled by the use of a probe with large liquid surface area (~2.8 mm(2) ) and with gravity to help direct ablated material vertically down towards the probe. The swirling vortex action of the liquid surface potentially enhanced capture and dissolution not only of particulates, but also of gaseous products of the laser ablation. The use of DIRECTOR(®) slides and PEN 1.0 (polyethylene naphthalate) membrane slides as sample substrates enabled effective ablation of a wide range of sample types (basic blue 7, polypropylene glycol, insulin and cyctochrome c) without photodamage using a UV laser. Imaging resolution of about 6 µm was demonstrated for stamped ink on DIRECTOR(®) slides based on the ability to distinguish features present both in the optical and in the chemical image. This imaging resolution was 20 times better than the previous best reported results with laser ablation/liquid sample capture mass spectrometry imaging. Using thin sections of brain tissue the chemical image of a selected lipid was obtained with an estimated imaging resolution of about 50 µm. A vertically aligned, transmission geometry laser ablation liquid vortex capture probe, electrospray ionization mass spectrometry system provides an effective means for spatially resolved spot sampling and imaging with mass spectrometry. Published in 2014. This article is a U.S. Government work and is in the public domain in the USA.

EGFR is involved in dermatofibrosarcoma protuberans progression to high grade sarcoma.

PubMed

Osio, Amélie; Xu, Shuo; El Bouchtaoui, Morad; Leboeuf, Christophe; Gapihan, Guillaume; Lemaignan, Christine; Bousquet, Guilhem; Lebbé, Céleste; Janin, Anne; Battistella, Maxime

2018-02-02

Dermatofibrosarcoma protuberans (DFSP), amounting to 6% of all soft tissue sarcomas, has a slow growth rate, contrasting with a likelihood for local recurrence and a 10-20% evolution to higher-grade sarcoma, or "transformed DFSP" (DFSP-T). At molecular level, the characteristic COL1A1-PDGFB rearrangement, leading to sustained PDGFR signaling, is not linked to the evolutive potential. Here, we studied EGFR, another tyrosine kinase receptor, using laser-microdissection to select the different histologic components of DFSP (DFSP center, DFSP infiltrative periphery, DFSP-T higher-grade sarcoma), in 22 patients followed over 3 to 156 months. EGFR protein and mRNA were expressed in 13/22 patients with DFSP or DFSP-T, and increased with tumor progression, both in microdissected areas of higher-grade sarcomas and in microdissected areas of local extension. No cancer-associated EGFR gene mutation or copy-number variation, nor any KRAS, BRAF, NRAS hotspot mutations were found in any microdissected area. Among epithelial-mesenchymal transition factors tested, SNAIL 1/2 had the same expression pattern as EGFR while ZEB1/2 or TWIST1/2 did not. Using a proteome profiler phospho-kinase array on 3 DFSP and 3 DFSP-T cryopreserved tissue samples, EGFR phosphorylation was detected in each case. Among EGFR downstream pathways, we found positive correlations between phosphorylation levels of EGFR and STAT5a/b (r = 0.87, p < 0.05) and TOR (r = 0.95, p < 0.01), but not ERK in the MAPK pathway (r = -0.18, p > 0.70). We thus demonstrated that in DFSP evolution to high grade sarcoma, EGFR and SNAIL were involved, with EGFR activation and signaling through TOR and STAT5a/b downstream effectors, which could lead on to new therapies for advanced DFSP.

Quantitative RT-PCR analysis of estrogen receptor gene expression in laser microdissected prostate cancer tissue.

PubMed

Walton, Thomas J; Li, Geng; McCulloch, Thomas A; Seth, Rashmi; Powe, Desmond G; Bishop, Michael C; Rees, Robert C

2009-06-01

Real-time quantitative RT-PCR analysis of laser microdissected tissue is considered the most accurate technique for determining tissue gene expression. The discovery of estrogen receptor beta (ERbeta) has focussed renewed interest on the role of estrogen receptors in prostate cancer, yet few studies have utilized the technique to analyze estrogen receptor gene expression in prostate cancer. Fresh tissue was obtained from 11 radical prostatectomy specimens and from 6 patients with benign prostate hyperplasia. Pure populations of benign and malignant prostate epithelium were laser microdissected, followed by RNA isolation and electrophoresis. Quantitative RT-PCR was performed using primers for androgen receptor (AR), estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), progesterone receptor (PGR) and prostate specific antigen (PSA), with normalization to two housekeeping genes. Differences in gene expression were analyzed using the Mann-Whitney U-test. Correlation coefficients were analyzed using Spearman's test. Significant positive correlations were seen when AR and AR-dependent PSA, and ERalpha and ERalpha-dependent PGR were compared, indicating a representative population of RNA transcripts. ERbeta gene expression was significantly over-expressed in the cancer group compared with benign controls (P < 0.01). In contrast, PGR expression was significantly down-regulated in the cancer group (P < 0.05). There were no significant differences in AR, ERalpha or PSA expression between the groups. This study represents the first to show an upregulation of ERbeta gene expression in laser microdissected prostate cancer specimens. In concert with recent studies the findings suggest differential production of ERbeta splice variants, which may play important roles in the genesis of prostate cancer. (c) 2009 Wiley-Liss, Inc.

Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.

PubMed

Bernsen, M R; Dijkman, H B; de Vries, E; Figdor, C G; Ruiter, D J; Adema, G J; van Muijen, G N

1998-10-01

Molecular analysis of small tissue samples has become increasingly important in biomedical studies. Using a laser dissection microscope and modified nucleic acid isolation protocols, we demonstrate that multiple mRNA as well as DNA sequences can be identified from a single-cell sample. In addition, we show that the specificity of procurement of tissue samples is not compromised by smear contamination resulting from scraping of the microtome knife during sectioning of lesions. The procedures described herein thus allow for efficient RT-PCR or PCR analysis of multiple nucleic acid sequences from small tissue samples obtained by laser-assisted microdissection.

A Treatise on the Application of Life Cycle Management Principles in Agricultural & Biological Engineering

USDA-ARS?s Scientific Manuscript database

Life Cycle Management (LCM) is a systematic approach, mindset and culture that considers economic, social, and environmental factors among other factors in the decision making process throughout various business or organizational decisions that affect both inputs and outputs of a product or service...

Class Extraction and Classification Accuracy in Latent Class Models

ERIC Educational Resources Information Center

Wu, Qiong

2009-01-01

Despite the increasing popularity of latent class models (LCM) in educational research, methodological studies have not yet accumulated much information on the appropriate application of this modeling technique, especially with regard to requirement on sample size and number of indicators. This dissertation study represented an initial attempt to…

Base Camp Life Cycle Management: Focusing on the Critical Elements

DTIC Science & Technology

2011-12-01

needs of the occupants, although “building” this infrastructure often meant cobbling together prefabricated buildings or tents as much as it meant...as System Boundaries.” Journal of Industrial Ecology 10, no. 1 (2006): 61-77. Rebitzer, G. and Hunkeler, D. Life Cycle Costing in LCM: Ambitions

Characterization of a Hybrid Optical Microscopy/Laser Ablation Liquid Vortex Capture/Electrospray Ionization System for Mass Spectrometry Imaging

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

Cahill, John F.; Kertesz, Vilmos; Van Berkel, Gary J.

Herein, a commercial optical microscope, laser microdissection instrument was coupled with an electrospray ionization mass spectrometer via a low profile liquid vortex capture probe to yield a hybrid optical microscopy/mass spectrometry imaging system. The instrument has bright-field and fluorescence microscopy capabilities in addition to a highly focused UV laser beam that is utilized for laser ablation of samples. With this system, material laser ablated from a sample using the microscope was caught by a liquid vortex capture probe and transported in solution for analysis by electrospray ionization mass spectrometry. Both lane scanning and spot sampling mass spectral imaging modes weremore » used. The smallest area the system was able to ablate was ~0.544 μm × ~0.544 μm, achieved by oversampling of the smallest laser ablation spot size that could be obtained (~1.9 μm). With use of a model photoresist surface, known features as small as ~1.5 μm were resolved. The capabilities of the system with real world samples were demonstrated first with a blended polymer thin film containing poly(2-vinylpyridine) and poly(N-vinylcarbazole). Using spot sampling imaging, sub-micrometer sized features (0.62, 0.86, and 0.98 μm) visible by optical microscopy were clearly distinguished in the mass spectral images. A second real world example showed the imaging of trace amounts of cocaine in mouse brain thin tissue sections. Lastly, with use of a lane scanning mode with ~6 μm × ~6 μm data pixels, features in the tissue as small as 15 μm in size could be distinguished in both the mass spectral and optical images.« less

Characterization of a Hybrid Optical Microscopy/Laser Ablation Liquid Vortex Capture/Electrospray Ionization System for Mass Spectrometry Imaging

DOE PAGES

Cahill, John F.; Kertesz, Vilmos; Van Berkel, Gary J.

2015-10-22

Herein, a commercial optical microscope, laser microdissection instrument was coupled with an electrospray ionization mass spectrometer via a low profile liquid vortex capture probe to yield a hybrid optical microscopy/mass spectrometry imaging system. The instrument has bright-field and fluorescence microscopy capabilities in addition to a highly focused UV laser beam that is utilized for laser ablation of samples. With this system, material laser ablated from a sample using the microscope was caught by a liquid vortex capture probe and transported in solution for analysis by electrospray ionization mass spectrometry. Both lane scanning and spot sampling mass spectral imaging modes weremore » used. The smallest area the system was able to ablate was ~0.544 μm × ~0.544 μm, achieved by oversampling of the smallest laser ablation spot size that could be obtained (~1.9 μm). With use of a model photoresist surface, known features as small as ~1.5 μm were resolved. The capabilities of the system with real world samples were demonstrated first with a blended polymer thin film containing poly(2-vinylpyridine) and poly(N-vinylcarbazole). Using spot sampling imaging, sub-micrometer sized features (0.62, 0.86, and 0.98 μm) visible by optical microscopy were clearly distinguished in the mass spectral images. A second real world example showed the imaging of trace amounts of cocaine in mouse brain thin tissue sections. Lastly, with use of a lane scanning mode with ~6 μm × ~6 μm data pixels, features in the tissue as small as 15 μm in size could be distinguished in both the mass spectral and optical images.« less

Software Technology for Adaptable, Reliable Systems (STARS)

DTIC Science & Technology

1994-03-25

Tmeline(3), SECOMO(3), SEER(3), GSFC Software Engineering Lab Model(l), SLIM(4), SEER-SEM(l), SPQR (2), PRICE-S(2), internally-developed models(3), APMSS(1...3 " Timeline - 3 " SASET (Software Architecture Sizing Estimating Tool) - 2 " MicroMan 11- 2 * LCM (Logistics Cost Model) - 2 * SPQR - 2 * PRICE-S - 2

Modeling end-of-life pathways of construction and demolition debris in the United States (poster for LCM 2017)

EPA Science Inventory

In 2013, the United States generated 530 million tons of construction and demolition debris (CDD), 90% of which was related to demolition. Despite this major contribution to national waste streams, Life Cycle Assessment (LCA) studies and product declarations of buildings and buil...

Rapid determination of five antibiotic residues in swine wastewater by online solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry.

PubMed

Tagiri-Endo, Misako; Suzuki, Shigeru; Nakamura, Tomoyuki; Hatakeyama, Takashi; Kawamukai, Kazuo

2009-02-01

A simple and quick online solid-phase extraction (SPE) coupled to liquid chromatography (LC)/tandem mass spectrometry (MS/MS) for the determination of the five antibiotics (florfenicol, FF; lincomycin, LCM; oxytetracyclin, OTC; tylosin, TS; valnemulin, VLM) in swine wastewater has been developed. After filtration, aliquots (100 microl) of wastewater samples were directly injected to a column-switching LC system. Some matrix interference was removed by washing up SPE column with 0.2% formic acid solution and acetonitrile. Antibiotics eluted from SPE column were separated on analytical column by converting switching valve and were detected by MS/MS. Calibration curves using the method of standard addition had very good correlation coefficients (r > 0.99) in the range of 0.1 to 2 ng/ml. The intra-day precision of the method was less than 12% and the inter-day precision was between 6 to 17%. The detection limits were 0.01-0.1 ng/ml. When this method was applied to wastewater samples in swine facilities, four compounds (LCM, OTC, TS, and VLM) were detected.

Targeting Pancreatic Islets with Phage Display Assisted by Laser Pressure Catapult Microdissection

PubMed Central

Yao, Virginia J.; Ozawa, Michael G.; Trepel, Martin; Arap, Wadih; McDonald, Donald M.; Pasqualini, Renata

2005-01-01

Heterogeneity of the microvasculature in different organs has been well documented by multiple methods including in vivo phage display. However, less is known about the diversity of blood vessels within functionally distinct regions of organs. Here, we combined in vivo phage display with laser pressure catapult microdissection to identify peptide ligands for vascular receptors in the islets of Langerhans in the murine pancreas. Protein database analyses of the peptides, CVSNPRWKC and CHVLWSTRC, showed sequence identity to two ephrin A-type ligand homologues, A2 and A4. Confocal microscopy confirmed that most immunoreactivity of CVSNPRWKC and CHVLWSTRC phage was associated with blood vessels in pancreatic islets. Antibodies recognizing EphA4, a receptor for ephrin-A ligands, were similarly associated with islet blood vessels. Importantly, binding of both islet-homing phage and anti-EphA4 antibody was strikingly increased in blood vessels of pancreatic islet tumors in RIP-Tag2 transgenic mice. These results indicate that endothelial cells of blood vessels in pancreatic islets preferentially express EphA4 receptors, and this expression is increased in tumors. Our findings show in vivo phage display and laser pressure catapult microdissection can be combined to reveal endothelial cell specialization within focal regions of the microvasculature. PMID:15681844

Increased expression of ADAM 9 and ADAM 15 mRNA in pancreatic cancer.

PubMed

Yamada, Daisuke; Ohuchida, Kenoki; Mizumoto, Kazuhiro; Ohhashi, Seiji; Yu, Jun; Egami, Takuya; Fujita, Hayato; Nagai, Eishi; Tanaka, Masao

2007-01-01

A disintegrin and metalloproteases (ADAMs) comprise a multifunctional family of membrane-anchored proteins. ADAM 9 and ADAM 15 are involved in cell migration and invasion. Expression of ADAM 9 and ADAM 15 was reported to be altered in several types of cancer. Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure the expression of ADAM 9 mRNA in bulk pancreatic tissues. Results showed no significant difference in the expression of ADAM 9 mRNA between pancreatic cancer and non-neoplastic pancreas. Primary cultured pancreatic fibroblasts also expressed ADAM 9 mRNA. Therefore, a laser microdissection and pressure catapulting technique was employed to isolate cancer cells from tumor tissues. The expression of ADAM 9 and ADAM 15 mRNA was measured in microdissected samples (cancer cells, n = 11; normal epithelial cells, n = 13 for ADAM 9; cancer cells, n = 9; normal epithelial cells, n = 9 for ADAM 15). Pancreatic cancer cells expressed significantly higher levels of ADAM 9 and ADAM 15 mRNA than did normal pancreatic epithelial cells (p = 0.016 for ADAM 9; p = 0.004 for ADAM 15). ADAM 9 and ADAM 15 are involved in pancreatic cancer. Microdissection-based analysis appears to be indispensable for the accurate analysis of the expression of certain ADAM family members in pancreatic cancer.

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

PubMed

Bova, G Steven; Eltoum, Isam A; Kiernan, John A; Siegal, Gene P; Frost, Andra R; Best, Carolyn J M; Gillespie, John W; Su, Gloria H; Emmert-Buck, Michael R

2005-02-01

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.

Optimal molecular profiling of tissue and tissue components: defining the best processing and microdissection methods for biomedical applications.

PubMed

Rodriguez-Canales, Jaime; Hanson, Jeffrey C; Hipp, Jason D; Balis, Ulysses J; Tangrea, Michael A; Emmert-Buck, Michael R; Bova, G Steven

2013-01-01

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This updated chapter reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high-quality, appropriately anatomically tagged scientific results. Improvement in this area will significantly increase life science quality and productivity. The chapter is divided into introduction, materials, protocols, and notes subheadings. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this chapter, readers are advised to read through the entire chapter first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.

The value of mutational profiling of the cytocentrifugation supernatant fluid from fine-needle aspiration of pancreatic solid mass lesions.

PubMed

Deftereos, Georgios; Finkelstein, Sydney D; Jackson, Sara A; Ellsworth, Eric M G; Krishnamurti, Uma; Liu, Yulin; Silverman, Jan F; Binkert, Candy R; Ujevich, Beth A; Mohanty, Alok

2014-04-01

Fine-needle aspiration (FNA) of pancreatic solid masses can be significantly impacted by sampling variation. Molecular analysis of tumor DNA can be an aid for more definitive diagnosis. The aim of this study was to evaluate how molecular analysis of the cell-free cytocentrifugation supernatant DNA can help reduce sampling variability and increase diagnostic yield. Twenty-three FNA smears from pancreatic solid masses were performed. Remaining aspirates were rinsed for preparation of cytocentrifuged slides or cell blocks. DNA was extracted from supernatant fluid and assessed for DNA quantity spectrophotometrically and for amplifiability by quantitative PCR (qPCR). Supernatants with adequate DNA were analyzed for mutations using PCR/capillary electrophoresis for a broad panel of markers (KRAS point mutation by sequencing, microsatellite fragment analysis for loss of heterozygosity (LOH) of 16 markers at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, and 22q). In selected cases, microdissection of stained cytology smears and/or cytocentrifugation cellular slides were analyzed and compared. In all, 5/23 samples cytologically confirmed as adenocarcinoma showed detectable mutations both in the microdissected slide-based cytology cells and in the cytocentrifugation supernatant. While most mutations detected were present in both microdissected slides and supernatant fluid specimens, the latter showed additional mutations supporting greater sensitivity for detecting relevant DNA damage. Clonality for individual marker mutations was higher in the supernatant fluid than in microdissected cells. Cytocentrifugation supernatant fluid contains levels of amplifiable DNA suitable for mutation detection and characterization. The finding of additional detectable mutations at higher clonality indicates that supernatant fluid may be enriched with tumor DNA. Molecular analysis of the supernatant fluid could serve as an adjunct method to reduce sampling variability and increase diagnostic yield, especially in cases with a high clinical suspicion for malignancy and limited number of atypical cells in the smears.

Radiation sources with planar wire arrays and planar foils for inertial confinement fusion and high energy density physics research

NASA Astrophysics Data System (ADS)

Kantsyrev, V. L.; Chuvatin, A. S.; Safronova, A. S.; Rudakov, L. I.; Esaulov, A. A.; Velikovich, A. L.; Shrestha, I.; Astanovitsky, A.; Osborne, G. C.; Shlyaptseva, V. V.; Weller, M. E.; Keim, S.; Stafford, A.; Cooper, M.

2014-03-01

This article reports on the joint success of two independent lines of research, each of them being a multi-year international effort. One of these is the development of innovative sources, such as planar wire arrays (PWAs). PWAs turned out to be a prolific radiator, which act mainly as a resistor, even though the physical mechanism of efficient magnetic energy conversion into radiation still remains unclear. We review the results of our extensive studies of PWAs. We also report the new results of the experimental comparison PWAs with planar foil liners (another promising alternative to wire array loads at multi-mega-ampere generators). Pioneered at UNR, the PWA Z-pinch loads have later been tested at the Sandia National Laboratories (SNL) on the Saturn generator, on GIT-12 machine in Russia, and on the QiangGuang-1 generator in China, always successfully. Another of these is the drastic improvement in energy efficiency of pulsed-power systems, which started in early 1980s with Zucker's experiments at Naval Research Laboratory (NRL). Successful continuation of this approach was the Load Current Multiplier (LCM) proposed by Chuvatin in collaboration with Rudakov and Weber from NRL. The 100 ns LCM was integrated into the Zebra generator, which almost doubled the plasma load current, from 0.9 to 1.7 MA. The two above-mentioned innovative approaches were used in combination to produce a new compact hohlraum radiation source for ICF, as jointly proposed by SNL and UNR [Jones et al., Phys. Rev. Lett. 104, 125001 (2010)]. The first successful proof-of-the-principle experimental implementation of new hohlraum concept at university-scale generator Zebra/LCM is demonstrated. A numerical simulation capability with VisRaD code (from PRISM Co.) established at UNR allowed for the study of hohlraum coupling physics and provides the possibility of optimization of a new hohlraum. Future studies are discussed.

Using System Mass (SM), Equivalent Mass (EM), Equivalent System Mass (ESM) or Life Cycle Mass (LCM) in Advanced Life Support (ALS) Reporting

NASA Technical Reports Server (NTRS)

Jones, Harry

2003-01-01

The Advanced Life Support (ALS) has used a single number, Equivalent System Mass (ESM), for both reporting progress and technology selection. ESM is the launch mass required to provide a space system. ESM indicates launch cost. ESM alone is inadequate for technology selection, which should include other metrics such as Technology Readiness Level (TRL) and Life Cycle Cost (LCC) and also consider perfom.arxe 2nd risk. ESM has proven difficult to implement as a reporting metric, partly because it includes non-mass technology selection factors. Since it will not be used exclusively for technology selection, a new reporting metric can be made easier to compute and explain. Systems design trades-off performance, cost, and risk, but a risk weighted cost/benefit metric would be too complex to report. Since life support has fixed requirements, different systems usually have roughly equal performance. Risk is important since failure can harm the crew, but it is difficult to treat simply. Cost is not easy to estimate, but preliminary space system cost estimates are usually based on mass, which is better estimated than cost. Amass-based cost estimate, similar to ESM, would be a good single reporting metric. The paper defines and compares four mass-based cost estimates, Equivalent Mass (EM), Equivalent System Mass (ESM), Life Cycle Mass (LCM), and System Mass (SM). EM is traditional in life support and includes mass, volume, power, cooling and logistics. ESM is the specifically defined ALS metric, which adds crew time and possibly other cost factors to EM. LCM is a new metric, a mass-based estimate of LCC measured in mass units. SM includes only the factors of EM that are originally measured in mass, the hardware and logistics mass. All four mass-based metrics usually give similar comparisons. SM is by far the simplest to compute and easiest to explain.

Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies

PubMed Central

2013-01-01

Background The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays. Methods Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at −80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection. Results The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA concentration of these samples ranged from 10.88 ng/12 μl to 25.8 ng/12 μl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded. Conclusions The optimised protocol for sample collection and laser microdissection improved the RNA yield of the insitu ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms. PMID:24160452

Pathogenesis of Rushton bodies: A novel hypothesis.

PubMed

Sarode, Gargi S; Sarode, Sachin C; Tupkari, Jagdish V; Deshmukh, Revati; Patil, Shankargouda

2016-08-01

Rushton bodies (RBs) are one of the characteristic features seen in the epithelial lining of odontogenic cysts mainly radicular, dentigerous and odontogenic keratocyst. It has two different histo-morphological appearances; granular and homogeneous. Although widely investigated, the exact pathogenesis and histogenesis of RBs is still an enigma. Many hypotheses were made in the literature but none has explained conceivably the two histo-morphological appearances of RBs and their association with inflammation. In the present paper the various pathogenesis for the formation of RBs proposed till date are discussed along with proposal for a novel hypothesis. The given hypothesis is mainly related to inflammation and its effect on pore size of basement membrane of odontogenic cystic epithelium. It explains RBs association with inflammation as well as existence of two histo-morphological appearances. The proposed hypothesis also justifies the RB's presence inside the lining epithelium of odontogenic cyst despite its hematogenous origin. Future studies are advocated for isolating RBs using laser capture microdissection and subsequent biochemical, histochemical and electron microscopic analysis to substantiate the proposed hypothesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Differential expression of growth factors at the cellular level in virus-infected brain

PubMed Central

Prosniak, Mikhail; Zborek, Anna; Scott, Gwen S.; Roy, Anirban; Phares, Timothy W.; Koprowski, Hilary; Hooper, D. Craig

2003-01-01

The contribution of host factors to rabies virus (RV) transcription/replication and axonal/transsynaptic spread is largely unknown. We previously identified several host genes that are up-regulated in the mouse brain during RV infection, including neuroleukin, which is involved in neuronal growth and survival, cell motility, and differentiation, and fibroblast growth factor homologous factor 4 (FHF4), which has been implicated in limb and nervous system development. In this study, we used real-time quantitative RT-PCR to assess the expression of mRNAs specific for neuroleukin, the two isoforms of FHF4 (FHF4-1a and -1b) encoded by the FHF4 gene, and N protein of RV in neurons and astrocytes isolated by laser capture microdissection from mouse brains infected with the laboratory-adapted RV strain CVS-N2c or with a street RV of silver-haired bat origin. Differences in the gene expression patterns suggest that the capacity of RV strains to infect nonneuronal cells and differentially modulate host gene expression may be important in virus replication and spread in the CNS. PMID:12736376

The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints

PubMed Central

Llosa, Nicolas J.; Cruise, Michael; Tam, Ada; Wick, Elizabeth C.; Hechenbleikner, Elizabeth M.; Taube, Janis M.; Blosser, Lee; Fan, Hongni; Wang, Hao; Luber, Brandon; Zhang, Ming; Papadopoulos, Nickolas; Kinzler, Kenneth W.; Vogelstein, Bert; Sears, Cynthia L.; Anders, Robert A.; Pardoll, Drew M.; Housseau, Franck

2014-01-01

We examined the immune microenvironment of primary colorectal cancer (CRC) using immunohistochemistry, laser capture microdissection/qRT-PCR, flow cytometry and functional analysis of tumor infiltrating lymphocytes. A subset of CRC displayed high infiltration with activated CD8+ CTL as well as activated Th1 cells characterized by IFN-γ production and the Th1 transcription factor Tbet. Parallel analysis of tumor genotypes revealed that virtually all of the tumors with this active Th1/CTL microenvironment had defects in mismatch repair, as evidenced by microsatellite instability (MSI). Counterbalancing this active Th1/CTL microenvironment, MSI tumors selectively demonstrated highly up-regulated expression of multiple immune checkpoints, including five – PD-1, PD-L1, CTLA-4, LAG-3 and IDO – currently being targeted clinically with inhibitors. These findings link tumor genotype with the immune microenvironment, and explain why MSI tumors are not naturally eliminated despite a hostile Th1/CTL microenvironment. They further suggest that blockade of specific checkpoints may be selectively efficacious in the MSI subset of CRC. PMID:25358689

Gene expression profiling at early organogenesis reveals both common and diverse mechanisms in foregut patterning

PubMed Central

Fagman, Henrik; Amendola, Elena; Parrillo, Luca; Zoppoli, Pietro; Marotta, Pina; Scarfò, Marzia; De Luca, Pasquale; de Carvalho, Denise Pires; Ceccarelli, Michele; De Felice, Mario; Di Lauro, Roberto

2011-01-01

The thyroid and lungs originate as neighboring bud shaped outgrowths from the midline of the embryonic foregut. When and how organ specific programs regulate development into structures of distinct shapes, positions and functions is incompletely understood. To characterize, at least in part, the genetic basis of these events, we have employed laser capture microdissection and microarray analysis to define gene expression in the mouse thyroid and lung primordia at E10.5. By comparing the transcriptome of each bud to that of the whole embryo as well as to each other, we broadly describe the genes that are preferentially expressed in each developing organ as well as those with an enriched expression common to both. The results thus obtained provide a valuable resource for further analysis of genes previously unrecognized to participate in thyroid and lung morphogenesis and to discover organ specific as well as common developmental mechanisms. As an initial step in this direction we describe a regulatory pathway involving the anti-apoptotic gene Bcl2 that controls cell survival in early thyroid development. PMID:21924257

The HVAC Challenges of Upgrading an Old Lab for High-end Light Microscopes

PubMed Central

Richard, R.; Martone, P.; Callahan, L.M.

