Defining disease with laser precision: laser capture microdissection in gastroenterology.

PubMed

Blatt, Richard; Srinivasan, Shanthi

2008-08-01

Laser capture microdissection (LCM) is an efficient and precise method for obtaining pure cell populations or specific cells of interest from a given tissue sample. LCM has been applied to animal and human gastroenterology research in analyzing the protein, DNA, and RNA from all organs of the gastrointestinal system. There are numerous potential applications for this technology in gastroenterology research, including malignancies of the esophagus, stomach, colon, biliary tract, and liver. This technology can also be used to study gastrointestinal infections, inflammatory bowel disease, pancreatitis, motility, malabsorption, and radiation enteropathy. LCM has multiple advantages when compared with conventional methods of microdissection, and this technology can be exploited to identify precursors to disease, diagnostic biomarkers, and therapeutic interventions.

Application of the laser capture microdissection technique for molecular definition of skeletal cell differentiation in vivo.

PubMed

Benayahu, Dafna; Socher, Rina; Shur, Irena

2008-01-01

Laser capture microdissection (LCM) method allows selection of individual or clustered cells from intact tissues. This technology enables one to pick cells from tissues that are difficult to study individually, sort the anatomical complexity of these tissues, and make the cells available for molecular analyses. Following the cells' extraction, the nucleic acids and proteins can be isolated and used for multiple applications that provide an opportunity to uncover the molecular control of cellular fate in the natural microenvironment. Utilization of LCM for the molecular analysis of cells from skeletal tissues will enable one to study differential patterns of gene expression in the native intact skeletal tissue with reliable interpretation of function for known genes as well as to discover novel genes. Variability between samples may be caused either by differences in the tissue samples (different areas isolated from the same section) or some variances in sample handling. LCM is a multi-task technology that combines histology, microscopy work, and dedicated molecular biology. The LCM application will provide results that will pave the way toward high throughput profiling of tissue-specific gene expression using Gene Chip arrays. Detailed description of in vivo molecular pathways will make it possible to elaborate on control systems to apply for the repair of genetic or metabolic diseases of skeletal tissues.

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

Evaluation of two-dimensional electrophoresis and liquid chromatography – tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samples

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

Schad, Martina; Lipton, Mary S.; Giavalisco, Patrick

2005-07-14

Laser microdissection (LM) allows the collection of homogeneous tissue- and cell specific plant samples. The employment of this technique with subsequent protein analysis has thus far not been reported for plant tissues, probably due to the difficulties associated with defining a reasonable cellular morphology and, in parallel, allowing efficient protein extraction from tissue samples. The relatively large sample amount needed for successful proteome analysis is an additional issue that complicates protein profiling on a tissue- or even cell-specific level. In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies, there ismore » as yet no amplification procedure for proteins available. In the current study, we compared different tissue preparation techniques prior to LM/laser pressure catapulting (LMPC) with respect to their suitability for protein retrieval. Cryosectioning was identified as the best compromise between tissue morphology and effective protein extraction. After collection of vascular bundles from Arabidopsis thaliana stem tissue by LMPC, proteins were extracted and subjected to protein analysis, either by classical two-dimensional gel electrophoresis (2-DE), or by high-efficiency liquid chromatography (LC) in conjunction with tandem mass spectrometry (MS/MS). Our results demonstrate that both methods can be used with LMPC collected plant material. But because of the significantly lower sample amount required for LC-MS/MS than for 2-DE, the combination of LMPC and LC-MS/MS has a higher potential to promote comprehensive proteome analysis of specific plant tissues.« less

Defining disease with laser precision: laser capture microdissection in gastroenterology

PubMed Central

Blatt, Richard; Srinivasan, Shanthi

2013-01-01

Laser capture microdissection (LCM) is an efficient and precise method for obtaining pure cell populations or specific cells of interest from a given tissue sample. LCM has been applied to animal and human gastroenterology research in analyzing the protein, DNA and RNA from all organs of the gastrointestinal system. There are numerous potential applications for this technology in gastroenterology research including malignancies of the esophagus, stomach, colon, biliary tract and liver. This technology can also be used to study gastrointestinal infections, inflammatory bowel disease, pancreatitis, motility, malabsorption and radiation enteropathy. LCM has multiple advantages when compared to conventional methods of microdissection, and this technology can be exploited to identify precursors to disease, diagnostic biomarkers, and therapeutic interventions. PMID:18619446

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

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

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

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

Laser capture microdissection to study flower morphogenesis

NASA Astrophysics Data System (ADS)