2014-01-01

The University of Rochester Medical Center forms the centerpiece of the University of Rochester's health research, teaching, patient care, and community outreach missions. Within this large facility of over 5 million square feet, demolition and remodeling of existing spaces is a constant activity. With more than $145 million in federal research funding, lab space is frequently repurposed and renovated to support this work. The URMC Medical Center Facilities Organization supporting small to medium space renovations is constantly challenged and constrained by the existing mechanical infrastructure and budgets to deliver a renovated space that functions within the equipment environmental parameters. One recent project, sponsored by the URMC Shared Resources Laboratory, demonstrates these points. The URMC Light Microscopy Shared Resource Laboratory requested renovation of a 121 sq. ft. room in a 40 year old building which would enable placement of a laser capture microdissection microscope and a Pascal 5 laser scanning confocal microscope with the instruments separated by a blackout curtain. This poster discusses the engineering approach implemented to bring an older lab into the environmental specifications needed for the proper operation of the high-end light microscopes.

Inflammatory signaling in human tuberculosis granulomas is spatially organized.

PubMed

Marakalala, Mohlopheni J; Raju, Ravikiran M; Sharma, Kirti; Zhang, Yanjia J; Eugenin, Eliseo A; Prideaux, Brendan; Daudelin, Isaac B; Chen, Pei-Yu; Booty, Matthew G; Kim, Jin Hee; Eum, Seok Yong; Via, Laura E; Behar, Samuel M; Barry, Clifton E; Mann, Matthias; Dartois, Véronique; Rubin, Eric J

2016-05-01

Granulomas are the pathological hallmark of tuberculosis (TB). However, their function and mechanisms of formation remain poorly understood. To understand the role of granulomas in TB, we analyzed the proteomes of granulomas from subjects with tuberculosis in an unbiased manner. Using laser-capture microdissection, mass spectrometry and confocal microscopy, we generated detailed molecular maps of human granulomas. We found that the centers of granulomas have a pro-inflammatory environment that is characterized by the presence of antimicrobial peptides, reactive oxygen species and pro-inflammatory eicosanoids. Conversely, the tissue surrounding the caseum has a comparatively anti-inflammatory signature. These findings are consistent across a set of six human subjects and in rabbits. Although the balance between systemic pro- and anti-inflammatory signals is crucial to TB disease outcome, here we find that these signals are physically segregated within each granuloma. From the protein and lipid snapshots of human and rabbit lesions analyzed here, we hypothesize that the pathologic response to TB is shaped by the precise anatomical localization of these inflammatory pathways during the development of the granuloma.

Immunohistochemical localization of hepatopancreatic phospholipase in gastropods mollusc, Littorina littorea and Buccinum undatum digestive cells

PubMed Central

2011-01-01

Background Among the digestive enzymes, phospholipase A2 (PLA2) hydrolyzes the essential dietary phospholipids in marine fish and shellfish. However, we know little about the organs that produce PLA2, and the ontogeny of the PLA2-cells. Accordingly, accurate localization of PLA2 in marine snails might afford a better understanding permitting the control of the quality and composition of diets and the mode of digestion of lipid food. Results We have previously producted an antiserum reacting specifically with mSDPLA2. It labeled zymogen granules of the hepatopancreatic acinar cells and the secretory materials of certain epithelial cells in the depths of epithelial crypts in the hepatopancreas of snail. To confirm this localization a laser capture microdissection was performed targeting stained cells of hepatopancreas tissue sections. A Western blot analysis revealed a strong signal at the expected size (30 kDa), probably corresponding to the PLA2. Conclusions The present results support the presence of two hepatopancreatic intracellular and extracellular PLA2 in the prosobranchs gastropods molluscs, Littorina littorea and Buccinum undatum and bring insights on their localizations. PMID:22114916

The detection of Propionibacterium acnes signatures in granulomas of lupus miliaris disseminatus faciei.

PubMed

Nishimoto, Junko; Amano, Masahiro; Setoyama, Mitsuru

2015-04-01

Lupus miliaris disseminatus faciei (LMDF) is a papular eruption that occurs on adults' faces, predominantly on the lower eyelids. Histologically, the granulomatous lesions are primarily situated around the hair follicles, particularly the superficial region/infundibula. Its etiology remains to be elucidated. Recently, Propionibacterium acnes (P. acnes) has been suspected as a cause of sarcoidosis. In light of the sarcoid-like reactions that are present in LMDF, we hypothesized that P. acnes may also be implicated in granulomas associated with the disease. We evaluated nine DNA samples from granulomatous lesions from the skin of patients with LMDF. We used laser capture microdissection to extract DNA from these regions. Polymerase chain reaction was performed to amplify segments of the 16S ribosomal RNA of P. acnes, and the P. acnes gene was clearly detectable in all nine DNA samples. The gene was also detected in samples from normal-appearing skin, but these bands were faint in all samples. The results of the present study suggest that P. acnes plays a pathogenetic roles in LMDF. © 2015 Japanese Dermatological Association.

Automated Image Analysis Corrosion Working Group Update: February 1, 2018

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

Wendelberger, James G.

These are slides for the automated image analysis corrosion working group update. The overall goals were: automate the detection and quantification of features in images (faster, more accurate), how to do this (obtain data, analyze data), focus on Laser Scanning Confocal Microscope (LCM) data (laser intensity, laser height/depth, optical RGB, optical plus laser RGB).

Defense ADP Acquisition Study.

DTIC Science & Technology

1981-11-30

Logistics ALS - Advanced Logistics System AMP - ADPS Master Plan ANSI - American National Standards Institute APR - Agency Procurement Request ASD(C...Computers IRM - Information Resources Management ISO - International Standards Organization L LCC - Life Cycle Costs LCM - Life Cycle Management LE...man- agement in the process * Lack of a mission orientation . Lack of systems management and life cycle perspectives * Lack of effective leadership

When Cognitive Diagnosis Meets Computerized Adaptive Testing: CD-CAT

ERIC Educational Resources Information Center

Cheng, Ying

2009-01-01

Computerized adaptive testing (CAT) is a mode of testing which enables more efficient and accurate recovery of one or more latent traits. Traditionally, CAT is built upon Item Response Theory (IRT) models that assume unidimensionality. However, the problem of how to build CAT upon latent class models (LCM) has not been investigated until recently,…

LC/MSMS STUDY OF BENZO[A]PYRENE-7,8-QUINONE ADDUCTION TO GLOBIN TRYPTIC PEPTIDES AND N-ACETYLAMINO ACIDS

EPA Science Inventory

Benzo[a]pyrene-7,8-quinone (BPQ) is regarded as a reactive genotoxic compound enzymatically formed from a xenobiotic precursor benzo[a]pyrene-7,8-diol by aldo-keto-reductase family of enzymes. Because BPQ, a Michael electrophile, was previously shown to react with oligonucleotide...

The Moderating Capacity of Racial Identity between Perceived Discrimination and Psychological Well-Being over Time among African American Youth

ERIC Educational Resources Information Center

Seaton, Eleanor K.; Neblett, Enrique W.; Upton, Rachel D.; Hammond, Wizdom Powell; Sellers, Robert M.

2011-01-01

This study examined the influence of racial identity in the longitudinal relation between perceptions of racial discrimination and psychological well-being for approximately 560 African American youth. Latent curve modeling (LCM) and parallel process multiple-indicator LCMs with latent moderators were used to assess whether perceptions of racial…

Efficient high-throughput sequencing of a laser microdissected chromosome arm

PubMed Central

2013-01-01

Background Genomic sequence assemblies are key tools for a broad range of gene function and evolutionary studies. The diploid amphibian Xenopus tropicalis plays a pivotal role in these fields due to its combination of experimental flexibility, diploid genome, and early-branching tetrapod taxonomic position, having diverged from the amniote lineage ~360 million years ago. A genome assembly and a genetic linkage map have recently been made available. Unfortunately, large gaps in the linkage map attenuate long-range integrity of the genome assembly. Results We laser dissected the short arm of X. tropicalis chromosome 7 for next generation sequencing and computational mapping to the reference genome. This arm is of particular interest as it encodes the sex determination locus, but its genetic map contains large gaps which undermine available genome assemblies. Whole genome amplification of 15 laser-microdissected 7p arms followed by next generation sequencing yielded ~35 million reads, over four million of which uniquely mapped to the X. tropicalis genome. Our analysis placed more than 200 previously unmapped scaffolds on the analyzed chromosome arm, providing valuable low-resolution physical map information for de novo genome assembly. Conclusion We present a new approach for improving and validating genetic maps and sequence assemblies. Whole genome amplification of 15 microdissected chromosome arms provided sufficient high-quality material for localizing previously unmapped scaffolds and genes as well as recognizing mislocalized scaffolds. PMID:23714049

The Cause of Death of a Child in the 18th Century Solved by Bone Microbiome Typing Using Laser Microdissection and Next Generation Sequencing.

PubMed

D'Argenio, Valeria; Torino, Marielva; Precone, Vincenza; Casaburi, Giorgio; Esposito, Maria Valeria; Iaffaldano, Laura; Malapelle, Umberto; Troncone, Giancarlo; Coto, Iolanda; Cavalcanti, Paolina; De Rosa, Gaetano; Salvatore, Francesco; Sacchetti, Lucia

2017-01-06

The history of medicine abounds in cases of mysterious deaths, especially by infectious diseases, which were probably unresolved because of the lack of knowledge and of appropriate technology. The aim of this study was to exploit contemporary technologies to try to identify the cause of death of a young boy who died from a putative "infection" at the end of the 18th century, and for whom an extraordinarily well-preserved minute bone fragment was available. After confirming the nature of the sample, we used laser microdissection to select the most "informative" area to be examined. Tissue genotyping indicated male gender, thereby confirming the notary's report. 16S ribosomal RNA sequencing showed that Proteobacteria and Actinobacteria were more abundant than Firmicutes and Bacteroidetes , and that Pseudomonas was the most abundant bacterial genus in the Pseudomonadaceae family. These data suggest that the patient most likely died from Pseudomonas osteomyelitis. This case is an example of how new technological approaches, like laser microdissection and next-generation sequencing, can resolve ancient cases of uncertain etiopathology. Lastly, medical samples may contain a wealth of information that may not be accessible until more sophisticated technology becomes available. Therefore, one may envisage the possibility of systematically storing medical samples for evaluation by future generations.

Anatomy of the temporomandibular joint in the cat: a study by microdissection, cryosection and vascular injection.

PubMed

Arredondo, Jorge; Agut, Amalia; Rodríguez, María Jesús; Sarriá, Ricardo; Latorre, Rafael

2013-02-01

The minute anatomy of the temporomandibular joint (TMJ) is of great clinical relevance in cats owing to a high number of lesions involving this articulation. However, the precise anatomy is poorly documented in textbooks and scientific articles. The aim of this study was to describe, in detail, the TMJ anatomy and its relationship with other adjacent anatomical structures in the cat. Different anatomical preparations, including vascular and articular injection, microdissection, cryosection and plastination, were performed in 12 cadaveric cats. All TMJ anatomical structures were identified and described in detail. A thorough understanding of the TMJ anatomy is essential to understand the clinical signs associated with TMJ disorders, to locate lesions precisely and to accurately interpret the results in all diagnostic imaging techniques.

UV-laser microdissection and mRNA expression analysis of individual neurons from postmortem Parkinson's disease brains.

PubMed

Gründemann, Jan; Schlaudraff, Falk; Liss, Birgit

2011-01-01

Cell specificity of gene expression analysis is essential to avoid tissue sample related artifacts, in particular when the relative number of target cells present in the compared tissues varies dramatically, e.g., when comparing dopamine neurons in midbrain tissues from control subjects with those from Parkinson's disease (PD) cases. Here, we describe a detailed protocol that combines contact-free UV-laser microdissection and quantitative PCR of reverse-transcribed RNA of individual neurons from postmortem human midbrain tissue from PD patients and unaffected controls. Among expression changes in a variety of dopamine neuron marker, maintenance, and cell-metabolism genes, we found that α-synuclein mRNA levels were significantly elevated in individual neuromelanin-positive dopamine midbrain neurons from PD brains when compared to those from matched controls.

Determination of differential gene expression profiles in superficial and deeper zones of mature rat articular cartilage using RNA sequencing of laser microdissected tissue specimens.

PubMed

Mori, Yoshifumi; Chung, Ung-Il; Tanaka, Sakae; Saito, Taku

2014-01-01

Superficial zone (SFZ) cells, which are morphologically and functionally distinct from chondrocytes in deeper zones, play important roles in the maintenance of articular cartilage. Here, we established an easy and reliable method for performance of laser microdissection (LMD) on cryosections of mature rat articular cartilage using an adhesive membrane. We further examined gene expression profiles in the SFZ and the deeper zones of articular cartilage by performing RNA sequencing (RNA-seq). We validated sample collection methods, RNA amplification and the RNA-seq data using real-time RT-PCR. The combined data provide comprehensive information regarding genes specifically expressed in the SFZ or deeper zones, as well as a useful protocol for expression analysis of microsamples of hard tissues.

Service-Oriented Architecture Approach to MAGTF Logistics Support Systems

DTIC Science & Technology

2013-09-01

Support System-Marine Corps IT Information Technology KPI Key Performance Indicators LCE Logistics Command Element ITV In-transit Visibility LCM...building blocks, options, KPI (key performance indicators), design decisions and the corresponding; the physical attributes which is the second attribute... KPI ) that they impact. h. Layer 8 (Information Architecture) The business intelligence layer and information architecture safeguards the inclusion

Single High Fidelity Geometric Data Sets for LCM - Model Requirements

DTIC Science & Technology

2006-11-01

are extensive single 3D CAD data models incorporating hull structure, propulsion, steering, piping , electrical, HVAC and other systems, which make...single 3D CAD data models incorporating hull structure, propulsion, steering, piping , electrical, HVAC and other systems. During this same period...be sufficiently flexible to accommodate the diverse requirements of various types of structural analyses. Section Properties & Material Data

Expectation Maximization and its Application in Modeling, Segmentation and Anomaly Detection

DTIC Science & Technology

2008-05-01

ocomplNc <la!a rrot>lcm,. ",., i’lCOll\\l>lc,c,ICSS of Ihc dala mayan "" IIuc lu missing dala. (J,,,,,,.,ed di,nibu!ions . elc . 0"" such c • ..- is a...Estimation Techniques in Computer Huiyan, Z., Yongfeng, C., Wen, Y. SAR Image Segmentation Using MPM Constrained Stochastic Relaxation. Civil Engineering

The Impact of Ignoring the Level of Nesting Structure in Nonparametric Multilevel Latent Class Models

ERIC Educational Resources Information Center

Park, Jungkyu; Yu, Hsiu-Ting

2016-01-01

The multilevel latent class model (MLCM) is a multilevel extension of a latent class model (LCM) that is used to analyze nested structure data structure. The nonparametric version of an MLCM assumes a discrete latent variable at a higher-level nesting structure to account for the dependency among observations nested within a higher-level unit. In…

The use of advanced steam reforming technology for hydrogen production

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

Abbishaw, J.B.; Cromarty, B.J.

1996-12-01

The demand for supplementary hydrogen production in refineries is growing significantly world-wide as environmental legislation concerning cleaner gasoline and diesel fuels is introduced. The main manufacturing method is by steam reforming. The process has been developed both to reduce the capital cost and increase efficiency, reliability and ease of operation. ICI Katalco`s Leading Concept Hydrogen or LCH process continues this process of improvement by replacing the conventional fired steam reformer with a type of heat exchange reformer known as the Gas Heated Reformer or GHR. The GHR was first used in the Leading Concept Ammonia process, LCA at ICI`s manufacturingmore » site at Severnside, England and commissioned in 1988 and later in the Leading Concept Methanol (LCM) process for methanol at Melbourne, Australia and commissioned in 1994. The development of the LCH process follows on from both LCA and LCM processes. This paper describes the development and use of the GHR in steam reforming, and shows how the GHR can be used in LCH. A comparison between the LCH process and a conventional hydrogen plant is given, showing the benefits of the LCH process in certain circumstances.« less

Air compressor battery duration with mechanical ventilation in a field anesthesia machine.

PubMed

Szpisjak, Dale F; Giberman, Anthony A

2015-05-01

Compressed air to power field anesthesia machine ventilators may be supplied by air compressor with battery backup. This study determined the battery duration when the compPAC ventilator's air compressor was powered by NiCd battery to ventilate the Vent Aid Training Test Lung modeling high (HC = 0.100 L/cm H2O) and low (LC = 0.020 L/cm H2O) pulmonary compliance. Target tidal volumes (VT) were 500, 750, and 1,000 mL. Respiratory rate = 10 bpm, inspiratory-to-expiratory time ratio = 1:2, and fresh gas flow = 1 L/min air. N = 5 in each group. Control limits were determined from the first 150 minutes of battery power for each run and lower control limit = mean VT - 3SD. Battery depletion occurred when VT was below the lower control limit. Battery duration ranged from 185.8 (±3.2) minutes in the LC-1000 group to 233.3 (±3.6) minutes in the HC-750 group. Battery duration of the LC-1000 group was less than all others (p = 0.027). The differences among the non-LC-1000 groups were not clinically significant. Reprint & Copyright © 2015 Association of Military Surgeons of the U.S.

Translating Thermal Response of Triblock Copolymer Assemblies in Dilute Solution to Macroscopic Gelation and Phase Separation

DOE PAGES

Sun, Zhe; Tian, Ye; Hom, Wendy L.; ...

2016-12-28

The thermal response of semi-dilute solutions (5 w/w%) of two amphiphilic thermoresponsive poly(ethylene oxide)-b-poly(N,N-diethylacrylamide)-b-poly(N,N-dibutylacrylamide) (PEO 45-PDEAm x-PDBAm 12) triblock copolymers, which differ only in the size of the central responsive block, in water was examined in this paper. Aqueous PEO45-PDEAm41-PDBAm12 solutions, which undergo a thermally induced sphere-to-worm transition in dilute solution, were found to reversibly form soft (G'≈10 Pa) free-standing physical gels after 10 min at 55 °C. PEO 45-PDEAm 89-PDBAm 12 copolymer solutions, which undergo a thermally induced transition from spheres to large compound micelles (LCM) in dilute solution, underwent phase separation after heating at 55 °C for 10more » min owing to sedimentation of LCMs. The reversibility of LCM formation was investigated as a non-specific method for removal of a water-soluble dye from aqueous solution. Finally, the composition and size of the central responsive block in these polymers dictate the microscopic and macroscopic response of the polymer solutions as well as the rates of transition between assemblies.« less

Individual crypt genetic heterogeneity and the origin of metaplastic glandular epithelium in human Barrett’s oesophagus

PubMed Central

Leedham, S J; Preston, S L; McDonald, S A C; Elia, G; Bhandari, P; Poller, D; Harrison, R; Novelli, M R; Jankowski, J A; Wright, N A

2008-01-01

Objectives: Current models of clonal expansion in human Barrett’s oesophagus are based upon heterogenous, flow-purified biopsy analysis taken at multiple segment levels. Detection of identical mutation fingerprints from these biopsy samples led to the proposal that a mutated clone with a selective advantage can clonally expand to fill an entire Barrett’s segment at the expense of competing clones (selective sweep to fixation model). We aimed to assess clonality at a much higher resolution by microdissecting and genetically analysing individual crypts. The histogenesis of Barrett’s metaplasia and neo-squamous islands has never been demonstrated. We investigated the oesophageal gland squamous ducts as the source of both epithelial sub-types. Methods: Individual crypts across Barrett’s biopsy and oesophagectomy blocks were dissected. Determination of tumour suppressor gene loss of heterozygosity patterns, p16 and p53 point mutations were carried out on a crypt-by-crypt basis. Cases of contiguous neo-squamous islands and columnar metaplasia with oesophageal squamous ducts were identified. Tissues were isolated by laser capture microdissection and genetically analysed. Results: Individual crypt dissection revealed mutation patterns that were masked in whole biopsy analysis. Dissection across oesophagectomy specimens demonstrated marked clonal heterogeneity, with multiple independent clones present. We identified a p16 point mutation arising in the squamous epithelium of the oesophageal gland duct, which was also present in a contiguous metaplastic crypt, whereas neo-squamous islands arising from squamous ducts were wild-type with respect to surrounding Barrett’s dysplasia. Conclusions: By studying clonality at the crypt level we demonstrate that Barrett’s heterogeneity arises from multiple independent clones, in contrast to the selective sweep to fixation model of clonal expansion previously described. We suggest that the squamous gland ducts situated throughout the oesophagus are the source of a progenitor cell that may be susceptible to gene mutation resulting in conversion to Barrett’s metaplastic epithelium. Additionally, these data suggest that wild-type ducts may be the source of neo-squamous islands. PMID:18305067

CMAPS_ASPS_DATA-LCM_GTC

EPA Pesticide Factsheets

Total mercury in precipitation collected using ASPS automated wet-only instrument and analyzed by cold vapor atomic fluorescence spectroscopy.This dataset is associated with the following publication:Lynam, M., J.T. Dvonch, J. Barres, M. Landis , and A. Kamal. Investigating the impact of local urban sources on total atmospheric mercury wet deposition in Cleveland, Ohio, USA. ATMOSPHERIC ENVIRONMENT. Elsevier Science Ltd, New York, NY, USA, 127: 262-271, (2016).

The Impact of Different CD4 Cell-Count Monitoring and Switching Strategies on Mortality in HIV-Infected African Adults on Antiretroviral Therapy: An Application of Dynamic Marginal Structural Models

PubMed Central

Ford, Deborah; Robins, James M.; Petersen, Maya L.; Gibb, Diana M.; Gilks, Charles F.; Mugyenyi, Peter; Grosskurth, Heiner; Hakim, James; Katabira, Elly; Babiker, Abdel G.; Walker, A. Sarah

2015-01-01

In Africa, antiretroviral therapy (ART) is delivered with limited laboratory monitoring, often none. In 2003–2004, investigators in the Development of Antiretroviral Therapy in Africa (DART) Trial randomized persons initiating ART in Uganda and Zimbabwe to either laboratory and clinical monitoring (LCM) or clinically driven monitoring (CDM). CD4 cell counts were measured every 12 weeks in both groups but were only returned to treating clinicians for management in the LCM group. Follow-up continued through 2008. In observational analyses, dynamic marginal structural models on pooled randomized groups were used to estimate survival under different monitoring-frequency and clinical/immunological switching strategies. Assumptions included no direct effect of randomized group on mortality or confounders and no unmeasured confounders which influenced treatment switch and mortality or treatment switch and time-dependent covariates. After 48 weeks of first-line ART, 2,946 individuals contributed 11,351 person-years of follow-up, 625 switches, and 179 deaths. The estimated survival probability after a further 240 weeks for post-48-week switch at the first CD4 cell count less than 100 cells/mm3 or non-Candida World Health Organization stage 4 event (with CD4 count <250) was 0.96 (95% confidence interval (CI): 0.94, 0.97) with 12-weekly CD4 testing, 0.96 (95% CI: 0.95, 0.97) with 24-weekly CD4 testing, 0.95 (95% CI: 0.93, 0.96) with a single CD4 test at 48 weeks (baseline), and 0.92 (95% CI: 0.91, 0.94) with no CD4 testing. Comparing randomized groups by 48-week CD4 count, the mortality risk associated with CDM versus LCM was greater in persons with CD4 counts of <100 (hazard ratio = 2.4, 95% CI: 1.3, 4.3) than in those with CD4 counts of ≥100 (hazard ratio = 1.1, 95% CI: 0.8, 1.7; interaction P = 0.04). These findings support a benefit from identifying patients immunologically failing first-line ART at 48 weeks. PMID:26316598

Sources of Uncertainty in the Prediction of LAI / fPAR from MODIS

NASA Technical Reports Server (NTRS)

Dungan, Jennifer L.; Ganapol, Barry D.; Brass, James A. (Technical Monitor)

2002-01-01

To explicate the sources of uncertainty in the prediction of biophysical variables over space, consider the general equation: where z is a variable with values on some nominal, ordinal, interval or ratio scale; y is a vector of input variables; u is the spatial support of y and z ; x and u are the spatial locations of y and z , respectively; f is a model and B is the vector of the parameters of this model. Any y or z has a value and a spatial extent which is called its support. Viewed in this way, categories of uncertainty are from variable (e.g. measurement), parameter, positional. support and model (e.g. structural) sources. The prediction of Leaf Area Index (LAI) and the fraction of absorbed photosynthetically active radiation (fPAR) are examples of z variables predicted using model(s) as a function of y variables and spatially constant parameters. The MOD15 algorithm is an example of f, called f(sub 1), with parameters including those defined by one of six biome types and solar and view angles. The Leaf Canopy Model (LCM)2, a nested model that combines leaf radiative transfer with a full canopy reflectance model through the phase function, is a simpler though similar radiative transfer approach to f(sub 1). In a previous study, MOD15 and LCM2 gave similar results for the broadleaf forest biome. Differences between these two models can be used to consider the structural uncertainty in prediction results. In an effort to quantify each of the five sources of uncertainty and rank their relative importance for the LAI/fPAR prediction problem, we used recent data for an EOS Core Validation Site in the broadleaf biome with coincident surface reflectance, vegetation index, fPAR and LAI products from the Moderate Resolution Imaging Spectrometer (MODIS). Uncertainty due to support on the input reflectance variable was characterized using Landsat ETM+ data. Input uncertainties were propagated through the LCM2 model and compared with published uncertainties from the MOD15 algorithm.

Cost Effectiveness Analysis of Clinically Driven versus Routine Laboratory Monitoring of Antiretroviral Therapy in Uganda and Zimbabwe

PubMed Central

Medina Lara, Antonieta; Kigozi, Jesse; Amurwon, Jovita; Muchabaiwa, Lazarus; Nyanzi Wakaholi, Barbara; Mujica Mota, Ruben E.; Walker, A. Sarah; Kasirye, Ronnie; Ssali, Francis; Reid, Andrew; Grosskurth, Heiner; Babiker, Abdel G.; Kityo, Cissy; Katabira, Elly; Munderi, Paula; Mugyenyi, Peter; Hakim, James; Darbyshire, Janet; Gibb, Diana M.; Gilks, Charles F.

2012-01-01

Background Despite funding constraints for treatment programmes in Africa, the costs and economic consequences of routine laboratory monitoring for efficacy and toxicity of antiretroviral therapy (ART) have rarely been evaluated. Methods Cost-effectiveness analysis was conducted in the DART trial (ISRCTN13968779). Adults in Uganda/Zimbabwe starting ART were randomised to clinically-driven monitoring (CDM) or laboratory and clinical monitoring (LCM); individual patient data on healthcare resource utilisation and outcomes were valued with primary economic costs and utilities. Total costs of first/second-line ART, routine 12-weekly CD4 and biochemistry/haematology tests, additional diagnostic investigations, clinic visits, concomitant medications and hospitalisations were considered from the public healthcare sector perspective. A Markov model was used to extrapolate costs and benefits 20 years beyond the trial. Results 3316 (1660LCM;1656CDM) symptomatic, immunosuppressed ART-naive adults (median (IQR) age 37 (32,42); CD4 86 (31,139) cells/mm3) were followed for median 4.9 years. LCM had a mean 0.112 year (41 days) survival benefit at an additional mean cost of $765 [95%CI:685,845], translating into an adjusted incremental cost of $7386 [3277,dominated] per life-year gained and $7793 [4442,39179] per quality-adjusted life year gained. Routine toxicity tests were prominent cost-drivers and had no benefit. With 12-weekly CD4 monitoring from year 2 on ART, low-cost second-line ART, but without toxicity monitoring, CD4 test costs need to fall below $3.78 to become cost-effective (<3xper-capita GDP, following WHO benchmarks). CD4 monitoring at current costs as undertaken in DART was not cost-effective in the long-term. Conclusions There is no rationale for routine toxicity monitoring, which did not affect outcomes and was costly. Even though beneficial, there is little justification for routine 12-weekly CD4 monitoring of ART at current test costs in low-income African countries. CD4 monitoring, restricted to the second year on ART onwards, could be cost-effective with lower cost second-line therapy and development of a cheaper, ideally point-of-care, CD4 test. PMID:22545079

Isolation of guard cells from fresh epidermis using a piezo-power micro-dissection system with vibration-attenuated needles.

PubMed

Terpitz, Ulrich; Zimmermann, Dirk

2010-01-01

The Eppendorf Piezo-Power Microdissection (PPMD) system uses a tungsten needle (MicroChisel) oscillating in a forward-backward (vertical) mode to cut cells from surrounding tissue. This technology competes with laser-based dissection systems, which offer high accuracy and precision, but are more expensive and require fixed tissue. In contrast, PPMD systems can dissect freshly prepared tissue, but their accuracy and precision is lower due to unwanted lateral vibrations of the MicroChisel. Especially in tissues where elasticity is high, these vibrations can limit the cutting resolution or hamper the dissection. Here we describe a cost-efficient and simple glass capillary-encapsulation modification of MicroChisels for effective attenuation of lateral vibrations. The use of modified MicroChisels enables accurate and precise tissue dissection from highly elastic material.

Shared liver-like transcriptional characteristics in liver metastases and corresponding primary colorectal tumors.

PubMed

Cheng, Jun; Song, Xuekun; Ao, Lu; Chen, Rou; Chi, Meirong; Guo, You; Zhang, Jiahui; Li, Hongdong; Zhao, Wenyuan; Guo, Zheng; Wang, Xianlong

2018-01-01

Background & Aims : Primary tumors of colorectal carcinoma (CRC) with liver metastasis might gain some liver-specific characteristics to adapt the liver micro-environment. This study aims to reveal potential liver-like transcriptional characteristics associated with the liver metastasis in primary colorectal carcinoma. Methods: Among the genes up-regulated in normal liver tissues versus normal colorectal tissues, we identified "liver-specific" genes whose expression levels ranked among the bottom 10% ("unexpressed") of all measured genes in both normal colorectal tissues and primary colorectal tumors without metastasis. These liver-specific genes were investigated for their expressions in both the primary tumors and the corresponding liver metastases of seven primary CRC patients with liver metastasis using microdissected samples. Results: Among the 3958 genes detected to be up-regulated in normal liver tissues versus normal colorectal tissues, we identified 12 liver-specific genes and found two of them, ANGPTL3 and CFHR5 , were unexpressed in microdissected primary colorectal tumors without metastasis but expressed in both microdissected liver metastases and corresponding primary colorectal tumors (Fisher's exact test, P < 0.05). Genes co-expressed with ANGPTL3 and CFHR5 were significantly enriched in metabolism pathways characterizing liver tissues, including "starch and sucrose metabolism" and "drug metabolism-cytochrome P450". Conclusions: For primary CRC with liver metastasis, both the liver metastases and corresponding primary colorectal tumors may express some liver-specific genes which may help the tumor cells adapt the liver micro-environment.

In situ phosphorylation of proteins in MCTs microdissected from rat kidney: Effect of AVP

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

Homma, S.; Gapstur, S.M.; Yusufi, N.K.

1988-04-01

Adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP)-dependent protein phosphorylation is considered a key step in the cellular action of vasopressin (AVP) to regulate water permeability in collecting tubules. However, the proteins serving as a substrate(s) for phosphorylation in undisrupted cells have not yet been identified. In the present study, the authors developed a method for investigation of in situ phosphorylation of microdissected segments of medullary collecting tubules (MCT) from rat kidney. Incubation of microdissected MCT segments with low concentrations of saponin, semipermeabilization, increased permeability of the membrane for ATP but did not allow leakage of macromolecules such as lactate dehydrogenase. This treatment alsomore » did not cause major disruption of cell structure, or impairment of AVP-sensitive adenylate cyclase. Incubation of semipermeabilized MCT with {gamma}-({sup 32}P)ATP resulted in corporation of {sup 32}P{sub i} into two major protein bands detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent autoradiography. Similar incubation of tubules disrupted by hyposmotic solutions and a stronger detergent Triton X-100 resulted in {sup 32}P{sub i} incorporation into multiple protein bands. These findings demonstrate a novel method for identification of endogenous protein substrate(s) for cAMP-dependent protein kinase and other protein kinases and phosphatases that are probably involved in post-cAMP steps in the cellular action of AVP in the intact cells of collecting tubules.« less

A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens.

PubMed

Del Prete, D; Forino, M; Gambaro, G; D'Angelo, A; Baggio, B; Anglani, F

1998-01-01

Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.

The increase in the expression and hypomethylation of MUC4 gene with the progression of pancreatic ductal adenocarcinoma.

PubMed

Zhu, Yi; Zhang, Jing-jing; Zhu, Rong; Zhu, Yan; Liang, Wen-biao; Gao, Wen-tao; Yu, Jun-bo; Xu, Ze-kuan; Miao, Yi

2011-12-01

The MUC4 gene could have a key role in the progression of pancreatic cancer, but the quantitative measurement of its expression in clinical tissue samples remains a challenge. The correlations between MUC4 promoter methylation status in vivo and either pancreatic cancer progression or MUC4 mRNA expression need to be demonstrated. We used the techniques of quantitative real-time PCR and DNA methylation-specific PCR combined microdissection to precisely detect MUC4 expression and promoter methylation status in 116 microdissected foci from 57 patients with pancreatic ductal adenocarcinoma. Both mRNA expression and hypomethylation frequency increased from normal to precancerous lesions to pancreatic cancer. Multivariate Cox regression analysis showed that high-level MUC4 expression (P = 0.008) and tumor-node-metastasis staging (P = 0.038) were significant independent risk factors for predicting the prognosis of 57 patients. The MUC4 mRNA expression was not significantly correlated with promoter methylation status in 30 foci of pancreatic ductal adenocarcinoma. These results suggest that high mRNA expression and hypomethylation of the MUC4 gene could be involved in carcinogenesis and in the malignant development of pancreatic ductal adenocarcinoma. The MUC4 mRNA expression may become a new prognostic marker for pancreatic cancer. Microdissection-based quantitative real-time PCR and methylation-specific PCR contribute to the quantitative detection of MUC4 expression in clinical samples and reflect the epigenetic regulatory mechanisms of MUC4 in vivo.

Fungal Iron Availability during Deep Seated Candidiasis Is Defined by a Complex Interplay Involving Systemic and Local Events

PubMed Central

Potrykus, Joanna; Stead, David; MacCallum, Donna M.; Urgast, Dagmar S.; Raab, Andrea; van Rooijen, Nico; Feldmann, Jörg; Brown, Alistair J. P.

2013-01-01

Nutritional immunity – the withholding of nutrients by the host – has long been recognised as an important factor that shapes bacterial-host interactions. However, the dynamics of nutrient availability within local host niches during fungal infection are poorly defined. We have combined laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS), MALDI imaging and immunohistochemistry with microtranscriptomics to examine iron homeostasis in the host and pathogen in the murine model of systemic candidiasis. Dramatic changes in the renal iron landscape occur during disease progression. The infection perturbs global iron homeostasis in the host leading to iron accumulation in the renal medulla. Paradoxically, this is accompanied by nutritional immunity in the renal cortex as iron exclusion zones emerge locally around fungal lesions. These exclusion zones correlate with immune infiltrates and haem oxygenase 1-expressing host cells. This local nutritional immunity decreases iron availability, leading to a switch in iron acquisition mechanisms within mature fungal lesions, as revealed by laser capture microdissection and qRT-PCR analyses. Therefore, a complex interplay of systemic and local events influences iron homeostasis and pathogen-host dynamics during disease progression. PMID:24146619

Identification of a core set of rhizobial infection genes using data from single cell-types.

PubMed

Chen, Da-Song; Liu, Cheng-Wu; Roy, Sonali; Cousins, Donna; Stacey, Nicola; Murray, Jeremy D

2015-01-01

Genome-wide expression studies on nodulation have varied in their scale from entire root systems to dissected nodules or root sections containing nodule primordia (NP). More recently efforts have focused on developing methods for isolation of root hairs from infected plants and the application of laser-capture microdissection technology to nodules. Here we analyze two published data sets to identify a core set of infection genes that are expressed in the nodule and in root hairs during infection. Among the genes identified were those encoding phenylpropanoid biosynthesis enzymes including Chalcone-O-Methyltransferase which is required for the production of the potent Nod gene inducer 4',4-dihydroxy-2-methoxychalcone. A promoter-GUS analysis in transgenic hairy roots for two genes encoding Chalcone-O-Methyltransferase isoforms revealed their expression in rhizobially infected root hairs and the nodule infection zone but not in the nitrogen fixation zone. We also describe a group of Rhizobially Induced Peroxidases whose expression overlaps with the production of superoxide in rhizobially infected root hairs and in nodules and roots. Finally, we identify a cohort of co-regulated transcription factors as candidate regulators of these processes.

Photostick: a method for selective isolation of target cells from culture† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c4sc03676j Click here for additional data file.

PubMed Central

Chien, Miao-Ping; Werley, Christopher A.; Farhi, Samouil L.

2015-01-01

Sorting of target cells from a heterogeneous pool is technically difficult when the selection criterion is complex, e.g. a dynamic response, a morphological feature, or a combination of multiple parameters. At present, mammalian cell selections are typically performed either via static fluorescence (e.g. fluorescence activated cell sorter), via survival (e.g. antibiotic resistance), or via serial operations (flow cytometry, laser capture microdissection). Here we present a simple protocol for selecting cells based on any static or dynamic property that can be identified by video microscopy and image processing. The “photostick” technique uses a cell-impermeant photochemical crosslinker and digital micromirror array-based patterned illumination to immobilize selected cells on the culture dish. Other cells are washed away with mild protease treatment. The crosslinker also labels the selected cells with a fluorescent dye and a biotin for later identification. The photostick protocol preserves cell viability, permits genetic profiling of selected cells, and can be performed with complex functional selection criteria such as neuronal firing patterns. PMID:25705368

Impact of CCR1 silencing on the assembly of lignified secondary walls in Arabidopsis thaliana.

PubMed

Ruel, Katia; Berrio-Sierra, Jimmy; Derikvand, Mohammad Mir; Pollet, Brigitte; Thévenin, Johanne; Lapierre, Catherine; Jouanin, Lise; Joseleau, Jean-Paul

2009-01-01

A cinnamoyl-CoA reductase 1 knockout mutant in Arabidopsis thaliana was investigated for the consequences of lignin synthesis perturbation on the assembly of the cell walls. The mutant displayed a dwarf phenotype and a strong collapse of its xylem vessels corresponding to lower lignin content and a loss of lignin units of the noncondensed type. Transmission electron microscopy revealed that the transformation considerably impaired the capacity of interfascicular fibers and vascular bundles to complete the assembly of cellulose microfibrils in the S(2) layer, the S(1) layer remaining unaltered. Such disorder in cellulose was correlated with X-ray diffraction showing altered organization. Semi-quantitative immunolabeling of lignins showed that the patterns of distribution were differentially affected in interfascicular fibers and vascular bundles, pointing to the importance of noncondensed lignin structures for the assembly of a coherent secondary wall. The use of laser capture microdissection combined with the microanalysis of lignins and polysaccharides allowed these polymers to be characterized into specific cell types. Wild-type A. thaliana displayed a two-fold higher syringyl to guaiacyl ratio in interfascicular fibers compared with vascular bundles, whereas this difference was less marked in the cinnamoyl-CoA reductase 1 knockout mutant.

Human hair follicle organ culture: theory, application and perspectives.

PubMed

Langan, Ewan A; Philpott, Michael P; Kloepper, Jennifer E; Paus, Ralf

2015-12-01

For almost a quarter of a century, ex vivo studies of human scalp hair follicles (HFs) have permitted major advances in hair research, spanning diverse fields such as chronobiology, endocrinology, immunology, metabolism, mitochondrial biology, neurobiology, pharmacology, pigmentation and stem cell biology. Despite this, a comprehensive methodological guide to serum-free human HF organ culture (HFOC) that facilitates the selection and analysis of standard HF biological parameters and points out both research opportunities and pitfalls to newcomers to the field is still lacking. The current methods review aims to close an important gap in the literature and attempts to promote standardisation of human HFOC. We provide basic information outlining the establishment of HFOC through to detailed descriptions of the analysis of standard read-out parameters alongside practical examples. The guide closes by pointing out how serum-free HFOC can be utilised optimally to obtain previously inaccessible insights into human HF biology and pathology that are of interest to experimental dermatologists, geneticists, developmental biologists and (neuro-) endocrinologists alike and by highlighting novel applications of the model, including gene silencing and gene expression profiling of defined, laser capture-microdissected HF compartments. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

High frequency of PTEN mutations in nevi and melanomas from xeroderma pigmentosum patients.

PubMed

Masaki, Taro; Wang, Yun; DiGiovanna, John J; Khan, Sikandar G; Raffeld, Mark; Beltaifa, Senda; Hornyak, Thomas J; Darling, Thomas N; Lee, Chyi-Chia R; Kraemer, Kenneth H

2014-05-01

We examined nevi and melanomas in 10 xeroderma pigmentosum (XP) patients with defective DNA repair. The lesions had a lentiginous appearance with markedly increased numbers of melanocytes. Using laser capture microdissection, we performed DNA sequencing of 18 benign and atypical nevi and 75 melanomas (melanoma in situ and invasive melanomas). The nevi had a similar high frequency of PTEN mutations as melanomas [61% (11/18) versus 53% (39/73)]. Both had a very high proportion of UV-type mutations (occurring at adjacent pyrimidines) [91% (10/11) versus 92% (36/39)]. In contrast to melanomas in the general population, the frequency of BRAF mutations (11%, 7/61), NRAS mutations (21%, 13/62), and KIT mutations (21%, 6/28) in XP melanomas was lower than for PTEN. Phospho-S6 immunostaining indicated activation of the mTOR pathway in the atypical nevi and melanomas. Thus, the clinical and histological appearances and the molecular pathology of these UV-related XP nevi and melanomas were different from nevi and melanomas in the general population. © 2014 Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Depression-like behavior in rat: Involvement of galanin receptor subtype 1 in the ventral periaqueductal gray

PubMed Central

Wang, Peng; Li, Hui; Barde, Swapnali; Zhang, Ming-Dong; Sun, Jing; Wang, Tong; Zhang, Pan; Luo, Hanjiang; Wang, Yongjun; Yang, Yutao; Wang, Chuanyue; Svenningsson, Per; Theodorsson, Elvar; Hökfelt, Tomas G. M.; Xu, Zhi-Qing David

2016-01-01

The neuropeptide galanin coexists in rat brain with serotonin in the dorsal raphe nucleus and with noradrenaline in the locus coeruleus (LC), and it has been suggested to be involved in depression. We studied rats exposed to chronic mild stress (CMS), a rodent model of depression. As expected, these rats showed several endophenotypes relevant to depression-like behavior compared with controls. All these endophenotypes were normalized after administration of a selective serotonin reuptake inhibitor. The transcripts for galanin and two of its receptors, galanin receptor 1 (GALR1) and GALR2, were analyzed with quantitative real-time PCR using laser capture microdissection in the following brain regions: the hippocampal formation, LC, and ventral periaqueductal gray (vPAG). Only Galr1 mRNA levels were significantly increased, and only in the latter region. After knocking down Galr1 in the vPAG with an siRNA technique, all parameters of the depressive behavioral phenotype were similar to controls. Thus, the depression-like behavior in rats exposed to CMS is likely related to an elevated expression of Galr1 in the vPAG, suggesting that a GALR1 antagonist could have antidepressant effects. PMID:27457954

Perivascular Delivery of Notch 1 siRNA Inhibits Injury-Induced Arterial Remodeling

PubMed Central

Redmond, Eileen M.; Liu, Weimin; Hamm, Katie; Hatch, Ekaterina; Cahill, Paul A.; Morrow, David

2014-01-01

Objectives To determine the efficacy of perivascular delivery of Notch 1 siRNA in preventing injury-induced arterial remodeling. Methods and Results Carotid artery ligation was performed to induce arterial remodeling. After 14 days, morphometric analysis confirmed increased vSMC growth and subsequent media thickening and neointimal formation. Laser capture microdissection, quantitative qRT-PCR and immunoblot analysis of medial tissue revealed a significant increase in Notch1 receptor and notch target gene, Hrt 1 and 2 expression in the injured vessels. Perivascular delivery of Notch 1 siRNA by pluronic gel inhibited the injury-induced increase in Notch 1 receptor and target gene expression when compared to scrambled siRNA controls while concomitantly reducing media thickening and neointimal formation to pre-injury, sham-operated levels. Selective Notch 1 knockdown also reversed the injury-induced inhibition of pro-apoptotic Bax expression while decreasing injury-induced anti-apoptotic Bcl-XL expression to sham-operated control levels. In parallel experiments, proliferative cyclin levels, as measured by PCNA expression, were reversed to sham-operated control levels following selective Notch 1 knockdown. Conclusion These results suggest that injury-induced arterial remodeling can be successfully inhibited by localized perivascular delivery of Notch 1 siRNA. PMID:24416200

Tumor Heterogeneity, Single-Cell Sequencing, and Drug Resistance.

PubMed

Schmidt, Felix; Efferth, Thomas

2016-06-16

Tumor heterogeneity has been compared with Darwinian evolution and survival of the fittest. The evolutionary ecosystem of tumors consisting of heterogeneous tumor cell populations represents a considerable challenge to tumor therapy, since all genetically and phenotypically different subpopulations have to be efficiently killed by therapy. Otherwise, even small surviving subpopulations may cause repopulation and refractory tumors. Single-cell sequencing allows for a better understanding of the genomic principles of tumor heterogeneity and represents the basis for more successful tumor treatments. The isolation and sequencing of single tumor cells still represents a considerable technical challenge and consists of three major steps: (1) single cell isolation (e.g., by laser-capture microdissection), fluorescence-activated cell sorting, micromanipulation, whole genome amplification (e.g., with the help of Phi29 DNA polymerase), and transcriptome-wide next generation sequencing technologies (e.g., 454 pyrosequencing, Illumina sequencing, and other systems). Data demonstrating the feasibility of single-cell sequencing for monitoring the emergence of drug-resistant cell clones in patient samples are discussed herein. It is envisioned that single-cell sequencing will be a valuable asset to assist the design of regimens for personalized tumor therapies based on tumor subpopulation-specific genetic alterations in individual patients.

12th Annual Small Business Conference

DTIC Science & Technology

2008-11-13

Joint Combat support systems. CoL myers graduated from marshall University in huntington, west virginia with a bachelors degree in Communications. he...as director and Principal assistant responsible for Contracting (ParC) for the Us army Contracting Command’s rock island Contracting Center (riCC...sustainment Command (asC) and the Joint munitions & Lethality Life Cycle management Command (Jm&L-LCmC) – rock island . in this position, he advises

Anomalous interlayer vibrations in strongly coupled layered PdSe 2

DOE PAGES

Puretzky, Alexander A.; Oyedele, Akinola D.; Xiao, Kai; ...

2018-05-04

In this work, we show unusual effects of strong interlayer coupling on low-frequency (LF) Raman scattering in exfoliated PdSe 2 crystals with different number of layers. Unlike many other layered materials, it is found that the measured frequencies of the breathing modes cannot be simply described by a conventional linear chain model (LCM) that treats each layer as a single rigid object. By using first-principles calculations, we show that strong deviations from layer rigidity can occur for the LF breathing vibrations of PdSe 2, which accounts for the observed disagreement with the conventional LCM. The layer non-rigidity and strong interlayermore » coupling could also explain the unusual strong intensities of the LF breathing modes that are comparable with those of the high-frequency Raman modes. These strong intensities allowed us to use a set of the measured LF Raman lines as unique fingerprints for a precise assignment of the layer numbers. The assignment of the layer numbers was further confirmed using second harmonic generation that appeared only in the noncentrosymmetric even-layer PdSe 2 crystals. In conclusion, this work thus demonstrates a simple and fast approach for the determination of the number of layers in 2D materials with strong interlayer coupling and non-rigid interlayer vibrations.« less

Effect of calcination temperature on microstructure, dielectric, magnetic and optical properties of Ba0.7La0.3Fe11.7Co0.3O19 hexaferrites

NASA Astrophysics Data System (ADS)

Kaur, Talwinder; Kaur, Barjinder; Bhat, Bilal H.; Kumar, Sachin; Srivastava, A. K.

2015-01-01

M-type barium hexaferrite Ba0.7La0.3Fe11.7Co0.3O19 (BaLCM) powder, synthesized using sol gel auto combustion method, heat treated at 700, 900, 1100 and 1200 °C. X ray diffraction (XRD) powder patterns of heat treated samples show the formation of pure phase of M-type hexaferrite after 700 °C. Thermo gravimetric analysis (TGA) reveals that the weight loss of BaLCM becomes constant after 680 °C. The presence of two prominent peaks, at 432 cm-1 and 586 cm-1 in Fourier Transform Infrared Spectroscopy (FT-IR) spectra, gives the idea of formation of M-type hexaferrites. The M-H curve obtained from Vibrating Sample Magnetometer (VSM) were used to calculate saturation magnetization (MS), retentivity (Mr), squareness ration (SR) and coercivity (Hc). The maximum value of coercivity (5602 Oe) is found at 900 °C. The band gap dependency on temperature was studied using UV-vis NIR spectroscopy. The dielectric constant has been found to be high at low frequency but it decreases with increase in frequency. Such kind of dielectric behavior is explained on the basis of Koop's phenomenological theory and Maxwell Wagner theory.

Incentives for Blood Donation: A Discrete Choice Experiment to Analyze Extrinsic Motivation.

PubMed

Sadler, Andrew; Shi, Ling; Bethge, Susanne; Mühlbacher, Axel

2018-04-01

Background: Demographic trends affect size and age structure of populations. One of the consequences will be an increasing need for blood products to treat age-related diseases. Donation services rely on voluntariness and charitable motivation. It might be questioned whether there will be sufficient blood supply with voluntary donation. The present study focused on elicitation of preferences for incentives and aimed to contribute to the discussion on how to increase donation rates. Methods: A self-administered discrete choice experiment (DCE) was applied. Respondents were repeatedly asked to choose between hypothetical blood donation centers. In case of reluctance to receiving incentives a none-option was included. Random parameter logit (RPL) and latent class models (LCM) were used for analysis. Results: The study sample included 416 college students from the US and Germany. Choice decisions were significantly influenced by the characteristics of the donation center in the DCE. Incentives most preferred were monetary compensation, paid leave, and blood screening test. LCM identified subgroups with preference heterogeneity. Small subgroups indicated moderate to strong aversion to incentives. Conclusion: The majority of the sample positively responded to incentives and indicated a willingness to accept incentives. In face of future challenges, the judicious use and appropriate utilization of incentives might be an option to motivate potential donors and should be open to discussion.

Anomalous interlayer vibrations in strongly coupled layered PdSe 2

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

Puretzky, Alexander A.; Oyedele, Akinola D.; Xiao, Kai

In this work, we show unusual effects of strong interlayer coupling on low-frequency (LF) Raman scattering in exfoliated PdSe 2 crystals with different number of layers. Unlike many other layered materials, it is found that the measured frequencies of the breathing modes cannot be simply described by a conventional linear chain model (LCM) that treats each layer as a single rigid object. By using first-principles calculations, we show that strong deviations from layer rigidity can occur for the LF breathing vibrations of PdSe 2, which accounts for the observed disagreement with the conventional LCM. The layer non-rigidity and strong interlayermore » coupling could also explain the unusual strong intensities of the LF breathing modes that are comparable with those of the high-frequency Raman modes. These strong intensities allowed us to use a set of the measured LF Raman lines as unique fingerprints for a precise assignment of the layer numbers. The assignment of the layer numbers was further confirmed using second harmonic generation that appeared only in the noncentrosymmetric even-layer PdSe 2 crystals. In conclusion, this work thus demonstrates a simple and fast approach for the determination of the number of layers in 2D materials with strong interlayer coupling and non-rigid interlayer vibrations.« less

Army Medical Robotics Research

DTIC Science & Technology

2007-01-01

environment. These advances in microsurgery would make possible procedures such as small vessel anastomosis, nerve reconstruction , and microdissection and...System, Intuitive Surgical, Inc. 3 b. Telepresence “ Microsurgery ” System for Uniformed Services University of the Health Sciences (USUHS) - Stanford

Localization of new, microdissection- generated, anonymous markers and of the genes Pcsk1, Dhfr, Ndub13, and Ccnb1 to rat chromosome region 2q1.

PubMed

Quan, X; Laes, J F; Ravoet, M; Van Vooren, P; Szpirer, J; Szpirer, C

2000-01-01

The centromeric region of rat chromosome 2 (2q1) harbors unidentified quantitative trait loci of genes that control tumor growth or development. To improve the mapping of this chromosome region, we microdissected it and generated 10 new microsatellite markers, which we included in the linkage map and/or radiation hybrid map of 2q1, together with other known markers, including four genes: Pcsk1 (protein convertase 1), Dhfr (dihydrofolate reductase), Ndub13 (NADH ubiquinone oxidoreductase subunit b13), and Ccnb1 (cyclin B1). To generate anchor points between the different maps, the gene Ndub13 and the microsatellite markers D2Ulb25 and D2Mit1 were also localized cytogenetically. The radiation map generated in region 2q1 extends its centromeric end of about 150 cR. Copyright 2000 S. Karger AG, Basel

Reference spectra from squamous epithelium and connective tissue allow whole section proteomics analysis.

PubMed

Roesch-Ely, Mariana; Schnölzer, Martina; Nees, Matthias; Plinkert, Peter K; Bosch, Franz X

2010-01-01

We reasoned that micro-dissection of tumour cells for protein expression studies should be omitted since tumour-stroma interactions are an important part of the biology of solid tumours. To study such interactions in head and neck squamous cell carcinoma (HNSCC) development, we generated reference protein spectra for normal squamous epithelium and connective tissue by SELDI-TOF-MS. Calgranulins A and B, Annexin1 and Histone H4 were found to be strongly enriched in the epithelium. The alpha-defensins 1-3 and the haemoglobin subunits were identified in the connective tissue. Tumour-distant epithelia, representing early pre-malignant lesions, showed up-regulated expression of the stromal alpha-defensins, whereas the epithelial Annexin 1 was down-regulated. Thus, tumour microenvironment interactions occur very early in the carcinogenic process. These data demonstrate that omitting micro-dissection is actually beneficial for studying changes in protein expression during development and progression of solid tumours.

The Y chromosome of the Atelidae family (Platyrrhini): study by chromosome microdissection.

PubMed

Gifalli-Iughetti, C; Koiffmann, C P

2009-01-01

In order to study the intergeneric variability of the Y chromosome, we describe the hybridization of the Y chromosome of Brachytelesarachnoides, obtained by microdissection, to metaphases of Atelesbelzebuthmarginatus, Lagothrixlagothricha, and Alouatta male specimens. Brachytelesarachnoides (Atelinae) has 62 chromosomes and a very small Y chromosome. Our results showed that the Brachytelesarachnoides Y chromosome probe hybridized to Lagothrixlagothricha metaphases yielding one hybridization signal on only the tiny Y chromosome, and when hybridized with Atelesbelzebuthmarginatus metaphases it yielded one hybridization signal on two thirds of the small acrocentric Y chromosome. However, no hybridization signal was observed in Alouatta metaphases (subfamily Alouattinae), a closely related genus in the Atelidae family. Furthermore, our data support a close phylogenetic relationship among Brachyteles, Ateles, and Lagothrix and their placement in the Atelinae subfamily, but exclude Alouatta from this group indicating its placement as basal to this group. Copyright 2009 S. Karger AG, Basel.