Pawełkowicz, Magdalena Ewa; Skarzyńska, Agnieszka; Kowalczuk, Cezary; PlÄ der, Wojciech; Przybecki, Zbigniew

2017-08-01

Laser Capture Microdissection (LCM) is a sample preparation microscopic method that enables isolation of an interesting cell or cells population from human, animal or plant tissue. This technique allows for obtaining pure sample from heterogeneous mixture. From isolated cells, it is possible to obtain the appropriate quality material used for genomic research in transcriptomics, proteomics and metabolomics. We used LCM method to study flower morphogenesis and specific bud's organ organization and development. The genes expression level in developing flower buds of male (B10) and female (2gg) lines were analyzed with qPCR. The expression was checked for stamen and carpel primordia obtained with LCM and for whole flower buds at successive stages of growth.

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.

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.

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.

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.

Laser Capture Microdissection Revisited as a Tool for Transcriptomic Analysis: Application of an Excel-Based qPCR Preparation Software (PREXCEL-Q)

USDA-ARS?s Scientific Manuscript database

The ability to reliably analyze cellular and molecular profiles of normal or diseased tissues is frequently obfuscated by the inherent heterogeneous nature of tissues. Laser Capture Microdissection (LCM) is an innovative technique that allows the isolation and enrichment of pure subpopulations of c...

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.

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.

Adaptation of Laser Microdissection Technique for the Study of a Spontaneous Metastatic Mammary Carcinoma Mouse Model by NanoString Technologies

PubMed Central

Saylor, Karen L.; Anver, Miriam R.; Salomon, David S.; Golubeva, Yelena G.

2016-01-01

Laser capture microdissection (LCM) of tissue is an established tool in medical research for collection of distinguished cell populations under direct microscopic visualization for molecular analysis. LCM samples have been successfully analyzed in a number of genomic and proteomic downstream molecular applications. However, LCM sample collection and preparation procedure has to be adapted to each downstream analysis platform. In this present manuscript we describe in detail the adaptation of LCM methodology for the collection and preparation of fresh frozen samples for NanoString analysis based on a study of a model of mouse mammary gland carcinoma and its lung metastasis. Our adaptation of LCM sample preparation and workflow to the requirements of the NanoString platform allowed acquiring samples with high RNA quality. The NanoString analysis of such samples provided sensitive detection of genes of interest and their associated molecular pathways. NanoString is a reliable gene expression analysis platform that can be effectively coupled with LCM. PMID:27077656

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.

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.

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.

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

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

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.

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.

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.

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.

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.

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.

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.

Laser capture microdissection: Arcturus(XT) infrared capture and UV cutting methods.

PubMed

Gallagher, Rosa I; Blakely, Steven R; Liotta, Lance A; Espina, Virginia

2012-01-01

Laser capture microdissection (LCM) is a technique that allows the precise procurement of enriched cell populations from a heterogeneous tissue under direct microscopic visualization. LCM can be used to harvest the cells of interest directly or can be used to isolate specific cells by ablating the unwanted cells, resulting in histologically enriched cell populations. The fundamental components of laser microdissection technology are (a) visualization of the cells of interest via microscopy, (b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and (c) removal of cells of interest from the heterogeneous tissue section. Laser energy supplied by LCM instruments can be infrared (810 nm) or ultraviolet (355 nm). Infrared lasers melt thermolabile polymers for cell capture, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes the unique features of the Arcturus(XT) laser capture microdissection instrument, which incorporates both infrared capture and ultraviolet cutting technology in one instrument, using a proteomic downstream assay as a model.

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.

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.

The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.

PubMed

Cai, Jing; Li, Tao; Huang, Bangxing; Cheng, Henghui; Ding, Hui; Dong, Weihong; Xiao, Man; Liu, Ling; Wang, Zehua

2014-01-01

Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

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).

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.

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

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.

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.

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

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.

LC-MS/MS imaging with thermal film-based laser microdissection.