Cytochrome P450-2C11 mRNA is not expressed in endothelial cells dissected from rat renal arterioles.

PubMed

Heil, Sandra G; De Vriese, An S; Kluijtmans, Leo A J; Dijkman, Henry; van Strien, Denise; Akkers, Robert; Blom, Henk J

2005-01-01

Cytochrome P450 (CYP) isoenzymes (CYP2C and CYP2J) are involved in the production of epoxyeicosatrienoic acids, which are postulated as endothelium-derived hyperpolarizing factors (EDHFs). We hypothesized that if CYP2C11 is involved in the EDHF-mediated responses, its mRNA should be expressed in endothelial cells. We, therefore, examined the mRNA expression of CYP2C11 in endothelial cells of renal arterioles. Laser microdissection was applied to isolate endothelial cells from the renal arterioles of 4 male and 4 female Wistar rats. As a positive control of CYP2C11 expression, hepatocytes were also dissected from these rats. RNA was isolated and real-time quantitative polymerase chain reaction (Q-PCR) analysis was applied. Q-PCR analysis showed that CYP2C11 mRNA was not expressed in laser microdissected endothelial cells of renal arterioles of male and female rats. CYP2C11 mRNA expression was highly abundant in hepatocytes dissected from male livers, but in female livers hardly any CYP2C11 mRNA was detected. We have shown that endothelial cells can be dissected from small renal arterioles by laser microdissection to study the mRNA expression of specific genes by Q-PCR. Using this novel tool, we demonstrated that the CYP2C11 mRNA was not expressed in the endothelial cells of renal arterioles. Therefore, we speculate that CYP2C11 does not contribute to the EDHF-mediated responses in renal arterioles. Copyright (c) 2005 S. Karger AG, Basel.

Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis

PubMed Central

Falter, Christian; Ellinger, Dorothea; von Hülsen, Behrend; Heim, René; Voigt, Christian A.

2015-01-01

The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape – liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant–microbe interaction with their potential outreach into crop breeding. PMID:25870605

Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis.

PubMed

Falter, Christian; Ellinger, Dorothea; von Hülsen, Behrend; Heim, René; Voigt, Christian A

2015-01-01

The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape - liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant-microbe interaction with their potential outreach into crop breeding.

A microdissection approach to detect molecular markers during progression of prostate cancer.

PubMed Central

Berthon, P.; Dimitrov, T.; Stower, M.; Cussenot, O.; Maitland, N. J.

1995-01-01

To investigate the underlying mechanisms of carcinogenesis, we have developed a technique to determine the frequency of genetic changes in prostatic carcinoma tissue. We have demonstrated that at a ratio of between 1:4 and 1:9 mutant-normal alleles, the signal from a mutant TP53 allele is not apparent after polymerase chain reaction (PCR) amplification and further direct sequencing or single-strand conformation polymorphism (SSCP) analysis. To bypass this problem, which is inherent in the heterogeneity of the prostate tissue and of the tumour, we selected areas of graded prostate tumours (Gleason score) from cryosectioned preparations and microdissected these cells (20-100 cells). After anionic resin removal of proteins, PCR amplification of TP53 gene exons 5/6 and SSCP analysis, an abnormal SSCP band shift was observed in suspected tumour cells, compared with microdissected stromal cells used as an internal control, while (1) a crude preparation of tissue DNA carrying the tumour did not show any abnormality and (2) immunostaining by a set of monoclonal antibodies against TP53 protein remained negative. Nucleotide sequence analysis of the different bands confirmed the presence of a mutation in the TP53 gene exon 6 position 13,336 in an abnormal band for one specimen, while no mutation was detected in the normal SSCP band. By targeting recognised tumour cells we can find DNA mutations which are undetectable using the standard technique of whole-tissue DNA extraction, particularly in a heterogeneous tumour such as carcinoma of the prostate. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7547246

Combined gene expression analysis of whole-tissue and microdissected pancreatic ductal adenocarcinoma identifies genes specifically overexpressed in tumor epithelia.

PubMed

Badea, Liviu; Herlea, Vlad; Dima, Simona Olimpia; Dumitrascu, Traian; Popescu, Irinel

2008-01-01

The precise details of pancreatic ductal adenocarcinoma (PDAC) pathogenesis are still insufficiently known, requiring the use of high-throughput methods. However, PDAC is especially difficult to study using microarrays due to its strong desmoplastic reaction, which involves a hyperproliferating stroma that effectively "masks" the contribution of the minoritary neoplastic epithelial cells. Thus it is not clear which of the genes that have been found differentially expressed between normal and whole tumor tissues are due to the tumor epithelia and which simply reflect the differences in cellular composition. To address this problem, laser microdissection studies have been performed, but these have to deal with much smaller tissue sample quantities and therefore have significantly higher experimental noise. In this paper we combine our own large sample whole-tissue study with a previously published smaller sample microdissection study by Grützmann et al. to identify the genes that are specifically overexpressed in PDAC tumor epithelia. The overlap of this list of genes with other microarray studies of pancreatic cancer as well as with the published literature is impressive. Moreover, we find a number of genes whose over-expression appears to be inversely correlated with patient survival: keratin 7, laminin gamma 2, stratifin, platelet phosphofructokinase, annexin A2, MAP4K4 and OACT2 (MBOAT2), which are all specifically upregulated in the neoplastic epithelia, rather than the tumor stroma. We improve on other microarray studies of PDAC by putting together the higher statistical power due to a larger number of samples with information about cell-type specific expression and patient survival.

Experimental methods for testing the effects of neurotrophic peptide, ADNF-9, against alcohol-induced apoptosis during pregnancy in c57bl/6 mice.

PubMed

Sari, Youssef

2013-04-24

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This is mostly due to the difficulty of microdissection of fetal brains and their sectioning for determination of apoptotic cells caused by prenatal exposure to alcohol. The experiments described here provide visualized techniques from mice breeding to the identification of cell death in fetal brain tissue. This study used C57BL/6 mice as the animal model for studying fetal alcohol exposure and the role of trophic peptide against alcohol-induced apoptosis. The breeding consists of a 2-hr matting window to determine the exact stage of embryonic age. An established fetal alcohol exposure model has been used in this study to determine the effects of prenatal alcohol exposure in fetal brains. This involves free access to alcohol or pair-fed liquid diets as the sole source of nutrients for the pregnant mice. The techniques involving dissection of fetuses and microdissection of fetal brains are described carefully, since the latter can be challenging. Microdissection requires a stereomicroscope and ultra-fine forceps. Step-by-step procedures for dissecting the fetal brains are provided visually. The fetal brains are dissected from the base of the primordium olfactory bulb to the base of the metencephalon. For investigating apoptosis, fetal brains are first embedded in gelatin using a peel-away mold to facilitate their sectioning with a vibratome apparatus. Fetal brains embedded and fixed in paraformaldehyde are easily sectioned, and the free floating sections can be mounted in superfrost plus slides for determination of apoptosis or cell death. TUNEL (TdT-mediated dUTP Nick End Labeling; TdT: terminal deoxynucleotidyl transferase) assay has been used to identify cell death or apoptotic cells. It is noteworthy that apoptosis and cell-mediated cytotoxicity are characterized by DNA fragmentation. Thus, the visualized TUNEL-positive cells are indicative of cell death or apoptotic cells. The experimental designs here provide information about the use of an established liquid diet for studying the effects of alcohol and the role of neurotrophic peptides during pregnancy in fetal brains. This involves breeding and feeding pregnant mice, microdissecting fetal brains, and determining apoptosis. Together, these visual and textual techniques might be a source for investigating prenatal exposure of harmful agents in fetal brains.

The Bauschinger and Hardening Effects on Residual Stresses in an Autofrettaged Thick-Walled Cylinder

DTIC Science & Technology

1984-06-01

8217Large Caliber Weapon3 Systems Labrra-ory 1ý’ NUBE OF PAGES Dover, 1.7)70 14 "NITOING AGENCY N XME: A ADDRF!SS(Ir -ditI.. fromI Controlling Office. IS...US ARMY AMCCOM COMMANDER ATTN: DRSMC-LC(D) 1 US ARMY TANK-AUTMV COMD DRSMC-LCE(D) I ATTN: DRSTA-RC DRSMC-LCM(D) ( BLDC 321) 1 WARREN, MI 48090 DRSMC-LCS

Meroplankton Monitoring Data from a Fixed Platform in the Chesapeake Bay Mouth, 1982-1983.

DTIC Science & Technology

1984-11-01

the field in 10% formalin in seawater. During .., the sorting process in the laboratory, samples were split as required following the "CVS" method of...8217 -23- [3 =NUSTON 0 -- WACE cJ LLJ to LuJ Lcm *pJ SL O Q_* J FMRA M J JR A5O NO0 1983 Figure 3. Density of Anchoa mitchilli eggs by depth by semimonth

Prevention of Ovarian High-Grade Serous Carcinoma by Elucidating Its Early Change

DTIC Science & Technology

2013-10-01

burden of ovarian cancer through prevention. Finally, it is important to note that clear cell, endometrioid and mucinous carcinomas are clinically...were cut onto PEN membrane slides (Leica), and immediately stored on dry ice. An entire cross-section of fallopian tubes for each case underwent LCM...containing BRCA1, BRCA2 and non-BRCA cases, utilizing the following exclusion criteria: o Systemic steroid treatment for other conditions (ie SLE/ankylosing

FY83 Posture Report: Research, Development, Engineering and Acquisition.

DTIC Science & Technology

1983-01-01

to provide staff management and con- fluids, corrosion preventives, chemistry , chemical trol of Belvoir R&D Center’s force structure through the...vialiing boith FV82 antI FY83 fundsi. Early mianagenlerit r atar refletotrs anti c4tint racting fair sei-imi-generat itir iietlingp estabrlisliedi the phi...ft) to accommodate boats such as the fabrics. paper chemistry , preservatives, fuels, optical LCU, LCM-8. Ribbon Bridge Erection Boat, Small Tug. and

Characteristics of microRNAs enriched in specific cell types and primary tissue types in solid organs.

PubMed

Kriegel, Alison J; Liu, Yong; Liu, Pengyuan; Baker, Maria Angeles; Hodges, Matthew R; Hua, Xing; Liang, Mingyu

2013-12-01

Knowledge of miRNA expression and function in specific cell types in solid organs is limited because of difficulty in obtaining appropriate specimens. We used laser capture microdissection to obtain nine tissue regions from rats, including the nucleus of the solitary tract, hypoglossal motor nucleus, ventral respiratory column/pre-Bötzinger complex, and midline raphe nucleus from the brain stem, myocardium and coronary artery from the heart, and glomerulus, proximal convoluted tubule, and medullary thick ascending limb from the kidney. Each tissue region consists of or is enriched for a specific cell type. Differential patterns of miRNA expression obtained by deep sequencing of minute amounts of laser-captured cells were highly consistent with data obtained from real-time PCR analysis. miRNA expression patterns correctly clustered the specimens by tissue regions and then by primary tissue types (neural, muscular, or epithelial). The aggregate difference in miRNA profiles between tissue regions that contained the same primary tissue type was as large as one-half of the aggregate difference between primary tissue types. miRNAs differentially expressed between primary tissue types are more likely to be abundant miRNAs, while miRNAs differentially expressed between tissue regions containing the same primary tissue type were distributed evenly across the abundance spectrum. The tissue type-enriched miRNAs were more likely to target genes enriched for specific functional categories compared with either cell type-enriched miRNAs or randomly selected miRNAs. These data indicate that the role of miRNAs in determining characteristics of primary tissue types may be different than their role in regulating cell type-specific functions in solid organs.

Analysis of nodule senescence in pea (Pisum sativum L.) using laser microdissection, real-time PCR, and ACC immunolocalization.

PubMed

Serova, Tatiana A; Tikhonovich, Igor A; Tsyganov, Viktor E

2017-05-01

A delay in the senescence of symbiotic nodules could prolong active nitrogen fixation, resulting in improved crop yield and a reduced need for chemical fertilizers. The molecular genetic mechanisms underlying nodule senescence have not been extensively studied with a view to breeding varieties with delayed nodule senescence. In such studies, plant mutants with the phenotype of premature degradation of symbiotic structures are useful models to elucidate the genetic basis of nodule senescence. Using a dataset from transcriptome analysis of Medicago truncatula Gaertn. nodules and previous studies on pea (Pisum sativum L.) nodules, we developed a set of molecular markers based on genes that are known to be activated during nodule senescence. These genes encode cysteine proteases, a thiol protease, a bZIP transcription factor, enzymes involved in the biosynthesis of ethylene (ACS2 for ACC synthase and ACO1 for ACC oxidase) and ABA (AO3 for aldehyde oxidase), and an enzyme involved in catabolism of gibberellins (GA 2-oxidase). We analyzed the transcript levels of these genes in the nodules of two pea wild-types (cv. Sparkle and line Sprint-2) and two mutant lines, one showing premature nodule senescence (E135F (sym13)) and one showing no morphological signs of symbiotic structure degradation (Sprint-2Fix - (sym31)). Real-time PCR analyses revealed that all of the selected genes showed increased transcript levels during nodule aging in all phenotypes. Remarkably, at 4 weeks after inoculation (WAI), the transcript levels of all analyzed genes were significantly higher in the early senescent nodules of the mutant line E135F (sym13) and in nodules of the mutant Sprint-2Fix - (sym31) than in the active nitrogen-fixing nodules of wild-types. In contrast, the transcript levels of the same genes of both wild-types were significantly increased only at 6 WAI. We evaluated the expression of selected markers in the different histological nodule zones of pea cv. Sparkle and its mutant line E135F (sym13) by laser capture microdissection analysis. Finally, we analyzed ACC by immunolocalization in the nodules of both wild-type pea and their mutants. Together, the results indicate that nodule senescence is a general plant response to nodule ineffectiveness. Copyright © 2017 Elsevier GmbH. All rights reserved.

Microdissection of black widow spider silk-producing glands.

PubMed

Jeffery, Felicia; La Mattina, Coby; Tuton-Blasingame, Tiffany; Hsia, Yang; Gnesa, Eric; Zhao, Liang; Franz, Andreas; Vierra, Craig

2011-01-11

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion. Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands. Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk], tubuliform [synthesizes egg case silk], flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] and pyriform [produces attachment disc silk]. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Hypermethylation of the Human Glutathione S-Transferase-π Gene (GSTP1) CpG Island Is Present in a Subset of Proliferative Inflammatory Atrophy Lesions but Not in Normal or Hyperplastic Epithelium of the Prostate

PubMed Central

Nakayama, Masashi; Bennett, Christina J.; Hicks, Jessica L.; Epstein, Jonathan I.; Platz, Elizabeth A.; Nelson, William G.; De Marzo, Angelo M.

2003-01-01

Somatic inactivation of the glutathione S-transferase-π gene (GSTP1) via CpG island hypermethylation occurs early during prostate carcinogenesis, present in ∼70% of high-grade prostatic intraepithelial neoplasia (high-grade PIN) lesions and more than 90% of adenocarcinomas. Recently, there has been a resurgence of the concept that foci of prostatic atrophy (referred to as proliferative inflammatory atrophy or PIA) may be precursor lesions for the development of prostate cancer and/or high-grade PIN. Many of the cells within PIA lesions contain elevated levels of GSTP1, glutathione S-transferase-α (GSTA1), and cyclooxygenase-II proteins, suggesting a stress response. Because not all PIA cells are positive for GSTP1 protein, we hypothesized that some of the cells within these regions acquire GSTP1 CpG island hypermethylation, increasing the chance of progression to high-grade PIN and/or adenocarcinoma. Separate regions (n =199) from 27 formalin-fixed paraffin-embedded prostates were microdissected by laser-capture microdissection (Arcturus PixCell II). These regions included normal epithelium (n = 48), hyperplasticepithelium from benign prostatic hyperplasia nodules (n = 22), PIA (n = 64), high-grade PIN (n = 32), and adenocarcinoma (n = 33). Genomic DNA was isolated and assessed for GSTP1 CpG island hypermethylation by methylation-specific polymerase chain reaction. GSTP1 CpG island hypermethylation was not detected in normal epithelium (0 of 48) or in hyperplastic epithelium (0 of 22), but was found in 4 of 64 (6.3%) PIA lesions. The difference in the frequency of GSTP1 CpG island hypermethylation between normal or hyperplastic epithelium and PIA was statistically significant (P = 0.049). Similar to studies using nonmicrodissected cases, hypermethylation was found in 22 of 32 (68.8%) high-grade PIN lesions and in 30 of 33 (90.9%) adenocarcinoma lesions. Unlike normal or hyperplastic epithelium, GSTP1 CpG island hypermethylation can be detected in some PIA lesions. These data support the hypothesis that atrophic epithelium in a subset of PIA lesions may lead to high-grade PIN and/or adenocarcinoma. Because these atrophic lesions are so prevalent and extensive, even though only a small subset contains this somatic DNA alteration, the clinical impact may be substantial. PMID:12937133

Microdissection of Shoot Meristem Functional Domains

USDA-ARS?s Scientific Manuscript database

The shoot apical meristem (SAM) maintains a pool of indeterminate cells within the SAM proper, while lateral organs are initiated from the SAM periphery. Laser microdissection–microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes th...

Gene expression analysis of rheumatoid arthritis synovial lining regions by cDNA microarray combined with laser microdissection: up-regulation of inflammation-associated STAT1, IRF1, CXCL9, CXCL10, and CCL5

PubMed Central

Yoshida, S; Arakawa, F; Higuchi, F; Ishibashi, Y; Goto, M; Sugita, Y; Nomura, Y; Niino, D; Shimizu, K; Aoki, R; Hashikawa, K; Kimura, Y; Yasuda, K; Tashiro, K; Kuhara, S; Nagata, K; Ohshima, K

2012-01-01

Objectives The main histological change in rheumatoid arthritis (RA) is the villous proliferation of synovial lining cells, an important source of cytokines and chemokines, which are associated with inflammation. The aim of this study was to evaluate gene expression in the microdissected synovial lining cells of RA patients, using those of osteoarthritis (OA) patients as the control. Methods Samples were obtained during total joint replacement from 11 RA and five OA patients. Total RNA from the synovial lining cells was derived from selected specimens by laser microdissection (LMD) for subsequent cDNA microarray analysis. In addition, the expression of significant genes was confirmed immunohistochemically. Results The 14 519 genes detected by cDNA microarray were used to compare gene expression levels in synovial lining cells from RA with those from OA patients. Cluster analysis indicated that RA cells, including low- and high-expression subgroups, and OA cells were stored in two main clusters. The molecular activity of RA was statistically consistent with its clinical and histological activity. Expression levels of signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor 1 (IRF1), and the chemokines CXCL9, CXCL10, and CCL5 were statistically significantly higher in the synovium of RA than in that of OA. Immunohistochemically, the lining synovium of RA, but not that of OA, clearly expressed STAT1, IRF1, and chemokines, as was seen in microarray analysis combined with LMD. Conclusions Our findings indicate an important role for lining synovial cells in the inflammatory and proliferative processes of RA. Further understanding of the local signalling in structural components is important in rheumatology. PMID:22401175

New insights in the homotopic and heterotopic connectivity of the frontal portion of the human corpus callosum revealed by microdissection and diffusion tractography.

PubMed

De Benedictis, Alessandro; Petit, Laurent; Descoteaux, Maxime; Marras, Carlo Efisio; Barbareschi, Mattia; Corsini, Francesco; Dallabona, Monica; Chioffi, Franco; Sarubbo, Silvio

2016-12-01

Extensive studies revealed that the human corpus callosum (CC) plays a crucial role in providing large-scale bi-hemispheric integration of sensory, motor and cognitive processing, especially within the frontal lobe. However, the literature lacks of conclusive data regarding the structural macroscopic connectivity of the frontal CC. In this study, a novel microdissection approach was adopted, to expose the frontal fibers of CC from the dorsum to the lateral cortex in eight hemispheres and in one entire brain. Post-mortem results were then combined with data from advanced constrained spherical deconvolution in 130 healthy subjects. We demonstrated as the frontal CC provides dense inter-hemispheric connections. In particular, we found three types of fronto-callosal fibers, having a dorso-ventral organization. First, the dorso-medial CC fibers subserve homotopic connections between the homologous medial cortices of the superior frontal gyrus. Second, the ventro-lateral CC fibers subserve homotopic connections between lateral frontal cortices, including both the middle frontal gyrus and the inferior frontal gyrus, as well as heterotopic connections between the medial and lateral frontal cortices. Third, the ventro-striatal CC fibers connect the medial and lateral frontal cortices with the contralateral putamen and caudate nucleus. We also highlighted an intricate crossing of CC fibers with the main association pathways terminating in the lateral regions of the frontal lobes. This combined approach of ex vivo microdissection and in vivo diffusion tractography allowed demonstrating a previously unappreciated three-dimensional architecture of the anterior frontal CC, thus clarifying the functional role of the CC in mediating the inter-hemispheric connectivity. Hum Brain Mapp 37:4718-4735, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval.

PubMed

Smith, Ryan P; Lowe, Greg J; Kavoussi, Parviz K; Steers, William D; Costabile, Raymond A; Herr, John C; Shetty, Jagathpala; Lysiak, Jeffrey J

2012-05-01

Microdissection testicular sperm extraction markedly improves the sperm retrieval rates in men with nonobstructive azoospermia. However, localizing sperm foci can be time-consuming and it is not always successful. Fiberoptic confocal fluorescent microscopy offers the advantage of rapid in vivo detection of fluorescently labeled sperm in the seminiferous tubules. After establishing the feasibility of fiberoptic confocal fluorescent microscopy to identify antibody labeled sperm in vivo C57/B6 mice underwent intraperitoneal injection of busulfan to induce azoospermia. During spermatogenesis reestablishment at approximately 16 weeks the mice were anesthetized and the testes were delivered through a low midline incision. Fluorescein isothiocyanate labeled antibody to intra-acrosomal protein Hs-14 was injected retrograde into a single murine rete testis. The testes were imaged in vivo with fiberoptic confocal fluorescent microscopy and sperm foci were detected. The respective seminiferous tubules were excised and squash prepared for immunofluorescence microscopy. Sperm foci were identified in the testis injected with fluorescently tagged antibody by in vivo fiberoptic confocal fluorescence microscopy. The contralateral control testis of each mouse showed no specific signal. Immunofluorescence microscopy of the excised tubules provided morphological confirmation of the presence of labeled sperm with an absence in controls. Findings were consistent in the feasibility portion of the study and in the busulfan model of nonobstructive azoospermia. Fiberoptic confocal fluorescent microscopy was feasible during microdissection testicular sperm extraction in an azoospermic mouse model to identify fluorescently labeled sperm in vivo. Translation to the clinical setting could decrease operative time and improve the sperm harvest rate. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Mechanisms of Laser-Induced Dissection and Transport of Histologic Specimens

PubMed Central

Vogel, Alfred; Lorenz, Kathrin; Horneffer, Verena; Hüttmann, Gereon; von Smolinski, Dorthe; Gebert, Andreas

2007-01-01

Rapid contact- and contamination-free procurement of histologic material for proteomic and genomic analysis can be achieved by laser microdissection of the sample of interest followed by laser-induced transport (laser pressure catapulting). The dynamics of laser microdissection and laser pressure catapulting of histologic samples of 80 μm diameter was investigated by means of time-resolved photography. The working mechanism of microdissection was found to be plasma-mediated ablation initiated by linear absorption. Catapulting was driven by plasma formation when tightly focused pulses were used, and by photothermal ablation at the bottom of the sample when defocused pulses producing laser spot diameters larger than 35 μm were used. With focused pulses, driving pressures of several hundred MPa accelerated the specimen to initial velocities of 100–300 m/s before they were rapidly slowed down by air friction. When the laser spot was increased to a size comparable to or larger than the sample diameter, both driving pressure and flight velocity decreased considerably. Based on a characterization of the thermal and optical properties of the histologic specimens and supporting materials used, we calculated the evolution of the heat distribution in the sample. Selected catapulted samples were examined by scanning electron microscopy or analyzed by real-time reverse-transcriptase polymerase chain reaction. We found that catapulting of dissected samples results in little collateral damage when the laser pulses are either tightly focused or when the laser spot size is comparable to the specimen size. By contrast, moderate defocusing with spot sizes up to one-third of the specimen diameter may involve significant heat and ultraviolet exposure. Potential side effects are maximal when samples are catapulted directly from a glass slide without a supporting polymer foil. PMID:17766336

The Need for the United States Army to Possess a Landing Craft with Maneuver Capabilities

DTIC Science & Technology

2015-06-12

Personnel, Facilities and Policy FAA Functional Area Analysis FNA Functional Needs Analysis FSA Functional Solution Analysis HADR Humanitarian ...increase the options available to the JTFC.7 Within the last 25 years, the LCM-8 and other landing craft have been used numerous times for Humanitarian ...and coastal islands after the bridges were destroyed.8 The World Food Program (WFP) and other humanitarian aid providers perfected the use of military

Comparisons Between Experimental and Semi-theoretical Cutting Forces of CCS Disc Cutters

NASA Astrophysics Data System (ADS)

Xia, Yimin; Guo, Ben; Tan, Qing; Zhang, Xuhui; Lan, Hao; Ji, Zhiyong

2018-05-01

This paper focuses on comparisons between the experimental and semi-theoretical forces of CCS disc cutters acting on different rocks. The experimental forces obtained from LCM tests were used to evaluate the prediction accuracy of a semi-theoretical CSM model. The results show that the CSM model reliably predicts the normal forces acting on red sandstone and granite, but underestimates the normal forces acting on marble. Some additional LCM test data from the literature were collected to further explore the ability of the CSM model to predict the normal forces acting on rocks of different strengths. The CSM model underestimates the normal forces acting on soft rocks, semi-hard rocks and hard rocks by approximately 38, 38 and 10%, respectively, but very accurately predicts those acting on very hard and extremely hard rocks. A calibration factor is introduced to modify the normal forces estimated by the CSM model. The overall trend of the calibration factor is characterized by an exponential decrease with increasing rock uniaxial compressive strength. The mean fitting ratios between the normal forces estimated by the modified CSM model and the experimental normal forces acting on soft rocks, semi-hard rocks and hard rocks are 1.076, 0.879 and 1.013, respectively. The results indicate that the prediction accuracy and the reliability of the CSM model have been improved.

New compact hohlraum configuration research at the 1.7 MA Z-pinch generator

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

Kantsyrev, V. L., E-mail: victor@unr.edu; Shrestha, I. K.; Esaulov, A. A.

A new compact Z-pinch x-ray hohlraum design with parallel-driven x-ray sources was experimentally demonstrated in a full configuration with a central target and tailored shine shields (to provide a symmetric temperature distribution on the target) at the 1.7 MA Zebra generator. This presentation reports on the joint success of two independent lines of research. One of these was the development of new sources – planar wire arrays (PWAs). PWAs turned out to be a prolific radiator. Another success was the drastic improvement in energy efficiency of pulsed-power systems, such as the Load Current Multiplier (LCM). The Zebra/LCM generator almost doubledmore » the plasma load current to 1.7 MA. The two above-mentioned innovative approaches were used in combination to produce a new compact hohlraum design for ICF, as jointly proposed by SNL and UNR. Good agreement between simulated and measured radiation temperature of the central target is shown. Experimental comparison of PWAs with planar foil liners (PFL) - another viable alternative to wire array loads at multi-MA generators show promising data. Results of research at the University of Nevada Reno allowed for the study of hohlraum coupling physics at University-scale generators. The advantages of new hohlraum design applications for multi-MA facilities with W or Au double PWAs or PFL x-ray sources are discussed.« less

Fracture Toughness (KIC) of Lithography Based Manufactured Alumina Ceramic

NASA Astrophysics Data System (ADS)

Nindhia, T. G. T.; Schlacher, J.; Lube, T.

2018-04-01

Precision shaped ceramic components can be obtained by an emerging technique called Lithography based Ceramic Manufacturing (LCM). A green part is made from a slurry consisting of a ceramic powder in a photocurable binder with addition of dispersant and plasticizer. Components are built in a layer–by-layer way by exposing the desired cross- sections to light. The parts are subsequently sintered to their final density. It is a challenge to produce ceramic component with this method that yield the same mechanical properties in all direction. The fracture toughness (KIc) of of LCM-alumina (prepared at LITHOZ GmbH, Austria) was tested by using the Single-Edge-V-Notched Beam (SEVNB) method. Notches are made into prismatic bend-bars in all three direction X, Y and Z to recognize the value of fracture toughness of the material in all three directions. The microstructure was revealed with optical microscopy as well as Scanning Electron Microscopy (SEM). The results indicate that the fracture toughness in Y-direction has the highest value (3.10 MPam1/2) that is followed by the one in X-direction which is just a bit lower (2.90 MPam1/2). The Z-direction is found to have a similar fracture toughness (2.95 MPam1/2). This is supported by a homogeneous microstructure showing no hint of the layers used during production.

Intratidal recruitment/derecruitment persists at low and moderate positive end-expiratory pressure in paediatric patients.

PubMed

Wirth, Steffen; Artner, Lisa; Broß, Tobias; Lozano-Zahonero, Sara; Spaeth, Johannes; Schumann, Stefan

2016-12-01

In paediatric patients positive end-expiratory pressure (PEEP) is traditionally set lower than in adults. We investigated whether moderately higher PEEP improves respiratory mechanics and regional ventilation. Therefore, 40 children were mechanically ventilated with PEEP 2 and 5cmH 2 O. Volume-dependent compliance profiles were analysed as a measure of intratidal recruitment/derecruitment. Regional ventilation was assessed using electrical impedance tomography. Mean compliance was 17.9±9.9mLcmH 2 O -1 (PEEP 2cmH 2 O), and 19.0±10.9mLcmH 2 O -1 (PEEP 5 cmH 2 O, p<0.001). Strong intratidal recruitment/derecruitment occurred in 40% of children at PEEP 2 cmH 2 O, and 36% at PEEP 5 cmH 2 O. Children showing strong recruitment/derecruitment were 33 (PEEP 2 cmH 2 0) and 20 (PEEP 5 cmH 2 0) months younger than children showing moderate recruitment/derecruitment. A higher PEEP improved peripheral ventilation. In conclusion, mechanically ventilated paediatric patients undergo intratidal recruitment/derecruitment which occurs more prominently in younger than in older children. A PEEP of 5cmH 2 O does not fully prevent intratidal recruitment/derecruitment but homogenizes regional ventilation in comparison to 2cmH 2 O. Copyright © 2016 Elsevier B.V. All rights reserved.

Longitudinal Associations Between Parental Monitoring Discrepancy and Delinquency: An Application of the Latent Congruency Model.

PubMed

Ksinan, Albert J; Vazsonyi, Alexander T

2016-12-01

Studies have shown that discrepancies (relative concordance or discordance) between parent and adolescent ratings are predictive of problem behaviors; monitoring, in particular, has been consistently linked to them. The current study tested whether discrepancies in perceptions of maternal monitoring, rated by mothers and youth at age 12, foretold delinquency (rule breaking) at age 15, and whether parental closeness and conflict predicted higher discrepancies, and indirectly, higher delinquency. The final study sample used the NICHD longitudinal dataset with N = 966 youth (50.1 % female) and their mothers (80.1 % European American, 12.9 % African American, 7 % other ethnicity). The analytic approach consisted of an extension and application of the Latent Congruency Model (LCM) to estimate monitoring discrepancies as well as age 15 delinquency scores. Findings showed that age 12 monitoring discrepancy was predictive of age 15 delinquency for both boys and girls based on youth reports, but not for maternal reports. Age 11 closeness predicted age 12 monitoring discrepancy, which served as a mediator for its effect on age 15 adolescent-reported delinquency. Thus, based on the rigorous LCM analytic approach which seeks to minimize the effects by competing explanations and to maximize precision in providing robust estimates, rates of perceived discordance in parenting behaviors during early adolescence matter in understanding variability in adolescent delinquency during middle adolescence.

A fast Monte Carlo EM algorithm for estimation in latent class model analysis with an application to assess diagnostic accuracy for cervical neoplasia in women with AGC

PubMed Central

Kang, Le; Carter, Randy; Darcy, Kathleen; Kauderer, James; Liao, Shu-Yuan

2013-01-01

In this article we use a latent class model (LCM) with prevalence modeled as a function of covariates to assess diagnostic test accuracy in situations where the true disease status is not observed, but observations on three or more conditionally independent diagnostic tests are available. A fast Monte Carlo EM (MCEM) algorithm with binary (disease) diagnostic data is implemented to estimate parameters of interest; namely, sensitivity, specificity, and prevalence of the disease as a function of covariates. To obtain standard errors for confidence interval construction of estimated parameters, the missing information principle is applied to adjust information matrix estimates. We compare the adjusted information matrix based standard error estimates with the bootstrap standard error estimates both obtained using the fast MCEM algorithm through an extensive Monte Carlo study. Simulation demonstrates that the adjusted information matrix approach estimates the standard error similarly with the bootstrap methods under certain scenarios. The bootstrap percentile intervals have satisfactory coverage probabilities. We then apply the LCM analysis to a real data set of 122 subjects from a Gynecologic Oncology Group (GOG) study of significant cervical lesion (S-CL) diagnosis in women with atypical glandular cells of undetermined significance (AGC) to compare the diagnostic accuracy of a histology-based evaluation, a CA-IX biomarker-based test and a human papillomavirus (HPV) DNA test. PMID:24163493

Tunable water desalination across Graphene Oxide Framework membranes

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

Nicolai, Adrien; Sumpter, Bobby G; Meunier, V.

2014-01-01

The performance of graphene oxide framework (GOF) membranes for water desalination is assessed using classical molecular dynamics (MD) simulations. The coupling between water permeability and salt rejection GOF membranes is studied as a function of linker concentration n, thickness h and applied pressure DP. The simulations reveal that water permeability in GOF-(n,h) membranes can be tuned from 5 (n = 32 and h = 6.5 nm) to 400 L/cm2/day/MPa (n = 64 and h = 2.5 nm) and follows the law Cnh an . For a given pore size (n = 16 or 32), water permeability of GOF membranes increasesmore » when the pore spacing decreases, whereas for a given pore spacing (n = 32 or 64), water permeability increases by up to two orders of magnitude when the pore size increases. Furthermore, for linker concentrations n 32, the high water permeability corresponds to a 100% salt rejection, elevating this type of GOF membrane as an ideal candidate for water desalination. Compared to experimental performance of reverse osmosis membranes, our calculations suggest that under the same conditions of applied pressure and characteristics of membranes (DP 10 MPa and h 100 nm), one can expect a perfect salt rejection coupled to a water permeability two orders of magnitude higher than existing technologies, i.e., from a few cL/cm2/day/MPa to a few L/cm2/day/MPa.« less

Generation of a complete set of human telomeric band painting probes by chromosome microdissection.

PubMed

Hu, Liang; Sham, Jonathan S T; Tjia, Wai Mui; Tan, Yue-qiu; Lu, Guang-xiu; Guan, Xin-Yuan

2004-02-01

Chromosomal rearrangements involving telomeric bands have been frequently detected in many malignancies and congenital diseases. To develop a useful tool to study chromosomal rearrangements within the telomeric band effectively and accurately, a whole set of telomeric band painting probes (TBP) has been generated by chromosome microdissection. The intensity and specificity of these TBPs have been tested by fluorescence in situ hybridization and all TBPs showed strong and specific signals to target regions. TBPs of 6q and 17p were successfully used to detect the loss of the terminal band of 6q in a hepatocellular carcinoma cell line and a complex translocation involving the 17p terminal band in a melanoma cell line. Meanwhile, the TBP of 21q was used to detect a de novo translocation, t(12;21), and the breakpoint at 21q was located at 21q22.2. Further application of these TBPs should greatly facilitate the cytogenetic analysis of complex chromosome rearrangements involving telomeric bands.

Species identification of processed animal proteins (PAPs) in animal feed containing feed materials from animal origin.

PubMed

Axmann, Sonja; Adler, Andreas; Brandstettner, Agnes Josephine; Spadinger, Gabriela; Weiss, Roland; Strnad, Irmengard

2015-01-01

Since June 2013 the total feed ban of processed animal proteins (PAPs) was partially lifted. Now it is possible to mix fish feed with PAPs from non-ruminants (pig and poultry). To guarantee that fish feed, which contains non-ruminant PAPs, is free of ruminant PAPs, it has to be analysed with a ruminant PCR assay to comply with the total ban of feeding PAPs from ruminants. However, PCR analysis cannot distinguish between ruminant DNA, which originates from proteins such as muscle and bones, and ruminant DNA, which comes from feed materials of animal origin such as milk products or fat. Thus, there is the risk of obtaining positive ruminant PCR signals based on these materials. The paper describes the development of the combination of two analysis methods, micro-dissection and PCR, to eliminate the problem of 'false-positive' PCR signals. With micro-dissection, single particles can be isolated and subsequently analysed with PCR.

Micromolecular methods for diagnosis and therapeutic strategy: a case study

PubMed Central

Elbouchtaoui, Morad; Tengher, Iulia; Miquel, Catherine; Brugière, Charlotte; Benbara, Amélie; Zelek, Laurent; Ziol, Marianne; Bouhidel, Fatiha; Janin, Anne; Bousquet, Guilhem; Leboeuf, Christophe

2018-01-01

An intraductal carcinoma, 55 mm across, was diagnosed on a total mastectomy in a 45-year-old woman. The 2 micro-invasive areas found were too small for reliable immunostainings for estrogen, progesterone, and HER2 receptors. In the sentinel lymph-node, a subcapsular tumor embole of about 50 cancer cells was identified on the extemporaneous cryo-cut section, but not on further sections after paraffin-embedding of the sample. Considering this tumor metastatic potential, we decided to assess HER2 status on the metastatic embole using pathological and molecular micro-methods. We laser-microdissected the tumor cells, extracted their DNA, and performed droplet-digital-PCR (ddPCR) for HER2 gene copy number variation. The HER2/RNaseP allele ratio was 5.2 in the laser-microdissected tumor cells, similar to the 5.3 ratio in the HER2-overexpressing breast cancer cell line BT-474. We thus optimized the adjuvant treatment of our patient and she received a trastuzumab-based adjuvant chemotherapy. PMID:29854320

In Planta Stage-Specific Fungal Gene Profiling Elucidates the Molecular Strategies of Fusarium graminearum Growing inside Wheat Coleoptiles[W][OA

PubMed Central

Zhang, Xiao-Wei; Jia, Lei-Jie; Zhang, Yan; Jiang, Gang; Li, Xuan; Zhang, Dong; Tang, Wei-Hua

2012-01-01

The ascomycete Fusarium graminearum is a destructive fungal pathogen of wheat (Triticum aestivum). To better understand how this pathogen proliferates within the host plant, we tracked pathogen growth inside wheat coleoptiles and then examined pathogen gene expression inside wheat coleoptiles at 16, 40, and 64 h after inoculation (HAI) using laser capture microdissection and microarray analysis. We identified 344 genes that were preferentially expressed during invasive growth in planta. Gene expression profiles for 134 putative plant cell wall–degrading enzyme genes suggest that there was limited cell wall degradation at 16 HAI and extensive degradation at 64 HAI. Expression profiles for genes encoding reactive oxygen species (ROS)–related enzymes suggest that F. graminearum primarily scavenges extracellular ROS before a later burst of extracellular ROS is produced by F. graminearum enzymes. Expression patterns of genes involved in primary metabolic pathways suggest that F. graminearum relies on the glyoxylate cycle at an early stage of plant infection. A secondary metabolite biosynthesis gene cluster was specifically induced at 64 HAI and was required for virulence. Our results indicate that F. graminearum initiates infection of coleoptiles using covert penetration strategies and switches to overt cellular destruction of tissues at an advanced stage of infection. PMID:23266949

Biotechnological application of functional genomics towards plant-parasitic nematode control.

PubMed

Li, Jiarui; Todd, Timothy C; Lee, Junghoon; Trick, Harold N

2011-12-01

Plant-parasitic nematodes are primary biotic factors limiting the crop production. Current nematode control strategies include nematicides, crop rotation and resistant cultivars, but each has serious limitations. RNA interference (RNAi) represents a major breakthrough in the application of functional genomics for plant-parasitic nematode control. RNAi-induced suppression of numerous genes essential for nematode development, reproduction or parasitism has been demonstrated, highlighting the considerable potential for using this strategy to control damaging pest populations. In an effort to find more suitable and effective gene targets for silencing, researchers are employing functional genomics methodologies, including genome sequencing and transcriptome profiling. Microarrays have been used for studying the interactions between nematodes and plant roots and to measure both plants and nematodes transcripts. Furthermore, laser capture microdissection has been applied for the precise dissection of nematode feeding sites (syncytia) to allow the study of gene expression specifically in syncytia. In the near future, small RNA sequencing techniques will provide more direct information for elucidating small RNA regulatory mechanisms in plants and specific gene silencing using artificial microRNAs should further improve the potential of targeted gene silencing as a strategy for nematode management. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Root Type-Specific Reprogramming of Maize Pericycle Transcriptomes by Local High Nitrate Results in Disparate Lateral Root Branching Patterns1[OPEN

PubMed Central

Lithio, Andrew

2016-01-01

The adaptability of root system architecture to unevenly distributed mineral nutrients in soil is a key determinant of plant performance. The molecular mechanisms underlying nitrate dependent plasticity of lateral root branching across the different root types of maize are only poorly understood. In this study, detailed morphological and anatomical analyses together with cell type-specific transcriptome profiling experiments combining laser capture microdissection with RNA-seq were performed to unravel the molecular signatures of lateral root formation in primary, seminal, crown, and brace roots of maize (Zea mays) upon local high nitrate stimulation. The four maize root types displayed divergent branching patterns of lateral roots upon local high nitrate stimulation. In particular, brace roots displayed an exceptional architectural plasticity compared to other root types. Transcriptome profiling revealed root type-specific transcriptomic reprogramming of pericycle cells upon local high nitrate stimulation. The alteration of the transcriptomic landscape of brace root pericycle cells in response to local high nitrate stimulation was most significant. Root type-specific transcriptome diversity in response to local high nitrate highlighted differences in the functional adaptability and systemic shoot nitrogen starvation response during development. Integration of morphological, anatomical, and transcriptomic data resulted in a framework underscoring similarity and diversity among root types grown in heterogeneous nitrate environments. PMID:26811190

Anti-inflammatory effect of topical administration of tofacitinib on corneal inflammation.

PubMed

Sakimoto, Tohru; Ishimori, Akiko

2016-04-01

We evaluated an anti-inflammatory effect of topical administration of tofacitinib, janus kinase (JAK) blocker, on corneal inflammation. Topical instillation of either tofacitinib or PBS was applied after wounding BALB/c mice corneas with alkali burn. Topical instillation was performed until day 14 after injury and injured eye was analyzed. The vascularized area in the alkali burned cornea was significantly reduced in the tofacitinib group compared with that in the PBS group. The immunoreactivity of Gr-1, F4/80, IFN-γ, and phosphorylated STAT(signal transducer and activator of transcription)1 in corneal stroma was diminished significantly in the tofacitinib group. Using laser capture microdissection system and quantitative PCR array analysis, the expression levels of CXCL9, CXCL5, CCL7, CCL2, MMP(matrix metalloproteinase)-9, and STAT1 in corneal stroma were down-regulated in the tofacitinib group. In in vitro study, human fibroblast pretreated by IFN-γ showed phosphorylation of STAT1, and this phosphorylation was down-regulated by adding tofacitinib to the culture medium. These results indicate the topical application of JAK inhibitor causes down-regulation of JAK- or IFN-γ-related molecules. Therefore, we deduce that application of JAK inhibitor for topical instillation may contribute to the treatment of corneal inflammation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cell- and Tissue-Specific Transcriptome Analyses of Medicago truncatula Root Nodules

PubMed Central

Limpens, Erik; Moling, Sjef; Hooiveld, Guido; Pereira, Patrícia A.; Bisseling, Ton; Becker, Jörg D.; Küster, Helge

2013-01-01

Legumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital “in situ”. This digital “in situ” offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies. PMID:23734198

Tachykinin-1 in the central nervous system regulates adiposity in rodents.

PubMed

Trivedi, Chitrang; Shan, Xiaoye; Tung, Yi-Chun Loraine; Kabra, Dhiraj; Holland, Jenna; Amburgy, Sarah; Heppner, Kristy; Kirchner, Henriette; Yeo, Giles S H; Perez-Tilve, Diego

2015-05-01

Ghrelin is a circulating hormone that targets the central nervous system to regulate feeding and adiposity. The best-characterized neural system that mediates the effects of ghrelin on energy balance involves the activation of neuropeptide Y/agouti-related peptide neurons, expressed exclusively in the arcuate nucleus of the hypothalamus. However, ghrelin receptors are expressed in other neuronal populations involved in the control of energy balance. We combined laser capture microdissection of several nuclei of the central nervous system expressing the ghrelin receptor (GH secretagoge receptor) with microarray gene expression analysis to identify additional neuronal systems involved in the control of central nervous system-ghrelin action. We identified tachykinin-1 (Tac1) as a gene negatively regulated by ghrelin in the hypothalamus. Furthermore, we identified neuropeptide k as the TAC1-derived peptide with more prominent activity, inducing negative energy balance when delivered directly into the brain. Conversely, loss of Tac1 expression enhances the effectiveness of ghrelin promoting fat mass gain both in male and in female mice and increases the susceptibility to diet-induced obesity in ovariectomized mice. Taken together, our data demonstrate a role TAC1 in the control energy balance by regulating the levels of adiposity in response to ghrelin administration and to changes in the status of the gonadal function.

Clonal analysis of human embryonic stem cell differentiation into teratomas.

PubMed

Blum, Barak; Benvenisty, Nissim

2007-08-01

Differentiation of human embryonic stem cells (HESCs) can be studied in vivo through the induction of teratomas in immune-deficient mice. Cells within the teratomas differentiate into all three embryonic germ layers. However, the exact nature of the proliferation and differentiation of HESCs within the teratoma is not fully characterized, and it is not clear whether the differentiation is cell autonomous or affected by neighboring cells. Here, we establish a genetic approach to study the clonality of differentiation in teratomas using a mixture of HESC lines. We first demonstrate, by means of 5-bromo-2'-deoxyuridine incorporation, that cell proliferation occurs throughout the teratoma, and that there are no clusters of undifferentiated-proliferating cells. Using a combination of laser capture microdissection and DNA fingerprinting analysis, we show that different cell lines contribute mutually to the same distinctive tissue structures. Further support for the nonclonal differentiation within the teratoma was achieved by fluorescence in situ hybridization analysis of sex chromosomes. We therefore suggest that in vivo differentiation of HESCs is polyclonal and, thus, may not be cell autonomous, stressing the need for a three-dimensional growth in order to achieve complex differentiation of HESCs. Disclosure of potential conflicts of interest is found at the end of this article.

Decreased expression of liver X receptor-α in macrophages infected with Chlamydia pneumoniae in human atherosclerotic arteries in situ.

PubMed

Bobryshev, Yuri V; Orekhov, Alexander N; Killingsworth, Murray C; Lu, Jinhua

2011-01-01

In in vitro experiments, Chlamydia pneumoniae has been shown to infect macrophages and to accelerate foam cell formation. It has been hypothesized that the C. pneumoniae infection affects foam cell formation by suppressing the expression of liver X receptors (LXR), but whether such an event occurs in human atherosclerosis is not known. In this study we examined carotid artery segments, obtained by endarterectomy, in which the presence of C. pneumoniae was confirmed by both polymerase chain reaction and immunohistochemistry. The expression of LXR-α in macrophages infected with C. pneumoniae and macrophages that were not infected was compared using a quantitative immunohistochemical analysis. The analysis revealed a 2.2-fold reduction in the expression of LXR-α in C. pneumoniae-infected cells around the lipid cores in atherosclerotic plaques. In the cytoplasm of laser-capture microdissected cells that were immunopositive for C. pneumoniae, electron microscopy demonstrated the presence of structures with the appearance of elementary, reticulate and aberrant bodies of C. pneumoniae. We conclude that LXR-α expression is reduced in C. pneumoniae-infected macrophages in human atherosclerotic lesions which supports the hypothesis that C. pneumoniae infection might suppress LXR expression in macrophages transforming into foam cells. Copyright © 2011 S. Karger AG, Basel.

Recent advances in genomic profiling of adenosquamous carcinoma of the pancreas.

PubMed

Marcus, Rebecca; Maitra, Anirban; Roszik, Jason

2017-11-01

Adenosquamous carcinoma of the pancreas (ASCP) is a mixed tumor type which contains squamous cell carcinoma and also ductal adenocarcinoma components. Due to the rarity of this malignancy, only very limited genomic profiling has been performed. A recent paper by Fang et al. published in The Journal of Pathology contributed to our knowledge of genomic alterations by performing whole-genome and -exome sequencing of 17 ASCP tumors. They found major genomic similarities to pancreatic ductal adenocarcinoma; however, the p53 pathway was altered in a greater proportion of cases, while a high frequency of 3p loss was a distinct copy number alteration pattern observed in ASCP. Laser capture microdissection revealed that adenocarcinoma and squamous carcinoma components of ASCP harbor similar genomic variations, indicating that the origin of tumor components is the same or similar. Although the study published by Fang et al. increases our knowledge of this rare mixed tumor type, further investigation, including RNA sequencing, will be needed to fully characterize this malignancy and to aid the development of novel treatment approaches. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Isothermal multiple displacement amplification: a methodical approach enhancing molecular routine diagnostics of microcarcinomas and small biopsies.

PubMed

Mairinger, Fabian D; Walter, Robert Fh; Vollbrecht, Claudia; Hager, Thomas; Worm, Karl; Ting, Saskia; Wohlschläger, Jeremias; Zarogoulidis, Paul; Zarogoulidis, Konstantinos; Schmid, Kurt W

2014-01-01

Isothermal multiple displacement amplification (IMDA) can be a powerful tool in molecular routine diagnostics for homogeneous and sequence-independent whole-genome amplification of notably small tumor samples, eg, microcarcinomas and biopsies containing a small amount of tumor. Currently, this method is not well established in pathology laboratories. We designed a study to confirm the feasibility and convenience of this method for routine diagnostics with formalin-fixed, paraffin-embedded samples prepared by laser-capture microdissection. A total of 250 μg DNA (concentration 5 μg/μL) was generated by amplification over a period of 8 hours with a material input of approximately 25 cells, approximately equivalent to 175 pg of genomic DNA. In the generated DNA, a representation of all chromosomes could be shown and the presence of elected genes relevant for diagnosis in clinical samples could be proven. Mutational analysis of clinical samples could be performed without any difficulty and showed concordance with earlier diagnostic findings. We established the feasibility and convenience of IMDA for routine diagnostics. We also showed that small amounts of DNA, which were not analyzable with current molecular methods, could be sufficient for a wide field of applications in molecular routine diagnostics when they are preamplified with IMDA.

Haploinsufficiency of Anx7 tumor suppressor gene and consequent genomic instability promotes tumorigenesis in the Anx7(+/-) mouse

PubMed Central

Srivastava, Meera; Montagna, Cristina; Leighton, Ximena; Glasman, Mirta; Naga, Shanmugam; Eidelman, Ofer; Ried, Thomas; Pollard, Harvey B.

2003-01-01

Annexin 7 (ANX7) acts as a tumor suppressor gene in prostate cancer, where loss of heterozygosity and reduction of ANX7 protein expression is associated with aggressive metastatic tumors. To investigate the mechanism by which this gene controls tumor development, we have developed an Anx7(+/-) knockout mouse. As hypothesized, the Anx7(+/-) mouse has a cancer-prone phenotype. The emerging tumors express low levels of Anx7 protein. Nonetheless, the wild-type Anx7 allele is detectable in laser-capture microdissection-derived tumor tissue cells. Genome array analysis of hepatocellular carcinoma tissue indicates that the Anx7(+/-) genotype is accompanied by profound reductions of expression of several other tumor suppressor genes, DNA repair genes, and apoptosis-related genes. In situ analysis by tissue imprinting from chromosomes in the primary tumor and spectral karyotyping analysis of derived cell lines identify chromosomal instability and clonal chromosomal aberrations. Furthermore, whereas 23% of the mutant mice develop spontaneous neoplasms, all mice exhibit growth anomalies, including gender-specific gigantism and organomegaly. We conclude that haploinsufficiency of Anx7 expression appears to drive disease progression to cancer because of genomic instability through a discrete signaling pathway involving other tumor suppressor genes, DNA-repair genes, and apoptosis-related genes. PMID:14608035

Tissue distribution and cell tropism of Brucella canis in naturally infected canine foetuses and neonates.

PubMed

de Souza, Tayse Domingues; de Carvalho, Tatiane Furtado; Mol, Juliana Pinto da Silva; Lopes, João Vítor Menezes; Silva, Monique Ferreira; da Paixão, Tatiane Alves; Santos, Renato Lima

2018-05-08

Brucella canis infection is an underdiagnosed zoonotic disease. Knowledge about perinatal brucellosis in dogs is extremely limited, although foetuses and neonates are under risk of infection due to vertical transmission. In this study, immunohistochemistry was used to determine tissue distribution and cell tropism of B. canis in canine foetuses and neonates. Diagnosis of B. canis in tissues of naturally infected pups was based on PCR and sequencing of amplicons, bacterial isolation, and immunohistochemistry, whose specificity was confirmed by laser capture microdissection. PCR positivity among 200 puppies was 21%, and nine isolates of B. canis were obtained. Tissues from 13 PCR-positive puppies (4 stillborn and 9 neonates) presented widespread immunolabeling. Stomach, intestines, kidney, nervous system, and umbilicus were positive in all animals tested. Other frequently infected organs included the liver (92%), lungs (85%), lymph nodes (69%), and spleen (62%). Immunolabeled coccobacilli occurred mostly in macrophages, but they were also observed in erythrocytes, epithelial cells of gastrointestinal mucosa, renal tubules, epidermis, adipocytes, choroid plexus, ependyma, neuroblasts, blood vessels endothelium, muscle cells, and in the intestinal lumen. These results largely expand our knowledge about perinatal brucellosis in the dog, clearly demonstrating a pantropic distribution of B. canis in naturally infected foetuses and neonates.

Mutational analysis of multiple lung cancers: Discrimination between primary and metastatic lung cancers by genomic profile.

PubMed

Goto, Taichiro; Hirotsu, Yosuke; Mochizuki, Hitoshi; Nakagomi, Takahiro; Shikata, Daichi; Yokoyama, Yujiro; Oyama, Toshio; Amemiya, Kenji; Okimoto, Kenichiro; Omata, Masao

2017-05-09

In cases of multiple lung cancers, individual tumors may represent either a primary lung cancer or both primary and metastatic lung cancers. Treatment selection varies depending on such features, and this discrimination is critically important in predicting prognosis. The present study was undertaken to determine the efficacy and validity of mutation analysis as a means of determining whether multiple lung cancers are primary or metastatic in nature. The study involved 12 patients who underwent surgery in our department for multiple lung cancers between July 2014 and March 2016. Tumor cells were collected from formalin-fixed paraffin-embedded tissues of the primary lesions by using laser capture microdissection, and targeted sequencing of 53 lung cancer-related genes was performed. In surgically treated patients with multiple lung cancers, the driver mutation profile differed among the individual tumors. Meanwhile, in a case of a solitary lung tumor that appeared after surgery for double primary lung cancers, gene mutation analysis using a bronchoscopic biopsy sample revealed a gene mutation profile consistent with the surgically resected specimen, thus demonstrating that the tumor in this case was metastatic. In cases of multiple lung cancers, the comparison of driver mutation profiles clarifies the clonal origin of the tumors and enables discrimination between primary and metastatic tumors.