PubMed

Oya, Michiko; Suzuki, Hiromi; Anas, Andrea Roxanne J; Oishi, Koichi; Ono, Kenji; Yamaguchi, Shun; Eguchi, Megumi; Sawada, Makoto

2018-01-01

Mass spectrometry (MS) imaging is a useful tool for direct and simultaneous visualization of specific molecules. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to evaluate the abundance of molecules in tissues using sample homogenates. To date, however, LC-MS/MS has not been utilized as an imaging tool because spatial information is lost during sample preparation. Here we report a new approach for LC-MS/MS imaging using a thermal film-based laser microdissection (LMD) technique. To isolate tissue spots, our LMD system uses a 808-nm near infrared laser, the diameter of which can be freely changed from 2.7 to 500 μm; for imaging purposes in this study, the diameter was fixed at 40 μm, allowing acquisition of LC-MS/MS images at a 40-μm resolution. The isolated spots are arranged on a thermal film at 4.5-mm intervals, corresponding to the well spacing on a 384-well plate. Each tissue spot is handled on the film in such a manner as to maintain its spatial information, allowing it to be extracted separately in its individual well. Using analytical LC-MS/MS in combination with the spatial information of each sample, we can reconstruct LC-MS/MS images. With this imaging technique, we successfully obtained the distributions of pilocarpine, glutamate, γ-aminobutyric acid, acetylcholine, and choline in a cross-section of mouse hippocampus. The protocol we established in this study is applicable to revealing the neurochemistry of pilocarpine model of epilepsy. Our system has a wide range of uses in fields such as biology, pharmacology, pathology, and neuroscience. Graphical abstract Schematic Indication of LMD-LC-MS/MS imaging.

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.

Cold-Temperature Plastic Resin Embedding of Liver for DNA- and RNA-Based Genotyping

PubMed Central

Finkelstein, Sydney D.; Dhir, Rajiv; Rabinovitz, Mordechai; Bischeglia, Michelle; Swalsky, Patricia A.; DeFlavia, Petrina; Woods, Jeffrey; Bakker, Anke; Becich, Michael

1999-01-01

The standard practice of tissue fixation in 10% formalin followed by embedding in paraffin wax preserves cellular morphology at the expense of availability and quality of DNA and RNA. The negative effect on cellular constituents results from a combination of extensive cross-linking and strand scission of DNA, RNA, and proteins induced by formaldehyde as well as RNA loss secondary to ubiquitous RNase activity and negative effects of high temperature exposure during paraffin melting, microscopic section collection, and tissue adherence to glass slides. An effective strategy to correlate cellular phenotype with molecular genotype involves microdissection of tissue sections based on specific histopathological features followed by genotyping of minute representative samples for specific underlying molecular alterations. Currently, this approach is limited to short-length polymerase chain reaction amplification (<250 bp) of DNA, due to the negative effects of standard tissue fixation and processing. To overcome this obstacle and permit both cellular morphology and nucleic acid content to be preserved to the fullest extent, we instituted a system of cold-temperature plastic resin embedding based on the use of the water-miscible methyl methacrylate polymer known as Immunobed (Polysciences, Warminster, PA). The system is simple, easy to adapt to clinical practice, and cost-effective. Immunobed tissue sections demonstrate a cellular appearance equivalent or even superior to that of standard tissue sections. Moreover, thin sectioning (0.5–1.0 μm thickness) renders ultrastructural evaluation feasible on plastic-embedded blocks. Tissue microdissection is readily performed, yielding high levels of long DNA and RNA for genomic and transcription-based correlative molecular analysis. We recommend the use of Immunobed or similar products for use in molecular anatomical pathology. PMID:11272904

Laser capture microdissection: should an ultraviolet or infrared laser be used?

PubMed

Vandewoestyne, Mado; Goossens, Karen; Burvenich, Christian; Van Soom, Ann; Peelman, Luc; Deforce, Dieter

2013-08-15

Laser capture microdissection (LCM) is a well-established cell separation technique. It combines microscopy with laser beam technology and allows targeting of specific cells or tissue regions that need to be separated from others. Consequently, this biological material can be used for genome or transcriptome analyses. Appropriate methods of sample preparation, however, are crucial for the success of downstream molecular analysis. The aim of this study was to objectively compare the two main LCM systems, one based on an ultraviolet (UV) laser and the other based on an infrared (IR) laser, on different criteria ranging from user-friendliness to sample quality. The comparison was performed on two types of samples: peripheral blood mononuclear cells and blastocysts. The UV laser LCM system had several advantages over the IR laser LCM system. Not only does the UV system allow faster and more precise sample collection, but also the obtained samples-even single cell samples-can be used for DNA extraction and downstream polymerase chain reaction (PCR) applications. RNA-based applications are more challenging for both LCM systems. Although sufficient RNA can be extracted from as few as 10 cells for reverse transcription quantitative PCR (RT-qPCR) analysis, the low RNA quality should be taken into account when designing the RT-qPCR assays. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Analysis of DNA methylation in FFPE tissues using the MethyLight technology.