Somatostatin, neuronal vulnerability and behavioral emotionality.

PubMed

Lin, L C; Sibille, E

2015-03-01

Somatostatin (SST) deficits are common pathological features in depression and other neurological disorders with mood disturbances, but little is known about the contribution of SST deficits to mood symptoms or causes of these deficits. Here we show that mice lacking SST (Sst(KO)) exhibit elevated behavioral emotionality, high basal plasma corticosterone and reduced gene expression of Bdnf, Cortistatin and Gad67, together recapitulating behavioral, neuroendocrine and molecular features of human depression. Studies in Sst(KO) and heterozygous (Sst(HZ)) mice show that elevated corticosterone is not sufficient to reproduce the behavioral phenotype, suggesting a putative role for Sst cell-specific molecular changes. Using laser capture microdissection, we show that cortical SST-positive interneurons display significantly greater transcriptome deregulations after chronic stress compared with pyramidal neurons. Protein translation through eukaryotic initiation factor 2 (EIF2) signaling, a pathway previously implicated in neurodegenerative diseases, was most affected and suppressed in stress-exposed SST neurons. We then show that activating EIF2 signaling through EIF2 kinase inhibition mitigated stress-induced behavioral emotionality in mice. Taken together, our data suggest that (1) low SST has a causal role in mood-related phenotypes, (2) deregulated EIF2-mediated protein translation may represent a mechanism for vulnerability of SST neurons and (3) that global EIF2 signaling has antidepressant/anxiolytic potential.

Somatostatin, neuronal vulnerability and behavioral emotionality

PubMed Central

Lin, LC; Sibille, E

2014-01-01

Somatostatin (SST) deficits are common pathological features in depression and other neurological disorders with mood disturbances, but little is known about the contribution of SST deficits to mood symptoms or causes of these deficits. Here we show that mice lacking Sst (SstKO) exhibit elevated behavioral emotionality, high basal plasma corticosterone and reduced gene expression of Bdnf, Cortistatin, and Gad67, together recapitulating behavioral, neuroendocrine and molecular features of human depression. Studies in SstKO and heterozygous (SstHZ) mice show that elevated corticosterone is not sufficient to reproduce the behavioral phenotype, suggesting a putative role for Sst cell-specific molecular changes. Using laser-capture microdissection, we show that cortical SST-positive interneurons display significantly greater transcriptome deregulations after chronic stress compared to pyramidal neurons. Protein translation through eukaryotic initiation factor 2 (EIF2) signaling, a pathway previously implicated in neurodegenerative diseases, was most affected and suppressed in stress-exposed SST neurons. We then show that activating EIF2 signaling through EIF2 kinase inhibition mitigated stress-induced behavioral emotionality in mice. Together, our data suggest that (1) low SST plays a causal role in mood-related phenotypes, (2) deregulated EIF2-mediated protein translation may represent a mechanism for vulnerability of SST neurons, and (3) that global EIF2 signaling has antidepressant/anxiolytic potential. PMID:25600109

Predominant mucosal expression of 5-HT4(+h) receptor splice variants in pig stomach and colon

PubMed Central

Priem, Evelien KV; De Maeyer, Joris H; Vandewoestyne, Mado; Deforce, Dieter; Lefebvre, Romain A

2013-01-01

AIM: To investigate cellular 5-HT4(-h/+h) receptor distribution, particularly in the epithelial layer, by laser microdissection and polymerase chain reaction (PCR) in porcine gastrointestinal (GI) tissues. METHODS: A stepwise approach was used to evaluate RNA quality and to study cell-specific 5-HT4 receptor mRNA expression in the porcine gastric fundus and colon descendens. After freezing, staining and laser microdissection and pressure catapulting (LMPC), RNA quality was evaluated by the Experion automated electrophoresis system. 5-HT4 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expressions were examined by endpoint reverse transcription (RT)-PCR in mucosal and muscle-myenteric plexus (MMP) tissue fractions, in mucosal and MMP parts of hematoxylin and eosin (HE) stained tissue sections and in microdissected patches of the epithelial and circular smooth muscle cell layer in these sections. Pig gastric fundus tissue sections were also stained immunohistochemically (IHC) for enterochromaffin cells (EC cells; MAB352); these cells were isolated by LMPC and examined by endpoint RT-PCR. RESULTS: After HE staining, the epithelial and circular smooth muscle cell layer of pig colon descendens and the epithelial cell layer of gastric fundus were identified morphologically and isolated by LMPC. EC cells of pig gastric fundus were successfully stained by IHC and isolated by LMPC. Freezing, HE and IHC staining, and LMPC had no influence on RNA quality. 5-HT4 receptor and GAPDH mRNA expressions were detected in mucosa and MMP tissue fractions, and in mucosal and MMP parts of HE stained tissue sections of pig colon descendens and gastric fundus. In the mucosa tissue fractions of both GI regions, the expression of h-exon containing receptor [5-HT4(+h) receptor] mRNA was significantly higher (P < 0.01) compared to 5-HT4(-h) receptor expression, and a similar trend was obtained in the mucosal part of HE stained tissue sections. Large microdissected patches of the epithelial and circular smooth muscle cell layer of pig colon descendens and of the epithelial cell layer of pig gastric fundus, also showed 5-HT4 receptor and GAPDH mRNA expression. No 5-HT4 receptor mRNA expression was detected in gastric LMPC-isolated EC cells from IHC stained tissues, which cells were positive for GAPDH. CONCLUSION: Porcine GI mucosa predominantly expresses 5-HT4(+h) receptor splice variants, suggesting their contribution to the 5-HT4 receptor-mediated mucosal effects of 5-HT. PMID:23840113

NASA/Howard University Large Space Structures Institute

NASA Technical Reports Server (NTRS)

Broome, T. H., Jr.

1984-01-01

Basic research on the engineering behavior of large space structures is presented. Methods of structural analysis, control, and optimization of large flexible systems are examined. Topics of investigation include the Load Correction Method (LCM) modeling technique, stabilization of flexible bodies by feedback control, mathematical refinement of analysis equations, optimization of the design of structural components, deployment dynamics, and the use of microprocessors in attitude and shape control of large space structures. Information on key personnel, budgeting, support plans and conferences is included.

DoD Life Cycle Management (LCM) and Product Support Manager (PSM) Rapid Deployment Training

DTIC Science & Technology

2010-10-01

fielding, sustainment, and disposal of a DOD system across its life cycle.” (JCIDS Operation Manual) • “The PM shall be the single point of...devote more funds to development and procurement in order to modernize weapon systems . But, in fact, growth in operating and support costs has limited the...Requirements Differently could Reduce Weapon Systems ’ Total Ownership Costs The DoD “Death Spiral” (Source: Dr. Jacques S. Gansler, USD(A&T

Final EIS for the Proposed Homeporting of Additional Surface Ships at Naval Station, Mayport, FL. Volume 1. Final Environmental Impact Statement

DTIC Science & Technology

2008-11-21

to air squadrons. 4. USS John F. Kennedy was decommissioned in 2007. Source: Adapted from DoN 2006a Final EIS for the Proposed Homeporting of...Tugs Slot Small Craft 650 35 YD/YC/LCM Source: Adapted from Naval Facilities Engineering Service Center 2002 MCM = Mine Countermeasures DDG...It would include nonradiologically controlled spaces for administrative and other support functions. The design would be a site- adapted replication of

Studies on the Inhalation Toxicity of Dyes Present in Colored Smoke Munitions. Phase IV. 90-Day Inhalation Exposures of Rats to Dye Aerosols

DTIC Science & Technology

1985-10-01

virus , rat corona - virus /Sialodacryodenitis virus (RCV/SOAV), Ir Reovirus type 3 (Reo 3), Theiler’s encephalomyelitis virus (GD VII), Toolan’s H-I virus (H...1), Kilham rat virus (KRV). Lymphocytic choriomeningitis (LCM) and Mvcoplasma pulmonis. Bacterial cultures are also performed on swabs or tissue... Cats _____ ___ -.--oci of Mononuclear Cells11 .:._Lubular Mineral ization 12221122 33 -LUNG __ __ SHemorrhage Pipment Deposition ::Hyperplasia of

Microdissection and chromosome painting of the alien chromosome in an addition line of wheat-Thinopyrum intermedium

USDA-ARS?s Scientific Manuscript database

The chromosome painting is an efficient tool for chromosome research. However, plant chromosome painting is relatively underdeveloped. In this study, chromosome painting was developed and used to identify alien chromosomes in TAi-27, a wheat-Thinopyrum intermedium addition line, and chromosomes of...

Exploitation of olive mill wastewater and liquid cow manure for biogas production

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

Dareioti, Margarita A.; Dokianakis, Spyros N.; Stamatelatou, Katerina

2010-10-15

Co-digestion of organic waste streams is an innovative technology for the reduction of methane/greenhouse gas emissions. Different organic substrates are combined to generate a homogeneous mixture as input to the anaerobic reactor in order to increase process performance, realize a more efficient use of equipment and cost-sharing by processing multiple waste streams in a single facility. In this study, the potential of anaerobic digestion for the treatment of a mixture containing olive mill wastewater (OMW) and liquid cow manure (LCM) using a two-stage process has been evaluated by using two continuously stirred tank reactors (CSTRs) under mesophilic conditions (35 {supmore » o}C) in order to separately monitor and control the processes of acidogenesis and methanogenesis. The overall process was studied with a hydraulic retention time (HRT) of 19 days. The digester was continuously fed with an influent composed (v/v) of 20% OMW and 80% LCM. The average removal of dissolved and total COD was 63.2% and 50%, respectively. The volatile solids (VS) removal was 34.2% for the examined mixture of feedstocks operating the system at an overall OLR of 3.63 g CODL{sub reactor}{sup -1}d{sup -1}. Methane production rate at the steady state reached 0.91 L CH{sub 4}L{sub reactor}{sup -1}d{sup -1} or 250.9 L CH{sub 4} at standard temperature and pressure conditions (STP) per kg COD fed to the system.« less

A Flexible Framework Hydrological Informatic Modeling System - HIMS

NASA Astrophysics Data System (ADS)

WANG, L.; Wang, Z.; Changming, L.; Li, J.; Bai, P.

2017-12-01

Simulating water cycling process temporally and spatially fitting for the characteristics of the study area was important for floods prediction and streamflow simulation with high accuracy, as soil properties, land scape, climate, and land managements were the critical factors influencing the non-linear relationship of rainfall-runoff at watershed scales. Most existing hydrological models cannot simulate water cycle process at different places with customized mechanisms with fixed single structure and mode. This study develops Hydro-Informatic Modeling System (HIMS) model with modular of each critical hydrological process with multiple choices for various scenarios to solve this problem. HIMS has the structure accounting for two runoff generation mechanisms of infiltration excess and saturation excess and estimated runoff with different methods including Time Variance Gain Model (TVGM), LCM which has good performance at ungauged areas, besides the widely used Soil Conservation Service-Curve Number (SCS-CN) method. Channel routing model contains the most widely used Muskingum, and kinematic wave equation with new solving method. HIMS model performance with its symbolic runoff generation model LCM was evaluated through comparison with the observed streamflow datasets of Lasha river watershed at hourly, daily, and monthly time steps. Comparisons between simulational and obervational streamflows were found with NSE higher than 0.87 and WE within ±20%. Water balance analysis about precipitation, streamflow, actual evapotranspiration (ET), and soil moisture change was conducted temporally at annual time step and it has been proved that HIMS model performance was reliable through comparison with literature results at the Lhasa River watershed.

Deforestation projections for carbon-rich peat swamp forests of Central Kalimantan, Indonesia.

PubMed

Fuller, Douglas O; Hardiono, Martin; Meijaard, Erik

2011-09-01

We evaluated three spatially explicit land use and cover change (LUCC) models to project deforestation from 2005-2020 in the carbon-rich peat swamp forests (PSF) of Central Kalimantan, Indonesia. Such models are increasingly used to evaluate the impact of deforestation on carbon fluxes between the biosphere and the atmosphere. We considered both business-as-usual (BAU) and a forest protection scenario to evaluate each model's accuracy, sensitivity, and total projected deforestation and landscape-level fragmentation patterns. The three models, Dinamica EGO (DE), GEOMOD and the Land Change Modeler (LCM), projected similar total deforestation amounts by 2020 with a mean of 1.01 million ha (Mha) and standard deviation of 0.17 Mha. The inclusion of a 0.54 Mha strict protected area in the LCM simulations reduced projected loss to 0.77 Mha over 15 years. Calibrated parameterizations of the models using nearly identical input drivers produced very different landscape properties, as measured by the number of forest patches, mean patch area, contagion, and Euclidean nearest neighbor determined using Fragstats software. The average BAU outputs of the models suggests that Central Kalimantan may lose slightly less than half (45.1%) of its 2005 PSF by 2020 if measures are not taken to reduce deforestation there. The relatively small reduction of 0.24 Mha in deforestation found in the 0.54 Mha protection scenario suggests that these models can identify potential leakage effects in which deforestation is forced to occur elsewhere in response to a policy intervention.

Data model for the collaboration between land administration systems and agricultural land parcel identification systems.

PubMed

Inan, Halil Ibrahim; Sagris, Valentina; Devos, Wim; Milenov, Pavel; van Oosterom, Peter; Zevenbergen, Jaap

2010-12-01

The Common Agricultural Policy (CAP) of the European Union (EU) has dramatically changed after 1992, and from then on the CAP focused on the management of direct income subsidies instead of production-based subsidies. For this focus, Member States (MS) are expected to establish Integrated Administration and Control System (IACS), including a Land Parcel Identification System (LPIS) as the spatial part of IACS. Different MS have chosen different solutions for their LPIS. Currently, some MS based their IACS/LPIS on data from their Land Administration Systems (LAS), and many others use purpose built special systems for their IACS/LPIS. The issue with these different IACS/LPIS is that they do not have standardized structures; rather, each represents a unique design in each MS, both in the case of LAS based or special systems. In this study, we aim at designing a core data model for those IACS/LPIS based on LAS. For this purpose, we make use of the ongoing standardization initiatives for LAS (Land Administration Domain Model: LADM) and IACS/LPIS (LPIS Core Model: LCM). The data model we propose in this study implies the collaboration between LADM and LCM and includes some extensions. Some basic issues with the collaboration model are discussed within this study: registration of farmers, land use rights and farming limitations, geometry/topology, temporal data management etc. For further explanation of the model structure, sample instance level diagrams illustrating some typical situations are also included. Copyright © 2010 Elsevier Ltd. All rights reserved.

Effective isolation of primo vessels in lymph using sound- and ultrasonic-wave stimulation.

PubMed

Park, Do-Young; Lee, Hye-Rie; Rho, Min-Suk; Lee, Sang-Suk

2014-12-01

The effects of stimulation with sound and ultrasonic waves of a specific bandwidth on the microdissection of primo vessels in lymphatic vessels of rabbit were investigated. The primo vessels stained with alcian-blue dye injected in the lymph nodes were definitely visualized and more easily isolated by sound-wave vibration and ultrasonic stimulation applied to rabbits at various frequencies and intensities. With sound wave at 7 Hz and ultrasonic waves at 2 MHz, the probability of detecting the primo vessels was improved to 90%; however, without wave stimulation the probability of discovering primo vessels was about 50% only. Sound and ultrasonic waves at specific frequency bands should be effective for microdissection of the primo vessels in the abdominal lymph of rabbit. We suggest that oscillation of the primo vessels by sound and ultrasonic waves may be useful to visualize specific primo structure, and wave vibration can be a very supportive process for observation and isolation of the primo vessels of rabbits. Copyright © 2014. Published by Elsevier B.V.

MICRODISSECTION TESTICULAR SPERM EXTRACTION IN MEN WITH SERTOLI CELL ONLY TESTICULAR HISTOLOGY

PubMed Central

Berookhim, Boback M.; Palermo, Gianpiero D.; Zaninovic, Nikica; Rosenwaks, Zev; Schlegel, Peter N.

2015-01-01

Objective To study the outcomes of microdissection testicular sperm extraction (microTESE) among men with pure Sertoli cell only histology on diagnostic testicular biopsy. Design Retrospective cohort study. Setting Tertiary referral center. Patients 640 patients with pure Sertoli cell only histology on testicular biopsy who underwent microTESE by a single surgeon. Intervention MicroTESE. Main Outcome Measure Sperm retrieval rates. Results Overall, 44.5% of patients with Sertoli cell-only had sperm retrieved with microTESE. No difference was noted in sperm retrieval rates based on testis volume (≥ 15cc versus <15cc, 35.3% versus 46.1%, respectively). Patients with ≥ 15cc testicular volume and FSH 10-15 mU/mL had the worst prognosis, with a sperm retrieval rate of 6.7%. Conclusions Patients with previous testicular biopsy demonstrating Sertoli cell only histology can be counseled that they have a reasonable likelihood of sperm retrieval with the contemporary delivery of microTESE. Given this finding, the utility of testicular biopsy prior to microTESE is further questioned. PMID:25441063

Proteomic Analysis of Laser Microdissected Melanoma Cells from Skin Organ Cultures

PubMed Central

Hood, Brian L.; Grahovac, Jelena; Flint, Melanie S.; Sun, Mai; Charro, Nuno; Becker, Dorothea; Wells, Alan; Conrads, Thomas P

2010-01-01

Gaining insights into the molecular events that govern the progression from melanoma in situ to advanced melanoma, and understanding how the local microenvironment at the melanoma site influences this progression, are two clinically pivotal aspects that to date are largely unexplored. In an effort to identify key regulators of the crosstalk between melanoma cells and the melanoma-skin microenvironment, primary and metastatic human melanoma cells were seeded into skin organ cultures (SOCs), and grown for two weeks. Melanoma cells were recovered from SOCs by laser microdissection and whole-cell tryptic digests analyzed by nanoflow liquid chromatography-tandem mass spectrometry with an LTQ-Orbitrap. The differential protein abundances were calculated by spectral counting, the results of which provides evidence that cell-matrix and cell-adhesion molecules that are upregulated in the presence of these melanoma cells recapitulate proteomic data obtained from comparative analysis of human biopsies of invasive melanoma and a tissue sample of adjacent, non-involved skin. This concordance demonstrates the value of SOCs for conducting proteomic investigations of the melanoma microenvironment. PMID:20459140

Laser Microdissection and Spatiotemporal Pinoresinol-Lariciresinol Reductase Gene Expression Assign the Cell Layer-Specific Accumulation of Secoisolariciresinol Diglucoside in Flaxseed Coats.

PubMed

Fang, Jingjing; Ramsay, Aïna; Renouard, Sullivan; Hano, Christophe; Lamblin, Frédéric; Chabbert, Brigitte; Mesnard, François; Schneider, Bernd

2016-01-01

The concentration of secoisolariciresinol diglucoside (SDG) found in flaxseed ( Linum usitatissimum L.) is higher than that found in any other plant. It exists in flaxseed coats as an SDG-3-hydroxy-3-methylglutaric acid oligomer complex. A laser microdissection method was applied to harvest material from different cell layers of seed coats of mature and developing flaxseed to detect the cell-layer specific localization of SDG in flaxseed; NMR and HPLC were used to identify and quantify SDG in dissected cell layers after alkaline hydrolysis. The obtained results were further confirmed by a standard molecular method. The promoter of one pinoresinol-lariciresinol reductase gene of L. usitatissimum ( LuPLR1 ), which is a key gene involved in SDG biosynthesis, was fused to a β-glucuronidase ( GUS ) reporter gene, and the spatio-temporal regulation of LuPLR1 gene expression in flaxseed was determined by histochemical and activity assays of GUS . The result showed that SDG was synthesized and accumulated in the parenchymatous cell layer of the outer integument of flaxseed coats.

Laser Microdissection and Spatiotemporal Pinoresinol-Lariciresinol Reductase Gene Expression Assign the Cell Layer-Specific Accumulation of Secoisolariciresinol Diglucoside in Flaxseed Coats

PubMed Central

Fang, Jingjing; Ramsay, Aïna; Renouard, Sullivan; Hano, Christophe; Lamblin, Frédéric; Chabbert, Brigitte; Mesnard, François; Schneider, Bernd

2016-01-01

The concentration of secoisolariciresinol diglucoside (SDG) found in flaxseed (Linum usitatissimum L.) is higher than that found in any other plant. It exists in flaxseed coats as an SDG-3-hydroxy-3-methylglutaric acid oligomer complex. A laser microdissection method was applied to harvest material from different cell layers of seed coats of mature and developing flaxseed to detect the cell-layer specific localization of SDG in flaxseed; NMR and HPLC were used to identify and quantify SDG in dissected cell layers after alkaline hydrolysis. The obtained results were further confirmed by a standard molecular method. The promoter of one pinoresinol-lariciresinol reductase gene of L. usitatissimum (LuPLR1), which is a key gene involved in SDG biosynthesis, was fused to a β-glucuronidase (GUS) reporter gene, and the spatio-temporal regulation of LuPLR1 gene expression in flaxseed was determined by histochemical and activity assays of GUS. The result showed that SDG was synthesized and accumulated in the parenchymatous cell layer of the outer integument of flaxseed coats. PMID:27917190

Expression analysis of human salivary glands by laser microdissection: differences between submandibular and labial glands.

PubMed

Kouznetsova, Irina; Gerlach, Klaus L; Zahl, Christian; Hoffmann, Werner

2010-01-01

Both the major and minor salivary glands are the sources of saliva, a fluid vital for the maintenance of a healthy oral cavity. Here, the expression profiles of human submandibular (SMG) and labial glands (LG) were compared by RT-PCR analysis of laser microdissected mucous and serous cells, respectively. The focus was on trefoil factor family (TFF) genes, but also other genes encoding secretory proteins (mucins, lysozyme, amylase, statherin, and histatins) or aquaporin 5 were included. Immunofluorescence studies concerning TFF1-3, FCGBP, amylase, and lysozyme are also presented. It was shown that LGs clearly contain serous cells and that these cells differ in their expression profiles from serous SMG cells. Furthermore, all three TFF peptides, together with MUC5B, MUC7, MUC19, and FCGBP, were clearly detectable in mucous acini of both LGs and SMGs. In contrast, lysozyme was differentially expressed in LGs and SMGs. It can be expected that labial saliva may play a particularly important role for protecting the teeth. Copyright 2010 S. Karger AG, Basel.

IN SITU DEMONSTRATION OF DNA HYBRIDIZING WITH CHROMOSOMAL AND NUCLEAR SAP RNA IN CHIRONOMUS TENTANS

PubMed Central

Lambert, B.; Wieslander, L.; Daneholt, B.; Egyházi, E.; Ringborg, U.

1972-01-01

Cytological hybridization combined with microdissection of Chironomus tentans salivary gland cells was used to locate DNA complementary to newly synthesized RNA from chromosomes and nuclear sap and from a single chromosomal puff, the Balbiani ring 2 (BR 2). Salivary glands were incubated with tritiated nucleosides. The labeled RNA was extracted from microdissected nuclei and hybridized to denatured squash preparations of salivary gland cells under conditions which primarily allow repeated sequences to interact. The bound RNA, resistant to ribonuclease treatment, was detected radioautographically. It was found that BR 2 RNA hybridizes specifically with the BR 2 region of chromosome IV. Nuclear sap RNA was fractionated into high and low molecular-weight RNA; the former hybridizes with the BR 2 region of chromosome IV, the latter in a diffuse distribution over the whole chromosome set. RNA from chromosome I hybridizes diffusely with all chromosomes. Nucleolar RNA hybridizes specifically with the nucleolar organizers, contained in chromosomes II and III. It is concluded that the BR 2 region of chromosome IV contains repeated DNA sequences and that nuclear sap contains BR 2 RNA. PMID:5025107

Molecular pathology of primary intraocular lymphoma.

PubMed Central

Chan, Chi-Chao

2003-01-01

PURPOSE: To evaluate immunoglobulin heavy chain (IgH) gene rearrangements, cytokines and chemokines, and infectious agents in primary intraocular B-cell lymphoma (PIOL) cells, in order to better diagnose and understand PIOL. METHODS: We studied ocular specimens from 57 patients with PIOL at the National Eye Institute from 1991 to 2001. Specimens were analyzed for IgH gene rearrangements using microdissection and polymerase chain reaction (PCR). We measured vitreal interleukin (IL)-10 and IL-6 levels by enzyme-linked immunosorbent assay. IL-10 mRNA was studied in PIOL cells using microdissection and reverse transcribed (RT)-PCR. Chemokine and chemokine receptor expression was examined by using immunohistochemistry. Infectious DNA of human herpetic virus-8 (HHV-8), Epstein-Bar virus (EBV), and Toxoplasma gondii was detected by using microdissection and PCR and was confirmed with Southern blot hybridization. RESULTS: IgH rearrangement(s) were demonstrated in all 50 tested cases. Cytokine levels were measured in the vitreous of 39 patients. Thirty-one had measurable cytokine levels: 24 of 31 had elevation of IL-10 relative to that of IL-6, and, in contrast, only 7 of 31 had elevation of IL-6 relative to IL-10. IL-10 mRNA was abundant in lymphoma cells of 6 examined cases. Lymphoma cells expressed chemokine receptors of CXCR4 and CXCR5 in three tested cases. HHV-8 DNA was found in 6 of 32 cases (18.8%), EBV DNA in 2 of 21 (9.5%), and T gondii DNA in 2 of 16 (12.5%). CONCLUSIONS: Molecular analyses detecting IgH rearrangements and vitreal levels of IL-10 and IL-6 are useful adjuncts for PIOL diagnosis. A role for specific infectious agents is hypothesized in the pathogenesis of some cases of PIOL. B-cell chemokine is likely involved in attracting PIOL cells into the eye. PMID:14971583

Single Cell Immuno-Laser Microdissection Coupled to Label-Free Proteomics to Reveal the Proteotypes of Human Brain Cells After Ischemia.

PubMed

García-Berrocoso, Teresa; Llombart, Víctor; Colàs-Campàs, Laura; Hainard, Alexandre; Licker, Virginie; Penalba, Anna; Ramiro, Laura; Simats, Alba; Bustamante, Alejandro; Martínez-Saez, Elena; Canals, Francesc; Sanchez, Jean-Charles; Montaner, Joan

2018-01-01

Cerebral ischemia entails rapid tissue damage in the affected brain area causing devastating neurological dysfunction. How each component of the neurovascular unit contributes or responds to the ischemic insult in the context of the human brain has not been solved yet. Thus, the analysis of the proteome is a straightforward approach to unraveling these cell proteotypes. In this study, post-mortem brain slices from ischemic stroke patients were obtained corresponding to infarcted (IC) and contralateral (CL) areas. By means of laser microdissection, neurons and blood brain barrier structures (BBB) were isolated and analyzed using label-free quantification. MS data are available via ProteomeXchange with identifier PXD003519. Ninety proteins were identified only in neurons, 260 proteins only in the BBB and 261 proteins in both cell types. Bioinformatics analyses revealed that repair processes, mainly related to synaptic plasticity, are outlined in microdissected neurons, with nonexclusive important functions found in the BBB. A total of 30 proteins showing p < 0.05 and fold-change> 2 between IC and CL areas were considered meaningful in this study: 13 in neurons, 14 in the BBB and 3 in both cell types. Twelve of these proteins were selected as candidates and analyzed by immunohistofluorescence in independent brains. The MS findings were completely verified for neuronal SAHH2 and SRSF1 whereas the presence in both cell types of GABT and EAA2 was only validated in neurons. In addition, SAHH2 showed its potential as a prognostic biomarker of neurological improvement when analyzed early in the plasma of ischemic stroke patients. Therefore, the quantitative proteomes of neurons and the BBB (or proteotypes) after human brain ischemia presented here contribute to increasing the knowledge regarding the molecular mechanisms of ischemic stroke pathology and highlight new proteins that might represent putative biomarkers of brain ischemia or therapeutic targets. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Doping-Induced Type-II to Type-I Transition and Interband Optical Gain in InAs/AlSb Quantum Wells

NASA Technical Reports Server (NTRS)

Kolokolov, K. I.; Ning, C. Z.

2003-01-01

We show that proper doping of the barrier regions can convert the well-known type-II InAs/AlSb QWs to type I, producing strong interband transitions comparable to regular type-I QWs. The interband gain for TM mode is as high as 4000 l/cm, thus providing an important alternative material system in the mid-infrared wavelength range. We also study the TE and TM gain as functions of doping level and intrinsic electron-hole density.

Primary Radiometry for the mise-en-pratique: The Laser-Based Radiance Method Applied to a Pyrometer

NASA Astrophysics Data System (ADS)

Briaudeau, S.; Sadli, M.; Bourson, F.; Rougi, B.; Rihan, A.; Zondy, J.-J.

2011-12-01

A new setup has been implemented at LCM-LNE-CNAM for the determination "of the spectral responsivity of radiation thermometers for the determination" of the thermodynamic temperature of high-temperature blackbodies at the temperature of a metal-carbon eutectic phase transition. In this new setup, an innovative acoustic-optic modulator feedback loop is used to stabilize the radiance of a wavelength tunable laser. The effect of residual optical interferences on the calibration of a test pyrometer is analyzed. The full uncertainty budget is presented.

Microdissection of Black Widow Spider Silk-producing Glands

PubMed Central

Hsia, Yang; Gnesa, Eric; Zhao, Liang; Franz, Andreas; Vierra, Craig

2011-01-01

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion. Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands. Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments. PMID:21248709

78 FR 28229 - Prospective Grant of Exclusive License: Device and System for Expression Microdissection (xMD)

Federal Register 2010, 2011, 2012, 2013, 2014

2013-05-14

... applications and foreign counterparts to xMD Diagnostics, LLC, a company having a place of business in Maryland... limited to the following below. ``Devices, systems, kits and related consumables, and methods using.... Methods, kits, and related consumables that are used independent of the devices or systems by individual...

Lymphotoxin β receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE−/− mice

PubMed Central

Gräbner, Rolf; Lötzer, Katharina; Döpping, Sandra; Hildner, Markus; Radke, Dörte; Beer, Michael; Spanbroek, Rainer; Lippert, Beatrix; Reardon, Catherine A.; Getz, Godfrey S.; Fu, Yang-Xin; Hehlgans, Thomas; Mebius, Reina E.; van der Wall, Michael; Kruspe, Dagmar; Englert, Christoph; Lovas, Agnes; Hu, Desheng; Randolph, Gwendalyn J.; Weih, Falk; Habenicht, Andreas J.R.

2009-01-01

Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE−/− mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin β receptor (LTβR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE−/− mice with LTβR-Ig to interrupt LTβR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTβR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall. PMID:19139167

Lymphotoxin beta receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE-/- mice.

PubMed

Gräbner, Rolf; Lötzer, Katharina; Döpping, Sandra; Hildner, Markus; Radke, Dörte; Beer, Michael; Spanbroek, Rainer; Lippert, Beatrix; Reardon, Catherine A; Getz, Godfrey S; Fu, Yang-Xin; Hehlgans, Thomas; Mebius, Reina E; van der Wall, Michael; Kruspe, Dagmar; Englert, Christoph; Lovas, Agnes; Hu, Desheng; Randolph, Gwendalyn J; Weih, Falk; Habenicht, Andreas J R

2009-01-16

Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE(-/-) mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin beta receptor (LTbetaR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE(-/-) mice with LTbetaR-Ig to interrupt LTbetaR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTbetaR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

Thymidylate synthase (TS) gene expression in primary lung cancer patients: a large-scale study in Japanese population.

PubMed

Tanaka, F; Wada, H; Fukui, Y; Fukushima, M

2011-08-01

Previous small-sized studies showed lower thymidylate synthase (TS) expression in adenocarcinoma of the lung, which may explain higher antitumor activity of TS-inhibiting agents such as pemetrexed. To quantitatively measure TS gene expression in a large-scale Japanese population (n = 2621) with primary lung cancer, laser-captured microdissected sections were cut from primary tumors, surrounding normal lung tissues and involved nodes. TS gene expression level in primary tumor was significantly higher than that in normal lung tissue (mean TS/β-actin, 3.4 and 1.0, respectively; P < 0.01), and TS gene expression level was further higher in involved node (mean TS/β-actin, 7.7; P < 0.01). Analyses of TS gene expression levels in primary tumor according to histologic cell type revealed that small-cell carcinoma showed highest TS expression (mean TS/β-actin, 13.8) and that squamous cell carcinoma showed higher TS expression as compared with adenocarcinoma (mean TS/β-actin, 4.3 and 2.3, respectively; P < 0.01); TS gene expression was significantly increased along with a decrease in the grade of tumor cell differentiation. There was no significant difference in TS gene expression according to any other patient characteristics including tumor progression. Lower TS expression in adenocarcinoma of the lung was confirmed in a large-scale study.

Expression of the Wilm's tumor gene WT1 during diaphragmatic development in the nitrofen model for congenital diaphragmatic hernia.

PubMed

Dingemann, Jens; Doi, Takashi; Ruttenstock, Elke; Puri, Prem

2011-02-01

The nitrofen model of congenital diaphragmatic hernia (CDH) reproduces a typical diaphragmatic defect. However, the exact pathomechanism of CDH is still unknown. The Wilm's tumor 1 gene (WT1) is crucial for diaphragmatic development. Mutations in WT1 associated with CDH have been described in humans. Additionally, WT1(-/-) mice display CDH. Furthermore, WT1 is involved in the retinoid signaling pathway, a candidate pathway for CDH. We hypothesized that diaphragmatic WT1 gene expression is downregulated during diaphragmatic development in the nitrofen CDH model. Pregnant rats received vehicle or nitrofen on gestational day 9 (D9). Embryos were delivered on D13, D18 and D21. The pleuroperitoneal folds (PPFs) were dissected using laser capture microdissection (D13). Diaphragms of D18 and D21 were manually dissected. RNA was extracted and relative mRNA expression of WT1 was determined using real-time PCR. Immunofluorescence was performed to evaluate protein expression of WT1. Statistical significance was considered p < 0.05. Diaphragmatic mRNA expression of WT1 was significantly decreased in the nitrofen group on D13, D18 and D21. Intensity of immunofluorescencence of WT1 was markedly decreased in the CDH diaphragms on D13, D18 and D21. Downregulation of diaphragmatic WT1 gene expression may impair diaphragmatic development in the nitrofen CDH model.

The Pacific bluefin tuna (Thunnus orientalis) dead end gene is suitable as a specific molecular marker of type A spermatogonia.

PubMed

Yazawa, Ryosuke; Takeuchi, Yutaka; Morita, Tetsuro; Ishida, Masashi; Yoshizaki, Goro

2013-10-01

We developed a spermatogonial transplantation technique to produce donor-derived gametes in surrogate fish. Our ultimate aim is to establish surrogate broodstock that can produce bluefin tuna. We previously determined that only type A spermatogonia (ASG) could colonize recipient gonads in salmonids. Therefore, it is necessary to develop a precise molecular marker that can distinguish ASG in order to develop efficient spermatogonial transplantation methods. In this study, the Pacific bluefin tuna (Thunnus orientalis) dead end (BFTdnd) gene was identified as a specific marker for ASG. In situ hybridization and RT-PCR analysis with various types of spermatogenic cell populations captured by laser microdissection revealed that localization of BFTdnd mRNA was restricted to ASG, and not detected in other differentiated spermatogenic cells. In order to determine if BFTdnd can be used as a molecular marker to identify germ cells with high transplantability, transplantation of dissociated testicular cells isolated from juvenile, immature, and mature Pacific bluefin tuna, which have different proportions of dnd-positive ASG, were performed using chub mackerel as the surrogate recipient species. Colonization of transplanted donor germ cells was only successful with testicular cells from immature Pacific Bluefin tuna, which contained higher proportions of dnd-positive ASG than juvenile and mature fish. Thus, BFTdnd is a useful tool for identifying highly transplantable ASG for spermatogonial transplantation. © 2013 Wiley Periodicals, Inc.

Role of Cbl-PI3K Interaction during Skeletal Remodeling in a Murine Model of Bone Repair.

PubMed

Scanlon, Vanessa; Soung, Do Yu; Adapala, Naga Suresh; Morgan, Elise; Hansen, Marc F; Drissi, Hicham; Sanjay, Archana

2015-01-01

Mice in which Cbl is unable to bind PI3K (YF mice) display increased bone volume due to enhanced bone formation and repressed bone resorption during normal bone homeostasis. We investigated the effects of disrupted Cbl-PI3K interaction on fracture healing to determine whether this interaction has an effect on bone repair. Mid-diaphyseal femoral fractures induced in wild type (WT) and YF mice were temporally evaluated via micro-computed tomography scans, biomechanical testing, histological and histomorphometric analyses. Imaging analyses revealed no change in soft callus formation, increased bony callus formation, and delayed callus remodeling in YF mice compared to WT mice. Histomorphometric analyses showed significantly increased osteoblast surface per bone surface and osteoclast numbers in the calluses of YF fractured mice, as well as increased incorporation of dynamic bone labels. Furthermore, using laser capture micro-dissection of the fracture callus we found that cells lacking Cbl-PI3K interaction have higher expression of Osterix, TRAP, and Cathepsin K. We also found increased expression of genes involved in propagating PI3K signaling in cells isolated from the YF fracture callus, suggesting that the lack of Cbl-PI3K interaction perhaps results in enhanced PI3K signaling, leading to increased bone formation, but delayed remodeling in the healing femora.

Molecular Changes in the Nasal Cavity after N,N-Dimethyl-p-toluidine Exposure

PubMed Central

Dunnick, June K.; Merrick, B. Alex; Brix, Amy; Morgan, Daniel L.; Gerrish, Kevin; Wang, Yu; Flake, Gordon; Foley, Julie; Shockley, Keith R.

2016-01-01

N,N-Dimethyl-p-toluidine (DMPT) (Cas No. 99-97-8), an accelerant for methyl methacrylate monomers in medical devices, is a nasal cavity carcinogen in a 2-year cancer study in male and female F344/N rats, with the nasal tumors arising from the transitional cell epithelium. In this study we exposed male F344/N rats for five days to DMPT (0, 1, 6, 20, 60 or 120 mg/kg (oral gavage)) to explore early changes in the nasal cavity after short-term exposure. Lesions occurred in the nasal cavity including hyperplasia of transitional cell epithelium (60 and 120 mg/kg). Nasal tissue was rapidly removed and preserved for subsequent laser capture microdissection and isolation of the transitional cell epithelium (0 and 120 mg/kg) for transcriptomic studies. DMPT transitional cell epithelium gene transcript patterns were characteristic of an anti-oxidative damage response (e.g. Akr7a3, Maff, Mgst3), cell proliferation, and decrease in signals for apoptosis. Amino acid transporters transcripts were upregulated (e. g, Slc7a11). The DMPT nasal transcript expression pattern was similar to that found in the rat nasal cavity after formaldehyde exposure with over 1000 transcripts in common. Molecular changes in the nasal cavity after DMPT exposure suggest that oxidative damage is a mechanism for the DMPT toxic and/or carcinogenic effects. PMID:27099258

Intracellular Insulin-like Growth Factor-I Induces Bcl-2 Expression in Airway Epithelial Cells 1

PubMed Central

Chand, Hitendra S.; Harris, Jennifer Foster; Mebratu, Yohannes; Chen, Yangde; Wright, Paul S.; Randell, Scott H.; Tesfaigzi, Yohannes

2012-01-01

Bcl-2, a prosurvival protein, regulates programmed cell death during development and repair processes, and can be oncogenic when cell proliferation is deregulated. The present study investigated what factors modulate Bcl-2 expression in airway epithelial cells and identified the pathways involved. Microarray analysis of mRNA from airway epithelial cells captured by laser microdissection showed that increased expression of IL-1β and IGF-1 coincided with induced Bcl-2 expression compared to controls. Treatment of cultured airway epithelial cells with IL-1β and IGF-1 induced Bcl-2 expression by increasing Bcl-2 mRNA stability with no discernible changes in promoter activity. Silencing the IGF-1 expression using shRNA showed that intracellular (IC)-IGF-1 was increasing Bcl-2 expression. Blocking EGFR or IGF-1R activation also suppressed IC-IGF-1, and abolished the Bcl-2 induction. Induced expression and co-localization of IC-IGF-1 and Bcl-2 were observed in airway epithelial cells of mice exposed to LPS or cigarette smoke and of patients with cystic fibrosis and chronic bronchitis but not in the respective controls. These studies demonstrate that IC-IGF-1 induces Bcl-2 expression in epithelial cells via IGF-1R and EGFR pathways, and targeting IC-IGF-1 could be beneficial to treat chronic airway diseases. PMID:22461702

Bone-induced c-kit expression in prostate cancer: a driver of intraosseous tumor growth

PubMed Central

Mainetti, Leandro E.; Zhe, Xiaoning; Diedrich, Jonathan; Saliganan, Allen D.; Cho, Won Jin; Cher, Michael L.; Heath, Elisabeth; Fridman, Rafael; Kim, Hyeong-Reh Choi; Bonfil, R. Daniel

2014-01-01

Loss of BRCA2 function stimulates prostate cancer (PCa) cell invasion and is associated with more aggressive and metastatic tumors in PCa patients. Concurrently, the receptor tyrosine kinase c-kit is highly expressed in skeletal metastases of PCa patients and induced in PCa cells placed into the bone microenvironment in experimental models. However, the precise requirement of c-kit for intraosseous growth of PCa and its relation to BRCA2 expression remain unexplored. Here, we show that c-kit expression promotes migration and invasion of PCa cells. Alongside, we found that c-kit expression in PCa cells parallels BRCA2 downregulation. Gene rescue experiments with human BRCA2 transgene in c-kit-transfected PCa cells resulted in reduction of c-kit protein expression and migration and invasion, suggesting a functional significance of BRCA2 downregulation by c-kit. The inverse association between c-kit and BRCA2 gene expressions in PCa cells was confirmed using laser capture microdissection in experimental intraosseous tumors and bone metastases of PCa patients. Inhibition of bone-induced c-kit expression in PCa cells transduced with lentiviral short hairpin RNA reduced intraosseous tumor incidence and growth. Overall, our results provide evidence of a novel pathway that links bone-induced c-kit expression in PCa cells to BRCA2 downregulation and supports bone metastasis. PMID:24798488

Temporomandibular joint formation requires two distinct hedgehog-dependent steps.

PubMed

Purcell, Patricia; Joo, Brian W; Hu, Jimmy K; Tran, Pamela V; Calicchio, Monica L; O'Connell, Daniel J; Maas, Richard L; Tabin, Clifford J

2009-10-27

We conducted a genetic analysis of the developing temporo-mandibular or temporomandi-bular joint (TMJ), a highly specialized synovial joint that permits movement and function of the mammalian jaw. First, we used laser capture microdissection to perform a genome-wide expression analysis of each of its developing components. The expression patterns of genes identified in this screen were examined in the TMJ and compared with those of other synovial joints, including the shoulder and the hip joints. Striking differences were noted, indicating that the TMJ forms via a distinct molecular program. Several components of the hedgehog (Hh) signaling pathway are among the genes identified in the screen, including Gli2, which is expressed specifically in the condyle and in the disk of the developing TMJ. We found that mice deficient in Gli2 display aberrant TMJ development such that the condyle loses its growth-plate-like cellular organization and no disk is formed. In addition, we used a conditional strategy to remove Smo, a positive effector of the Hh signaling pathway, from chondrocyte progenitors. This cell autonomous loss of Hh signaling allows for disk formation, but the resulting structure fails to separate from the condyle. Thus, these experiments establish that Hh signaling acts at two distinct steps in disk morphogenesis, condyle initiation, and disk-condyle separation and provide a molecular framework for future studies of the TMJ.

Temporomandibular joint formation requires two distinct hedgehog-dependent steps

PubMed Central

Purcell, Patricia; Joo, Brian W.; Hu, Jimmy K.; Tran, Pamela V.; Calicchio, Monica L.; O'Connell, Daniel J.; Maas, Richard L.; Tabin, Clifford J.

2009-01-01

We conducted a genetic analysis of the developing temporo-mandibular or temporomandi-bular joint (TMJ), a highly specialized synovial joint that permits movement and function of the mammalian jaw. First, we used laser capture microdissection to perform a genome-wide expression analysis of each of its developing components. The expression patterns of genes identified in this screen were examined in the TMJ and compared with those of other synovial joints, including the shoulder and the hip joints. Striking differences were noted, indicating that the TMJ forms via a distinct molecular program. Several components of the hedgehog (Hh) signaling pathway are among the genes identified in the screen, including Gli2, which is expressed specifically in the condyle and in the disk of the developing TMJ. We found that mice deficient in Gli2 display aberrant TMJ development such that the condyle loses its growth-plate-like cellular organization and no disk is formed. In addition, we used a conditional strategy to remove Smo, a positive effector of the Hh signaling pathway, from chondrocyte progenitors. This cell autonomous loss of Hh signaling allows for disk formation, but the resulting structure fails to separate from the condyle. Thus, these experiments establish that Hh signaling acts at two distinct steps in disk morphogenesis, condyle initiation, and disk–condyle separation and provide a molecular framework for future studies of the TMJ. PMID:19815519

Phloem-exudate proteome analysis of response to insect brown plant-hopper in rice.

PubMed

Du, Ba; Wei, Zhe; Wang, Zhanqi; Wang, Xiaoxiao; Peng, Xinxin; Du, Bo; Chen, Rongzhi; Zhu, Lili; He, Guangcun

2015-07-01

Brown plant-hopper (Nilaparvata lugens Stål, BPH), one of the most devastating agricultural insect pests of rice throughout Asia, ingests nutrients from rice sieve tubes and causes a dramatic yield loss. Planting resistant variety is an efficient and economical way to control this pest. Understanding the mechanisms of host resistance is extremely valuable for molecular design of resistant rice variety. Here, we used an iTRAQ-based quantitative proteomics approach to perform analysis of protein expression profiles in the phloem exudates of BPH-resistant and susceptible rice plants following BPH infestation. A total of 238 proteins were identified, most of which were previously described to be present in the phloem of rice and other plants. The expression of genes for selected proteins was confirmed using a laser capture micro-dissection method and RT-PCR. The mRNAs for three proteins, RGAP, TCTP, and TRXH, were further analyzed by using in situ mRNA hybridization and localized in the phloem cells. Our results showed that BPH feeding induced significant changes in the abundance of proteins in phloem sap of rice involved in multiple pathways, including defense signal transduction, redox regulation, and carbohydrate and protein metabolism, as well as cell structural proteins. The results presented provide new insights into rice resistance mechanisms and should facilitate the breeding of novel elite BPH-resistant rice varieties. Copyright © 2015 Elsevier GmbH. All rights reserved.

Resources to handle childhood asthma in Spain: The role of plans and guides and the participation of nurses.

PubMed

Úbeda-Sansano, M I; Cano-Garcinuño, A; Rueda-Esteban, S; Praena-Crespo, M

2018-05-05

Describe the assistance provided to asthmatic patients by Primary Care Paediatricians (PCP) in Spain and the material and human resources available for diagnosis and follow-up. A cross-sectional descriptive study using an on-line survey, sent to PCP regarding the availability of diagnostic resources, carrying out programmed and educational activities, collaboration of nursing staff and their relationship with existing institutional plans to care for children with asthma. A latent class model (LCM) was used to describe the differences among paediatricians based on the variables studied. Of the 708 answers, 675 were considered valid; 76% of the paediatricians had a spirometer, 75% specific IgE, 17% prick-test, 95% had placebo inhalers and 97% inhalation chambers. 57% performed programmed activities with their patients, while 56% shared their care of asthmatic patients with their nursing staff, but only 25% of the nurses were involved in the follow-up and 12% in education. LCM identified four patterns. The two groups with greater access to diagnostic resources counted on institutional plans/guidelines. However, the only variable differentiating the groups with more programmed and educational activities was the participation of nurses. The availability of asthma plans/guidelines and resources for diagnosis and follow-up is not sufficient to improve important aspects of primary care for children with asthma. Organisational changes are necessary to include programmed asthma-related visits and paediatric teams with greater involvement of the nurses when caring for these patients. Copyright © 2018 SEICAP. Published by Elsevier España, S.L.U. All rights reserved.

DNA Sequences from Formalin-Fixed Nematodes: Integrating Molecular and Morphological Approaches to Taxonomy

PubMed Central

Thomas, W. Kelley; Vida, J. T.; Frisse, Linda M.; Mundo, Manuel; Baldwin, James G.

1997-01-01

To effectively integrate DNA sequence analysis and classical nematode taxonomy, we must be able to obtain DNA sequences from formalin-fixed specimens. Microdissected sections of nematodes were removed from specimens fixed in formalin, using standard protocols and without destroying morphological features. The fixed sections provided sufficient template for multiple polymerase chain reaction-based DNA sequence analyses. PMID:19274156

Noncontact laser microsurgery of three-dimensional living objects for use in reproductive and regenerative medicine

NASA Astrophysics Data System (ADS)

Sitnikov, D. S.; Ilina, I. V.; Kosheleva, N. V.; Khramova, Yu V.; Filatov, M. A.; Semenova, M. L.; Zurina, I. M.; Gorkun, A. A.; Saburina, I. N.

2018-01-01

Laser microsurgery has enabled us to make highly precise and delicate processing of living biological specimens. We present the results of using femtosecond (fs) laser pulses in assisted reproductive technologies. Femtosecond laser dissection of outer shells of embryos (so-called laser-assisted hatching) as well as laser-mediated detachment of the desired amount of trophectoderm cells (so-called embryo biopsy) required for preimplantaion genetic diagnosis were successfully performed. The parameters of laser radiation were optimized so as to efficiently perform embryo biopsy and preserve the viability of the treated embryos. Effects of application of fs-laser radiation in the infrared (1028 nm) and visible (514 nm) wavelength ranges were studied. We also applied laser microsurgery to develop a new simple reproducible model for studying repair and regeneration in vitro. Nanosecond laser pulses were applied to perform localized microdissection of cell spheroids. After microdissection, the edges of the wound surface opened, the destruction of the initial spheroid structure was observed in the wound area, with surviving cells changing their shape into a round one. It was shown that the spheroid form partially restored in the first six hours with subsequent complete restoration within seven days due to remodeling of surviving cells.

Loss of heterozygosity on chromosome 9q22.3 in microdissected basal cell carcinomas around the Semipalatinsk Nuclear Testing Site, Kazakhstan.

PubMed

Iwata, Kenji; Takamura, Noboru; Nakashima, Masahiro; Alipov, Gabit; Mine, Mariko; Matsumoto, Naomichi; Yoshiura, Koichiro; Prouglo, Yuriy; Sekine, Ichiro; Katayama, Ichiro; Yamashita, Shunichi

2004-04-01

A high incidence of skin cancers has been noted around the Semipalatinsk Nuclear Testing Site (SNTS) in Kazakhstan. Recently, basal cell carcinoma (BCC) susceptibility genes, human homolog of the Drosophila pathed gene (PTCH), and the xeroderma pigmentosa group A-complementing gene (XPA), have been cloned and localized on chromosome 9q22.3. To clarify the effect of low-dose irradiation on the occurrence of BCC, we used microdissection and polymerase chain reaction to identify loss of heterozygosity (LOH) at 9q22.3 using BCC samples obtained from this region. Ten Japanese samples were analyzed as controls. LOH with at least 1 marker was identified in 5 of 14 cases from around SNTS, whereas only 1 case with 1 marker was identified among the 10 Nagasaki cases. The total number of LOH alleles from SNTS (8 of 45) was significantly higher than the number from Nagasaki (1 of 26) (P = 0.03). The higher incidence of LOH on 9q22.3 in BCC from around SNTS suggests involvement of chronic low-dose irradiation by fallout from the test site as a factor in the cancers.

Anatomical study of the pigs temporal bone by microdissection.

PubMed

Garcia, Leandro de Borborema; Andrade, José Santos Cruz de; Testa, José Ricardo Gurgel

2014-01-01

Initial study of the pig`s temporal bone anatomy in order to enable a new experimental model in ear surgery. Dissection of five temporal bones of Sus scrofa pigs obtained from UNIFESP - Surgical Skills Laboratory, removed with hole saw to avoid any injury and stored in formaldehyde 10% for better conservation. The microdissection in all five temporal bone had the following steps: inspection of the outer part, external canal and tympanic membrane microscopy, mastoidectomy, removal of external ear canal and tympanic membrane, inspection of ossicular chain and middle ear. Anatomically it is located at the same position than in humans. Some landmarks usually found in humans are missing. The tympanic membrane of the pig showed to be very similar to the human, separating the external and the middle ear. The middle ear`s appearance is very similar than in humans. The ossicular chain is almost exactly the same, as well as the facial nerve, showing the same relationship with the lateral semicircular canal. The temporal bone of the pigs can be used as an alternative for training in ear surgery, especially due the facility to find it and its similarity with temporal bone of the humans.

Comparative proteomic analysis using samples obtained with laser microdissection and saturation dye labelling.

PubMed

Wilson, Kate E; Marouga, Rita; Prime, John E; Pashby, D Paul; Orange, Paul R; Crosier, Steven; Keith, Alexander B; Lathe, Richard; Mullins, John; Estibeiro, Peter; Bergling, Helene; Hawkins, Edward; Morris, Christopher M

2005-10-01

Comparative proteomic methods are rapidly being applied to many different biological systems including complex tissues. One pitfall of these methods is that in some cases, such as oncology and neuroscience, tissue complexity requires isolation of specific cell types and sample is limited. Laser microdissection (LMD) is commonly used for obtaining such samples for proteomic studies. We have combined LMD with sensitive thiol-reactive saturation dye labelling of protein samples and 2-D DIGE to identify protein changes in a test system, the isolated CA1 pyramidal neurone layer of a transgenic (Tg) rat carrying a human amyloid precursor protein transgene. Saturation dye labelling proved to be extremely sensitive with a spot map of over 5,000 proteins being readily produced from 5 mug total protein, with over 100 proteins being significantly altered at p < 0.0005. Of the proteins identified, all showed coherent changes associated with transgene expression. It was, however, difficult to identify significantly different proteins using PMF and MALDI-TOF on gels containing less than 500 mug total protein. The use of saturation dye labelling of limiting samples will therefore require the use of highly sensitive MS techniques to identify the significantly altered proteins isolated using methods such as LMD.

Tin accumulation in spermatozoa of the rats exposed to tributyltin chloride by synchrotron radiation X-ray fluorescence (SR-XRF) analysis with microprobe

NASA Astrophysics Data System (ADS)

Homma-Takeda, S.; Nishimura, Y.; Terada, Y.; Ueno, S.; Watanabe, Y.; Yukawa, M.