PubMed

Dallol, Ashraf; Al-Ali, Waleed; Al-Shaibani, Amina; Al-Mulla, Fahd

2011-01-01

Novel biomarkers are sought after by mining DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. Such tissues offer the great advantage of often having complete clinical data (including survival), as well as the tissues are amenable for laser microdissection targeting specific tissue areas. Downstream analysis of such DNA includes mutational screens and methylation profiling. Screening for mutations by sequencing requires a significant amount of DNA for PCR and cycle sequencing. This is self-inhibitory if the gene screened has a large number of exons. Profiling DNA methylation using the MethyLight technology circumvents this problem and allows for the mining of several biomarkers from DNA extracted from a single microscope slide of the tissue of interest. We describe in this chapter a detailed protocol for MethyLight and its use in the determination of CpG Island Methylator Phenotype status in FFPE colorectal cancer samples.

Laser capture microdissection of embryonic cells and preparation of RNA for microarray assays.

PubMed

Redmond, Latasha C; Pang, Christopher J; Dumur, Catherine; Haar, Jack L; Lloyd, Joyce A

2014-01-01

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice-isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure(®) LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM.

Laser Capture Microdissection of Embryonic Cells and Preparation of RNA for Microarray Assays

PubMed Central

Redmond, Latasha C.; Pang, Christopher J.; Dumur, Catherine; Haar, Jack L.; Lloyd, Joyce A.

2014-01-01

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice–isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure® LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM. PMID:24318813

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.
<...

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

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.

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 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.

Liver gene expression profiles of rats treated with clofibric acid: comparison of whole liver and laser capture microdissected liver.

PubMed

Michel, Cécile; Desdouets, Chantal; Sacre-Salem, Béatrice; Gautier, Jean-Charles; Roberts, Ruth; Boitier, Eric

2003-12-01

Clofibric acid (CLO) is a peroxisome proliferator (PP) that acts through the peroxisome proliferator activated receptor alpha, leading to hepatocarcinogenesis in rodents. CLO-induced hepatocarcinogenesis is a multi-step process, first transforming normal liver cells into foci. The combination of laser capture microdissection (LCM) and genomics has the potential to provide expression profiles from such small cell clusters, giving an opportunity to understand the process of cancer development in response to PPs. To our knowledge, this is the first evaluation of the impact of the successive steps of LCM procedure on gene expression profiling by comparing profiles from LCM samples to those obtained with non-microdissected liver samples collected after a 1 month CLO treatment in the rat. We showed that hematoxylin and eosin (H&E) staining and laser microdissection itself do not impact on RNA quality. However, the overall process of the LCM procedure affects the RNA quality, resulting in a bias in the gene profiles. Nonetheless, this bias did not prevent accurate determination of a CLO-specific molecular signature. Thus, gene-profiling analysis of microdissected foci, identified by H&E staining may provide insight into the mechanisms underlying non-genotoxic hepatocarcinogenesis in the rat by allowing identification of specific genes that are regulated by CLO in early pre-neoplastic foci.

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.

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

Transcriptional profiling of cork oak phellogenic cells isolated by laser microdissection.

PubMed

Teixeira, Rita Teresa; Fortes, Ana Margarida; Bai, Hua; Pinheiro, Carla; Pereira, Helena

2018-02-01

The phenylpropanoid pathway impacts the cork quality development. In cork of bad quality, the flavonoid route is favored, whereas in good quality, cork lignin and suberin production prevails. Cork oaks develop a thick cork tissue as a protective shield that results of the continuous activity of a secondary meristem, the cork cambium, or phellogen. Most studies applied to developmental processes do not consider the cell types from which the samples were extracted. Here, laser microdissection (LM) coupled with transcript profiling using RNA sequencing (454 pyrosequencing) was applied to phellogen cells of trees producing low- and good quality cork. Functional annotation and functional enrichment analyses showed that stress-related genes are enriched in samples extracted from trees producing good quality cork (GQC). This process is under tight transcriptional (transcription factors, kinases) regulation and also hormonal control involving ABA, ethylene, and auxins. The phellogen cells collected from trees producing bad quality cork (BQC) show a consistent up-regulation of genes belonging to the flavonoid pathway as a response to stress. They also display a different modulation of cell wall genes resulting into a thinner cork layer, i.e., less meristematic activity. Based on the analysis of the phenylpropanoid pathway regulating genes, in GQC, the synthesis of lignin and suberin is promoted, whereas in BQC, the same pathway favors the biosynthesis of free phenolic compounds. This study provided new insights of how cell-specific gene expression can determine tissue and organ morphology and physiology and identified robust candidate genes that can be used in breeding programs aiming at improving cork quality.

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.

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

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.

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

Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection.

PubMed

Loayza, María Fernanda; Villavicencio, Fernando Xavier; Santander, Stephanie Carolina; Baldeón, Manuel; Ponce, Lourdes Karina; Salvador, Iván; Vivar Díaz, Nicolás

2015-01-01

To assess the molecular events exerted by Helicobacter pylori interacting directly with gastric epithelial cells, an improved procedure for microbial DNA isolation from stained hematoxilin-eosin gastric biopsies was developed based on laser micro-dissection (LM) [1]. Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2]. To improve the yield and quality of DNA isolated from H. pylori contacting intestinal epithelial cells, the following conditions were established after modification of the QIAamp DNA Micro kit. •Use of at least 25 cut sections of 10-20 μm of diameter and 3 μm thick with more than 10 bacteria in each cut.•Lysis with 30 μL of tissue lysis buffer and 20 μL of proteinase K (PK) with the tube in an upside-down position.•The use of thin purification columns with 35 μL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/μL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.

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.

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.

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.

Exploring the potential of laser capture microdissection technology in integrated oral biosciences.

PubMed

Thennavan, A; Sharma, M; Chandrashekar, C; Hunter, K; Radhakrishnan, R

2017-09-01

Laser capture microdissection (LCM) is a high-end research and diagnostic technology that helps in obtaining pure cell populations for the purpose of cell- or lesion-specific genomic and proteomic analysis. Literature search on the application of LCM in oral tissues was made through PubMed. There is ample evidence to substantiate the utility of LCM in understanding the underlying molecular mechanism involving an array of oral physiological and pathological processes, including odontogenesis, taste perception, eruptive tooth movement, oral microbes, and cancers of the mouth and jaw tumors. This review is aimed at exploring the potential application of LCM in oral tissues as a high-throughput tool for integrated oral sciences. The indispensable application of LCM in the construction of lesion-specific genomic libraries with emphasis on some of the novel molecular markers thus discovered is also highlighted. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

Liver Gene Expression Profiles of Rats Treated with Clofibric Acid

PubMed Central

Michel, Cécile; Desdouets, Chantal; Sacre-Salem, Béatrice; Gautier, Jean-Charles; Roberts, Ruth; Boitier, Eric

2003-01-01

Clofibric acid (CLO) is a peroxisome proliferator (PP) that acts through the peroxisome proliferator activated receptor α, leading to hepatocarcinogenesis in rodents. CLO-induced hepatocarcinogenesis is a multi-step process, first transforming normal liver cells into foci. The combination of laser capture microdissection (LCM) and genomics has the potential to provide expression profiles from such small cell clusters, giving an opportunity to understand the process of cancer development in response to PPs. To our knowledge, this is the first evaluation of the impact of the successive steps of LCM procedure on gene expression profiling by comparing profiles from LCM samples to those obtained with non-microdissected liver samples collected after a 1 month CLO treatment in the rat. We showed that hematoxylin and eosin (H&E) staining and laser microdissection itself do not impact on RNA quality. However, the overall process of the LCM procedure affects the RNA quality, resulting in a bias in the gene profiles. Nonetheless, this bias did not prevent accurate determination of a CLO-specific molecular signature. Thus, gene-profiling analysis of microdissected foci, identified by H&E staining may provide insight into the mechanisms underlying non-genotoxic hepatocarcinogenesis in the rat by allowing identification of specific genes that are regulated by CLO in early pre-neoplastic foci. PMID:14633594

Tissue-specific metabolites profiling and quantitative analyses of flavonoids in the rhizome of Belamcanda chinensis by combining laser-microdissection with UHPLC-Q/TOF-MS and UHPLC-QqQ-MS.

PubMed

Chen, Yu Jie; Liang, Zhi Tao; Zhu, Yan; Xie, Guo Yong; Tian, Mei; Zhao, Zhong Zhen; Qin, Min Jian