2005-04-01

Organotin compounds are widely used in industry and its environmental contamination by these compounds has recently become a concern. It is known that they act as endocrine disruptors but details of the dynamics of Sn in reproductive organs are still unknown. In the present study, we attempted to determine Sn distribution in the testis of rats exposed to tributyltin chloride (TBTC) by inductively coupled argon plasma-mass spectrometry (ICP-MS) for microdissectioned seminiferous tubules and cell-selective metal determination of synchrotron radiation X-ray florescence (SR-XRF) analysis. TBTC was orally administered to rats at a dose of 45 μmol/kg per day for 3 days. One day later, Sn was detected in the microdissectioned seminiferous tubules at a level approximately equivalent to that in the testis. Significant stage-specificity of Sn accumulation was not observed in the experimental model. Sn was also detected in spermatozoa at the stage VIII seminiferous tubule, which are the final step of spermatogenesis in the testis. These data indicate that Sn accumulates in germ cells as well as in spermatozoa in a short period of TBTC exposure.

The role of high-energy synchrotron radiation in biomedical trace element research

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

Pounds, J.G.; Long, G.J.; Kwiatek, W.M.

1987-01-01

This paper will present the results of an investigation of the distribution of essential elements in the normal hepatic lobule. the liver is the organ responsible for metabolism and storage of most trace elements. Although parenchymal hepatocytes are rather uniform histologically, morphometry, histochemistry, immunohistochemistry, and microdissection with microchemical investigations have revealed marked heterogeneity on a functional and biochemical level. Hepatocytes from the periportal and perivenous zones of the liver parrenchyma differ in oxidative energy metabolism, glucose uptake and output, unreagenesis, biotransformation, bile acid secretion, and palsma protein synthesis and secretion. Although trace elements are intimately involved in the regulation andmore » maintenance of these functions, little is known regarding the heterogeneity of trace element localization of the liver parenchyma. Histochemical techniques for trace elements generally give high spatial resolution, but lack specificity and stoichiometry. Microdissection has been of marginal usefulness for trace element analyses due to the very small size of the dissected parenchyma. The characteristics of the high-energy x-ray microscope provide an effective approach for elucidating the trace element content of these small biological structures or regions. 5 refs., 1 fig., 1 tab.« less

Characterization of Crystallographic Structure and Internal Stress of Chromium Coatings Plated under Current Interruptions.

DTIC Science & Technology

1983-09-01

phase has a preferred orientation as most other electrodeposits, this peak found off the equatorial direction provided a way of measuring...40 (S)IoA) 1IliN3i0d , STANDARD HARD CHROM (KiC) INTERRUPTED PLATED CHROME (tC) -4’? 1001 1000X KC Chrome IC Chrome Bath Temperature *C 55 55 Current...COMMANDER ATTN: DRSNC- LC (D) I US ARMY TANK-AUTMV COWD DRSMC-LCE(D) 1 ATTN: DRSTA-RC DRSC-LCM(D) (BLDG 321) 1 WARREN, MI 48090 DRSMC- LCS (D) 1 DRSNC-LCU

Design of the ANTARES LCM-DAQ board test bench using a FPGA-based system-on-chip approach

NASA Astrophysics Data System (ADS)

Anvar, S.; Kestener, P.; Le Provost, H.

2006-11-01

The System-on-Chip (SoC) approach consists in using state-of-the-art FPGA devices with embedded RISC processor cores, high-speed differential LVDS links and ready-to-use multi-gigabit transceivers allowing development of compact systems with substantial number of IO channels. Required performances are obtained through a subtle separation of tasks between closely cooperating programmable hardware logic and user-friendly software environment. We report about our experience in using the SoC approach for designing the production test bench of the off-shore readout system for the ANTARES neutrino experiment.

LASH Ship Pretest Results of the Joint Logistics-Over-the-Shore (LOTS) Test and Evaluation Program.

DTIC Science & Technology

1977-03-07

75-05. 9 0 wU C-7) z C) S.- C)C 40 10 GA’THEFRING CO~l BARGE LIFTING POINT I , I I II I I . . .. .. KI- V~~ PS4 and 5. LCM8 LIFT BEAM IS ENGAGED...design but which could not be delivered in time by the vendor. The attachment of the struts proved to be a time- consuming effort because of too close...actually used in the pretest, the general behavior and characteristics are similar.) C-4 APPENDIX D BREAKBULK CARGO THROUGHPUT OPERATIONS As an adjunct to

Comparison of the transcriptpmes of long-tern label retaining-cells and C cells microdissected from mammary epithelium: an initial study to character potential stem/progenitor cells

USDA-ARS?s Scientific Manuscript database

Mammary stem cells (MaSC) account for the cell lineage of mammary epithelia and provide for mammary growth, development and tissue homeostasis. The presence of MaSC was clearly demonstrated by the generation of an entire mammary gland from a single cell implanted into epithelium-ablated mammary fat...

Donor-derived stem-cells and epithelial mesenchymal transition in squamous cell carcinoma in transplant recipients.

PubMed

Verneuil, Laurence; Leboeuf, Christophe; Bousquet, Guilhem; Brugiere, Charlotte; Elbouchtaoui, Morad; Plassa, Louis-François; Peraldi, Marie-Noelle; Lebbé, Celeste; Ratajczak, Philippe; Janin, Anne

2015-12-08

Skin squamous-cell-carcinoma (SCC), is the main complication in long-term kidney-transplant recipients, and it can include donor-derived cells. Preclinical models demonstrated the involvement of epithelial mesenchymal transition (EMT) in the progression of skin SCC, and the role of Snail, an EMT transcription factor, in cancer stem-cell survival and expansion.Here, we studied stem-cells and EMT expression in SCCs and concomitant actinic keratoses (AK) in kidney-transplant recipients. In SCC and AK in 3 female recipients of male kidney-transplants, donor-derived Y chromosome in epidermal stem cells was assessed using combined XY-FISH/CD133 immunostaining, and digital-droplet-PCR on laser-microdissected CD133 expressing epidermal cells.For EMT study, double immunostainings of CD133 with vimentin or snail and slug, electron microscopy and immunostainings of keratinocytes junctions were performed. Digital droplet PCR was used to check CDH1 (E-cadherin) expression level in laser-microdissected cells co-expressing CD133 and vimentin or snail and slug.The numbers of Y-chromosome were assessed using digital droplet PCR in laser-microdissected cells co-expressing CD133 and vimentin, or snail and slug, and in CD133 positive cells not expressing any EMT maker. We identified donor-derived stem-cells in basal layers and invasive areas in all skin SCCs and in concomitant AKs, but not in surrounding normal skin.The donor-derived stem-cells expressed the EMT markers, vimentin, snail and slug in SCCs but not in AKs. The expression of the EMT transcription factor, SNAI1, was higher in stem-cells when they expressed vimentin. They were located in invasive areas of SCCs. In these areas, the expressions of claudin-1 and desmoglein 1 were reduced or absent, and within the basal layer there were features of basal membrane disappearance.Donor-derived stem cells were in larger numbers in stem cells co-expressing vimentin or snail and slug than in stem cells not expressing any EMT marker. We identified here donor-derived stem cells within skin SCC in kidney-transplant recipients. They were located in invasive areas of SCC and had EMT characteristics.

Continuous Cough Monitoring Using Ambient Sound Recording During Convalescence from a COPD Exacerbation.

PubMed

Crooks, Michael G; den Brinker, Albertus; Hayman, Yvette; Williamson, James D; Innes, Andrew; Wright, Caroline E; Hill, Peter; Morice, Alyn H

2017-06-01

Cough is common in chronic obstructive pulmonary disease (COPD) and is associated with frequent exacerbations and increased mortality. Cough increases during acute exacerbations (AE-COPD), representing a possible metric of clinical deterioration. Conventional cough monitors accurately report cough counts over short time periods. We describe a novel monitoring system which we used to record cough continuously for up to 45 days during AE-COPD convalescence. This is a longitudinal, observational study of cough monitoring in AE-COPD patients discharged from a single teaching hospital. Ambient sound was recorded from two sites in the domestic environment and analysed using novel cough classifier software. For comparison, the validated hybrid HACC/LCM cough monitoring system was used on days 1, 5, 20 and 45. Patients were asked to record symptoms daily using diaries. Cough monitoring data were available for 16 subjects with a total of 568 monitored days. Daily cough count fell significantly from mean ± SEM 272.7 ± 54.5 on day 1 to 110.9 ± 26.3 on day 9 (p < 0.01) before plateauing. The absolute cough count detected by the continuous monitoring system was significantly lower than detected by the hybrid HACC/LCM system but normalised counts strongly correlated (r = 0.88, p < 0.01) demonstrating an ability to detect trends. Objective cough count and subjective cough scores modestly correlated (r = 0.46). Cough frequency declines significantly following AE-COPD and the reducing trend can be detected using continuous ambient sound recording and novel cough classifier software. Objective measurement of cough frequency has the potential to enhance our ability to monitor the clinical state in patients with COPD.

Theranostic self-assembly structure of gold nanoparticles for NIR photothermal therapy and X-Ray computed tomography imaging.

PubMed

Deng, Heng; Zhong, Yanqi; Du, Meihong; Liu, Qinjun; Fan, Zhanming; Dai, Fengying; Zhang, Xin

2014-01-01

The controllable self-assembly of amphiphilic mixed polymers grafted gold nanoparitcles (AuNPs) leads to strong interparticle plasmonic coupling, which can be tuned to the near-infrared (NIR) region for enhanced photothermal therapy (PTT). In this study, an improved thiolation method was adopted for ATRP and ROP polymer to obtain amphiphilic brushes of PMEO2MA-SH and PCL-SH. By anchoring PCL-SH and PMEO2MA-SH onto the 14 nm AuNPs, a smart hybrid building block for self-assembly was obtained. Increasing the PCL/PMEO2MA chain ratio from 0.8:1, 2:1 and 3:1 to 7:1, the structure of gold assemblies (GAs) was observed to transfer from vesicle to large compound micelle (LCM). Contributed to the special dense packed structure of gold nanoparticles in LCM, the absorption spectrometry of gold nanoparticles drastically red-shifted from 520 nm to 830 nm, which endowed the GAs remarkable NIR photothermal conversion ability. In addition, gold has high X-ray absorption coefficient which qualifies gold nanomaterial a potential CT contrast agent Herein, we obtain a novel gold assembly structure which can be utilized as potential photothermal therapeutic and CT contrast agents. In vitro and In vivo studies testified the excellent treatment efficacy of optimum GAs as a PTT and CT contrast agent. In vitro degradation test, MTT assay and histology study indicated that GAs was a safe, low toxic reagent with good biodegradability. Therefore, the optimum GAs with strong NIR absorption and high X-ray absorption coefficient could be used as a theranostic agent and the formation of novel gold large compound micelle might offers a new theory foundation for engineering design and synthesis of polymer grafted AuNPs for biomedical applications.

Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles

PubMed Central

Peel, Trisha N.; Dylla, Brenda L.; Hughes, John G.; Lynch, David T.; Greenwood-Quaintance, Kerryl E.; Cheng, Allen C.; Mandrekar, Jayawant N.

2016-01-01

ABSTRACT 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. PMID:26733067

Genetic divergence in the transcriptional engram of chronic alcohol abuse: A laser-capture RNA-seq study of the mouse mesocorticolimbic system.

PubMed

Mulligan, Megan K; Mozhui, Khyobeni; Pandey, Ashutosh K; Smith, Maren L; Gong, Suzhen; Ingels, Jesse; Miles, Michael F; Lopez, Marcelo F; Lu, Lu; Williams, Robert W

2017-02-01

Genetic factors that influence the transition from initial drinking to dependence remain enigmatic. Recent studies have leveraged chronic intermittent ethanol (CIE) paradigms to measure changes in brain gene expression in a single strain at 0, 8, 72 h, and even 7 days following CIE. We extend these findings using LCM RNA-seq to profile expression in 11 brain regions in two inbred strains - C57BL/6J (B6) and DBA/2J (D2) - 72 h following multiple cycles of ethanol self-administration and CIE. Linear models identified differential expression based on treatment, region, strain, or interactions with treatment. Nearly 40% of genes showed a robust effect (FDR < 0.01) of region, and hippocampus CA1, cortex, bed nucleus stria terminalis, and nucleus accumbens core had the highest number of differentially expressed genes after treatment. Another 8% of differentially expressed genes demonstrated a robust effect of strain. As expected, based on similar studies in B6, treatment had a much smaller impact on expression; only 72 genes (p < 0.01) are modulated by treatment (independent of region or strain). Strikingly, many more genes (415) show a strain-specific and largely opposite response to treatment and are enriched in processes related to RNA metabolism, transcription factor activity, and mitochondrial function. Over 3 times as many changes in gene expression were detected in D2 compared to B6, and weighted gene co-expression network analysis (WGCNA) module comparison identified more modules enriched for treatment effects in D2. Substantial strain differences exist in the temporal pattern of transcriptional neuroadaptation to CIE, and these may drive individual differences in risk of addiction following excessive alcohol consumption. Copyright © 2016 Elsevier Inc. All rights reserved.

Mitochondrial DNA copy numbers in pyramidal neurons are decreased and mitochondrial biogenesis transcriptome signaling is disrupted in Alzheimer's disease hippocampi.

PubMed

Rice, Ann C; Keeney, Paula M; Algarzae, Norah K; Ladd, Amy C; Thomas, Ravindar R; Bennett, James P

2014-01-01

Alzheimer's disease (AD) is the major cause of adult-onset dementia and is characterized in its pre-diagnostic stage by reduced cerebral cortical glucose metabolism and in later stages by reduced cortical oxygen uptake, implying reduced mitochondrial respiration. Using quantitative PCR we determined the mitochondrial DNA (mtDNA) gene copy numbers from multiple groups of 15 or 20 pyramidal neurons, GFAP(+) astrocytes and dentate granule neurons isolated using laser capture microdissection, and the relative expression of mitochondrial biogenesis (mitobiogenesis) genes in hippocampi from 10 AD and 9 control (CTL) cases. AD pyramidal but not dentate granule neurons had significantly reduced mtDNA copy numbers compared to CTL neurons. Pyramidal neuron mtDNA copy numbers in CTL, but not AD, positively correlated with cDNA levels of multiple mitobiogenesis genes. In CTL, but not in AD, hippocampal cDNA levels of PGC1α were positively correlated with multiple downstream mitobiogenesis factors. Mitochondrial DNA copy numbers in pyramidal neurons did not correlate with hippocampal Aβ1-42 levels. After 48 h exposure of H9 human neural stem cells to the neurotoxic fragment Aβ25-35, mtDNA copy numbers were not significantly altered. In summary, AD postmortem hippocampal pyramidal neurons have reduced mtDNA copy numbers. Mitochondrial biogenesis pathway signaling relationships are disrupted in AD, but are mostly preserved in CTL. Our findings implicate complex alterations of mitochondria-host cell relationships in AD.

ANG II receptor subtype 1a gene knockdown in the subfornical organ prevents increased drinking behavior in bile duct-ligated rats.

PubMed

Walch, Joseph D; Nedungadi, T Prashant; Cunningham, J Thomas

2014-09-15

Bile duct ligation (BDL) causes congestive liver failure that initiates hemodynamic changes, resulting in dilutional hyponatremia due to increased water intake and vasopressin release. This project tested the hypothesis that angiotensin signaling at the subfornical organ (SFO) augments drinking behavior in BDL rats. A genetically modified adeno-associated virus containing short hairpin RNA (shRNA) for ANG II receptor subtype 1a (AT1aR) gene was microinjected into the SFO of rats to knock down expression. Two weeks later, BDL or sham surgery was performed. Rats were housed in metabolic chambers for measurement of fluid and food intake and urine output. The rats were euthanized 28 days after BDL surgery for analysis. A group of rats was perfused for immunohistochemistry, and a second group was used for laser-capture microdissection for analysis of SFO AT1aR gene expression. BDL rats showed increased water intake that was attenuated in rats that received SFO microinjection of AT1aR shRNA. Among BDL rats treated with scrambled (control) and AT1aR shRNA, we observed an increased number of vasopressin-positive cells in the supraoptic nucleus that colocalized with ΔFosB staining, suggesting increased vasopressin release in both groups. These results indicate that angiotensin signaling through the SFO contributes to increased water intake, but not dilutional hyponatremia, during congestive liver failure. Copyright © 2014 the American Physiological Society.

Antibody αPEP13h Reacts With Lymphangioleiomyomatosis Cells in Lung Nodules

PubMed Central

Valencia, Julio C.; Steagall, Wendy K.; Zhang, Yi; Fetsch, Patricia; Abati, Andrea; Tsukada, Katsuya; Billings, Eric; Hearing, Vincent J.; Yu, Zu-Xi; Pacheco-Rodriguez, Gustavo

2015-01-01

BACKGROUND: Lymphangioleiomyomatosis (LAM) is characterized by the proliferation in the lung, axial lymphatics (eg, lymphangioleiomyomas), and kidney (eg, angiomyolipomas) of abnormal smooth muscle-like LAM cells, which express melanoma antigens such as Pmel17/gp100 and have dysfunctional tumor suppressor tuberous sclerosis complex (TSC) genes TSC2 or TSC1. Histopathologic diagnosis of LAM in lung specimens is based on identification of the Pmel17 protein with the monoclonal antibody HMB-45. METHODS: We compared the sensitivity of HMB-45 to that of antipeptide antibody αPEP13h, which reacts with a C-terminal peptide of Pmel17. LAM lung nodules were laser-capture microdissected to identify proteins by Western blotting. RESULTS: HMB-45 recognized approximately 25% of LAM cells within the LAM lung nodules, whereas αPEP13h identified > 82% of LAM cells within these structures in approximately 90% of patients. Whereas HMB-45 reacted with epithelioid but not with spindle-shaped LAM cells, αPEP13h identified both spindle-shaped and epithelioid LAM cells, providing greater sensitivity for detection of all types of LAM cells. HMB-45 recognized Pmel17 in premelanosomal organelles; αPEP13h recognized proteins in the cytoplasm as well as in premelanosomal organelles. Both antibodies recognized a Pmel17 variant of approximately 50 kDa. CONCLUSIONS: Based on its sensitivity and specificity, αPEP13h may be useful in the diagnosis of LAM and more sensitive than HMB-45. PMID:25411763

Lamina propria macrophage phenotypes in relation to Escherichia coli in Crohn's disease.

PubMed

Elliott, Timothy R; Rayment, Neil B; Hudspith, Barry N; Hands, Rebecca E; Taylor, Kirstin; Parkes, Gareth C; Prescott, Natalie J; Petrovska, Liljana; Hermon-Taylor, John; Brostoff, Jonathan; Boussioutas, Alex; Mathew, Christopher G; Bustin, Stephen A; Sanderson, Jeremy D

2015-07-03

Abnormal handling of E. coli by lamina propria (LP) macrophages may contribute to Crohn's disease (CD) pathogenesis. We aimed to determine LP macrophage phenotypes in CD, ulcerative colitis (UC) and healthy controls (HC), and in CD, to compare macrophage phenotypes according to E. coli carriage. Mucosal biopsies were taken from 35 patients with CD, 9 with UC and 18 HCs. Laser capture microdissection was used to isolate E. coli-laden and unladen LP macrophages from ileal or colonic biopsies. From these macrophages, mRNA was extracted and cytokine and activation marker expression measured using RT-qPCR. E. coli-laden LP macrophages were identified commonly in mucosal biopsies from CD patients (25/35, 71 %), rarely in UC (1/9, 11 %) and not at all in healthy controls (0/18). LP macrophage cytokine mRNA expression was greater in CD and UC than healthy controls. In CD, E. coli-laden macrophages expressed high IL-10 & CD163 and lower TNFα, IL-23 & iNOS irrespective of macroscopic inflammation. In inflamed tissue, E. coli-unladen macrophages expressed high TNFα, IL-23 & iNOS and lower IL-10 & CD163. In uninflamed tissue, unladen macrophages had low cytokine mRNA expression, closer to that of healthy controls. In CD, intra-macrophage E. coli are commonly found and LP macrophages express characteristic cytokine mRNA profiles according to E. coli carriage. Persistence of E. coli within LP macrophages may provide a stimulus for chronic inflammation.

Mesencephalic dopaminergic neurons express a repertoire of olfactory receptors and respond to odorant-like molecules.

PubMed

Grison, Alice; Zucchelli, Silvia; Urzì, Alice; Zamparo, Ilaria; Lazarevic, Dejan; Pascarella, Giovanni; Roncaglia, Paola; Giorgetti, Alejandro; Garcia-Esparcia, Paula; Vlachouli, Christina; Simone, Roberto; Persichetti, Francesca; Forrest, Alistair R R; Hayashizaki, Yoshihide; Carloni, Paolo; Ferrer, Isidro; Lodovichi, Claudia; Plessy, Charles; Carninci, Piero; Gustincich, Stefano

2014-08-27

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the Substantia Nigra (SN) (A9 neurons) and the Ventral Tegmental Area (VTA) (A10 cells). Selective degeneration of A9 neurons occurs in Parkinson's disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown. By taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca2+ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains. Our study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease.

Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate

PubMed Central

Pascal, Laura E; True, Lawrence D; Campbell, David S; Deutsch, Eric W; Risk, Michael; Coleman, Ilsa M; Eichner, Lillian J; Nelson, Peter S; Liu, Alvin Y

2008-01-01

Background: Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD) genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate – basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial – and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes. Results: Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63). Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50) but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance. Conclusion: Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers. PMID:18501003

Assessment of the quality of DNA from various formalin-fixed paraffin-embedded (FFPE) tissues and the use of this DNA for next-generation sequencing (NGS) with no artifactual mutation

PubMed Central

Einaga, Naoki; Yoshida, Akio; Noda, Hiroko; Suemitsu, Masaaki; Nakayama, Yuki; Sakurada, Akihisa; Kawaji, Yoshiko; Yamaguchi, Hiromi; Sasaki, Yasushi; Tokino, Takashi; Esumi, Mariko

2017-01-01

Formalin-fixed, paraffin-embedded (FFPE) tissues used for pathological diagnosis are valuable for studying cancer genomics. In particular, laser-capture microdissection of target cells determined by histopathology combined with FFPE tissue section immunohistochemistry (IHC) enables precise analysis by next-generation sequencing (NGS) of the genetic events occurring in cancer. The result is a new strategy for a pathological tool for cancer diagnosis: ‘microgenomics’. To more conveniently and precisely perform microgenomics, we revealed by systematic analysis the following three details regarding FFPE DNA compared with paired frozen tissue DNA. 1) The best quality of FFPE DNA is obtained by tissue fixation with 10% neutral buffered formalin for 1 day and heat treatment of tissue lysates at 95°C for 30 minutes. 2) IHC staining of FFPE tissues decreases the quantity and quality of FFPE DNA to one-fourth, and antigen retrieval (at 120°C for 15 minutes, pH 6.0) is the major reason for this decrease. 3) FFPE DNA prepared as described herein is sufficient for NGS. For non-mutated tissue specimens, no artifactual mutation occurs during FFPE preparation, as shown by precise comparison of NGS of FFPE DNA and paired frozen tissue DNA followed by validation. These results demonstrate that even FFPE tissues used for routine clinical diagnosis can be utilized to obtain reliable NGS data if appropriate conditions of fixation and validation are applied. PMID:28498833

Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer.

PubMed

Court, Colin M; Ankeny, Jacob S; Sho, Shonan; Hou, Shuang; Li, Qingyu; Hsieh, Carolyn; Song, Min; Liao, Xinfang; Rochefort, Matthew M; Wainberg, Zev A; Graeber, Thomas G; Tseng, Hsian-Rong; Tomlinson, James S

2016-09-01

To understand the potential and limitations of circulating tumor cell (CTC) sequencing for molecular diagnostics, we investigated the feasibility of identifying the ubiquitous KRAS mutation in single CTCs from pancreatic cancer (PC) patients. We used the NanoVelcro/laser capture microdissection CTC platform, combined with whole genome amplification and KRAS Sanger sequencing. We assessed both KRAS codon-12 coverage and the degree that allele dropout during whole genome amplification affected the detection of KRAS mutations from single CTCs. We isolated 385 single cells, 163 from PC cell lines and 222 from the blood of 12 PC patients, and obtained KRAS sequence coverage in 218 of 385 single cells (56.6%). For PC cell lines with known KRAS mutations, single mutations were detected in 67% of homozygous cells but only 37.4% of heterozygous single cells, demonstrating that both coverage and allele dropout are important causes of mutation detection failure from single cells. We could detect KRAS mutations in CTCs from 11 of 12 patients (92%) and 33 of 119 single CTCs sequenced, resulting in a KRAS mutation detection rate of 27.7%. Importantly, KRAS mutations were never found in the 103 white blood cells sequenced. Sequencing of groups of cells containing between 1 and 100 cells determined that at least 10 CTCs are likely required to reliably assess KRAS mutation status from CTCs. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Reconstitution of the ERG Gene Expression Network Reveals New Biomarkers and Therapeutic Targets in ERG Positive Prostate Tumors

PubMed Central

Dubovenko, Alexey; Serebryiskaya, Tatiana; Nikolsky, Yuri; Nikolskaya, Tatiana; Perlina, Ally; JeBailey, Lellean; Bureeva, Svetlana; Katta, Shilpa; Srivastava, Shiv; Dobi, Albert; Khasanova, Tatiana

2015-01-01

Background: Despite a growing number of studies evaluating cancer of prostate (CaP) specific gene alterations, oncogenic activation of the ETS Related Gene (ERG) by gene fusions remains the most validated cancer gene alteration in CaP. Prevalent gene fusions have been described between the ERG gene and promoter upstream sequences of androgen-inducible genes, predominantly TMPRSS2 (transmembrane protease serine 2). Despite the extensive evaluations of ERG genomic rearrangements, fusion transcripts and the ERG oncoprotein, the prognostic value of ERG remains to be better understood. Using gene expression dataset from matched prostate tumor and normal epithelial cells from an 80 GeneChip experiment examining 40 tumors and their matching normal pairs in 40 patients with known ERG status, we conducted a cancer signaling-focused functional analysis of prostatic carcinoma representing moderate and aggressive cancers stratified by ERG expression. Results: In the present study of matched pairs of laser capture microdissected normal epithelial cells and well-to-moderately differentiated tumor epithelial cells with known ERG gene expression status from 20 patients with localized prostate cancer, we have discovered novel ERG associated biochemical networks. Conclusions: Using causal network reconstruction methods, we have identified three major signaling pathways related to MAPK/PI3K cascade that may indeed contribute synergistically to the ERG dependent tumor development. Moreover, the key components of these pathways have potential as biomarkers and therapeutic target for ERG positive prostate tumors. PMID:26000039

Propiverine-induced accumulation of nuclear and cytosolic protein in F344 rat kidneys: Isolation and identification of the accumulating protein

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

Dietrich, D.R.; Heussner, A.H.; O'Brien, E.

2008-12-15

Male and female F344 rats but not B6C3F1 mice exposed for 104 weeks to propiverine hydrochloride (1-methylpiperid-4-yl 2,2-diphenyl-2-(1-propoxy)acetate hydrochloride), used for treatment of patients with neurogenic detrusor overactivity (NDO) and overactive bladder (OAB), presented with an accumulation of proteins in the cytosol and nuclei of renal proximal tubule epithelial cells, yet despite this, no increased renal tumor incidence was observed. In order to provide an improved interpretation of these findings and a better basis for human health risk assessment, male and female F344 rats were exposed for 16 weeks to 1000 ppm propiverine in the diet, the accumulating protein wasmore » isolated from the kidneys via cytosolic and nuclear preparations or laser-capture microdissection and analyzed using molecular weight determination and mass spectrometry. The accumulating protein was found to be D-amino acid oxidase (DAAO), an enzyme involved in amino and fatty acid metabolism. Subsequent reanalysis of kidney homogenate and nuclear samples as well as tissue sections using western blot and DAAO-immunohistochemistry, confirmed the presence and localization of DAAO in propiverine-treated male and female F344 rats. The accumulation of DAAO only in rats, and the limited similarity of rat DAAO with other species, including humans, suggests a rat-specific mechanism underlying the drug-induced renal DAAO accumulation with little relevance for patients chronically treated with propiverine.« less