2014-12-01

The rhizome of Belamcanda chinensis (L.) DC. is a traditionally used medicinal material in China. Due to increasing demand, B. chinensis has been cultivated widely, and thus the study on its rational utilization of medicinal part and guidelines for the optimal cultivation and harvest is an important issue. Considering flavonoids were the main bioactive secondary metabolites of B. chinensis, fluorescence microscopy, laser microdissection (LMD), ultra-high performance liquid chromatography-quadrupole/time-of-flight-mass spectrometry (UHPLC-Q/TOF-MS), and UHPLC coupled with triple quadrupole mass spectrometer (UHPLC-QqQ-MS) were applied to profile and determine flavonoids in various tissues in this study. Consequently, 43 peaks were detected by UHPLC-Q/TOF-MS, and 26 flavonoid compounds combined with seven triterpene compounds were identified or tentatively identified in the tissue extractions. The results indicated that the hydrophobic compounds, especially flavonoid or isoflavonoid aglycones and xanthone mainly accumulated in the cork, whereas the hydrophilic compounds, namely the flavonoid and isoflavonoid glycosides were usually found in the cortex or center (the part inside of endodermis). Samples of rhizomes from different growth ages and origins were simultaneously analyzed. It was shown that the bulb or lateral part of the rhizome generally possessed more total flavonoids than the vertical part or the primordium. The present study established a new practical method to evaluate the quality of the rhizome of B. chinensis and to explore the relationship between distribution patterns of secondary metabolites and growth years of plants, thus important information for cultivation and processing was provided. Copyright © 2014 Elsevier B.V. All rights reserved.

Laser Capture Microdissection of Feline Streptomyces spp Pyogranulomatous Dermatitis and Cellulitis.

PubMed

Traslavina, R P; Reilly, C M; Vasireddy, R; Samitz, E M; Stepnik, C T; Outerbridge, C; Affolter, V K; Byrne, B A; Lowenstine, L J; White, S D; Murphy, B

2015-11-01

Suspected Streptomyces spp infections were identified in 4 cats at UC Davis Veterinary Medical Teaching Hospital between 1982 and 2011. Three had ulcerated, dark red mycetomas involving the dermis, subcutis, and fascia with fistulous tracts and/or regional lymphadenopathy. One cat had pyogranulomatous mesenteric lymphadenitis. Granulomatous inflammation in all cats contained colonies of Gram-positive, non-acid-fast organisms. All 4 cats failed to respond to aggressive medical and surgical treatment and were euthanized. Laser capture microdissection (LCM) was used to selectively harvest DNA from the affected formalin-fixed, paraffin-embedded (FFPE) tissues. Cloned amplicons from LCM-derived tissue confirmed the presence of Streptomyces spp in the dermatitis cases. Amplicons from the remaining cat with peritoneal involvement aligned with the 16S ribosomal RNA gene for Actinomycetales. Usually considered a contaminant, Streptomyces spp can be associated with refractory pyogranulomatous dermatitis and cellulitis in cats with outdoor access. LCM is useful in the diagnosis of bacterial diseases where contamination may be an issue. © The Author(s) 2014.

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

PubMed

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

2014-02-28

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

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

PubMed Central

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

2014-01-01

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

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.

Combining laser-assisted microdissection (LAM) and RNA-seq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo.

PubMed

Sakai, Kaori; Taconnat, Ludivine; Borrega, Nero; Yansouni, Jennifer; Brunaud, Véronique; Paysant-Le Roux, Christine; Delannoy, Etienne; Martin Magniette, Marie-Laure; Lepiniec, Loïc; Faure, Jean Denis; Balzergue, Sandrine; Dubreucq, Bertrand

2018-01-01

Genome-wide characterization of tissue- or cell-specific gene expression is a recurrent bottleneck in biology. We have developed a sensitive approach based on ultra-low RNA sequencing coupled to laser assisted microdissection for analyzing different tissues of the small Arabidopsis embryo. We first characterized the number of genes detected according to the quantity of tissue yield and total RNA extracted. Our results revealed that as low as 0.02 mm 2 of tissue and 50 pg of total RNA can be used without compromising the number of genes detected. The optimised protocol was used to compare the epidermal versus mesophyll cell transcriptomes of cotyledons at the torpedo-shaped stage of embryo development. The approach was validated by the recovery of well-known epidermal genes such AtML1 or AtPDF2 and genes involved in flavonoid and cuticular waxes pathways. Moreover, the interest and sensitivity of this approach were highlighted by the characterization of several transcription factors preferentially expressed in epidermal cells. This technical advance unlocks some current limitations of transcriptomic analyses and allows to investigate further and efficiently new biological questions for which only a very small amounts of cells need to be isolated. For instance, it paves the way to increasing the spatial accuracy of regulatory networks in developing small embryo of Arabidopsis or other plant tissues.

Comparison of Different Buffers for Protein Extraction from Formalin-Fixed and Paraffin-Embedded Tissue Specimens.

PubMed

Shen, Kaini; Sun, Jian; Cao, Xinxin; Zhou, Daobin; Li, Jian

2015-01-01

We determined the best extraction buffer for proteomic investigation using formalin-fixation and paraffin-embedded (FFPE) specimens. A Zwittergent 3-16 based buffer, sodium dodecyl sulfate (SDS)-containing buffer with/without polyethylene glycol 20000 (PEG20000), urea-containing buffer, and FFPE-FASP protein preparation kit were compared for protein extraction from different types of rat FFPE tissues, including the heart, brain, liver, lung, and kidney. All of the samples were divided into two groups of laser microdissected (LMD) and non-LMD specimens. For both kinds of specimens, Zwittergent was the most efficient buffer for identifying peptides and proteins, was broadly applicable to different tissues without impairing the enzymatic digestion, and was well compatible with mass spectrometry analysis. As a high molecular weight carrier substance, PEG20000 improved the identification of peptides and proteins; however, such an advantage is limited to tissues containing submicrograms to micrograms of protein. Considering its low lytic strength, urea-containing buffer would not be the first alternative for protein recovery. In conclusion, Zwittergent 3-16 is an effective buffer for extracting proteins from FFPE specimens for downstream proteomics analysis.

Is gliomatosis peritonei derived from the associated ovarian teratoma?

PubMed

Kwan, Man-Yee; Kalle, Wouter; Lau, Gene T C; Chan, John K C

2004-06-01

Gliomatosis peritonei, a rare condition that occurs almost exclusively in the setting of ovarian immature teratoma, is characterized by the occurrence of nodules of mature glial tissues in the peritoneum. It is controversial whether glial tissues are derived from maturation of the associated teratomatous tissue that has implanted in the peritoneum, or glial differentiation of subperitoneal stem cells. In this study, we employed the unique genetic characteristics of ovarian teratomas (often with a duplicated set of maternal chromosomes and thus homozygous at many polymorphic microsatellite loci) versus normal tissues (heterozygous pattern due to presence of maternal and paternal genetic materials) to investigate the origin of gliomatosis peritonei. DNA samples were extracted from microdissected paraffin-embedded tissues, including the glial implants, the associated ovarian teratomas, and normal tissues, to determine their patterns of microsatellite loci in a multiplex polymerase chain reaction system. Two cases were not informative because the ovarian teratoma showed a heterozygous microsatellite pattern. In the 5 informative cases, the normal tissues showed a heterozygous pattern in the microsatellite loci, the associated teratomas showed a homozygous pattern, and the glial tissues showed a heterozygous pattern. Thus, gliomatosis peritonei is genetically unrelated to the associated teratoma but is probably derived from nonteratomatous cells, such as through metaplasia of submesothelial cells.

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.

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.

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.

Analysis of p53 gene mutations in human gliomas by polymerase chain reaction-based single-strand conformation polymorphism and DNA sequencing.

PubMed

Sarkar, F H; Kupsky, W J; Li, Y W; Sreepathi, P

1994-03-01

Mutations in the p53 gene have been recognized in brain tumors, and clonal expansion of p53 mutant cells has been shown to be associated with glioma progression. However, studies on the p53 gene have been limited by the need for frozen tissues. We have developed a method utilizing polymerase chain reaction (PCR) for the direct analysis of p53 mutation by single-strand conformation polymorphism (SSCP) and by direct DNA sequencing of the p53 gene using a single 10-microns paraffin-embedded tissue section. We applied this method to screen for p53 gene mutations in exons 5-8 in human gliomas utilizing paraffin-embedded tissues. Twenty paraffin blocks containing tumor were selected from surgical specimens from 17 different adult patients. Tumors included six anaplastic astrocytomas (AAs), nine glioblastomas (GBs), and two mixed malignant gliomas (MMGs). The tissue section on the stained glass slide was used to guide microdissection of an unstained adjacent tissue section to ensure > 90% of the tumor cell population for p53 mutational analysis. Simultaneously, microdissection of the tissue was also carried out to obtain normal tissue from adjacent areas as a control. Mutations in the p53 gene were identified in 3 of 17 (18%) patients by PCR-SSCP analysis and subsequently confirmed by PCR-based DNA sequencing. Mutations in exon 5 resulting in amino acid substitution were found in one thalamic AA (codon 158, CGC > CTT: Arg > Leu) and one cerebral hemispheric GB (codon 151, CCG > CTG: Pro > Leu).(ABSTRACT TRUNCATED AT 250 WORDS)

HOXB2 as a novel prognostic indicator for stage I lung adenocarcinomas.

PubMed

Inamura, Kentauro; Togashi, Yuki; Okui, Michiyo; Ninomiya, Hironori; Hiramatsu, Miyako; Satoh, Yukitoshi; Okumura, Sakae; Nakagawa, Ken; Shimoji, Takashi; Noda, Tetsuo; Ishikawa, Yuichi

2007-09-01

Outcomes of patients with lung adenocarcinomas can be predicted to some extent from the pathologic stage (p-stage). Although all attempts are made to fully remove cancer lesions, still a number of p-stage I patients without metastatic disease at the time of surgery develop recurrences and die of cancer. It is thus very important to identify p-stage I patients who are at risk of recurrence. Previously, using microdissected samples, we identified metastasis-related genes. Using real-time reverse-transcriptase polymerase chain reaction analysis, we investigated the transcriptional levels of the top metastasis-related genes using 96 independent test lung adenocarcinoma samples and investigated their correlations with the prognosis. We document evidence that p-stage I patients with HOXB2 up-regulation have a worse prognosis than those with HOXB2 down-regulation (p = 0.0065), whereas the HOXB2 status has no prognostic significance for p-stage II-IV patients. Comparing tumors and corresponding normal lung tissue, we confirmed HOXB2 up-regulated lesions to have much higher HOXB2 expression than the corresponding normal tissue. Confirmation with a larger number of samples is needed, with further research to clarify the molecular functions of HOXB2.

RHEB expression in fibroadenomas of the breast.

PubMed

Eom, Minseob; Han, Airi; Yi, Sang Yeop; Shin, John Junghun; Cui, Ying; Park, Kwang Hwa

2008-04-01

Although fibroadenoma is one of the most common types of benign breast tumor, genes specific to the tumor have not been identified. Microarrays were used to identify differentially expressed genes between fibroadenoma and infiltrating ductal carcinoma. The comparative expression of one of the identified genes, RAS homolog enriched in the brain (RHEB), was further explored using reverse transcriptase-polymerase chain reaction (RT-PCR). Microarray analysis was performed on tissue samples from five patients with fibroadenoma. In the fibroadenoma samples, the genes HDAC1, ROS1, TNFRSF10A, WASP2, TYRP1, WEE1, and RHEB were expressed at levels more than twofold higher than in the normal tissues. RT-PCR for RHEB indicated increased expression of RHEB in fibroadenoma compared to breast cancer. When studied with real-time PCR, the average RHEB/beta-actin ratio in fibroadenoma samples was 1.99, 2.46-fold greater than the average RHEB/beta-actin ratio in breast carcinoma of 0.81 (P < 0.01). Immunohistochemistry and PCR followed by microdissection shows increased expression of RHEB in epithelial cells compared to the stromal cells of fibroadenoma. Therefore, RHEB could be used cytopathologically to distinguish fibroadenoma from malignant breast carcinomas as a secondary diagnostic tool.

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.

Laser assisted microdissection, an efficient technique to understand tissue specific gene expression patterns and functional genomics in plants.

PubMed

Gautam, Vibhav; Sarkar, Ananda K

2015-04-01

Laser assisted microdissection (LAM) is an advanced technology used to perform tissue or cell-specific expression profiling of genes and proteins, owing to its ability to isolate the desired tissue or cell type from a heterogeneous population. Due to the specificity and high efficiency acquired during its pioneering use in medical science, the LAM technique has quickly been adopted for use in many biological researches. Today, it has become a potent tool to address a wide range of questions in diverse field of plant biology. Beginning with comparative transcriptome analysis of different tissues such as reproductive parts, meristems, lateral organs, roots etc., LAM has also been extensively used in plant-pathogen interaction studies, proteomics, and metabolomics. In combination with next generation sequencing and proteomics analysis, LAM has opened up promising opportunities in the area of large scale functional studies in plants. Ever since the advent of this technique, significant improvements have been achieved in term of its instrumentation and method, which has made LAM a more efficient tool applicable in wider research areas. Here, we discuss the advancement of LAM technique with special emphasis on its methodology and highlight its scope in modern research areas of plant biology. Although we put emphasis on use of LAM in transcriptome studies, which is mostly used, we also discuss its recent application and scope in proteome and metabolome studies.

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

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.

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.

Genetic heterogeneity in uveal melanoma assessed by multiplex ligation-dependent probe amplification.

PubMed

Dopierala, Justyna; Damato, Bertil E; Lake, Sarah L; Taktak, Azzam F G; Coupland, Sarah E

2010-10-01

To determine intratumor genetic heterogeneity in uveal melanoma (UM) by multiplex ligation-dependent probe amplification (MLPA) in formalin-fixed, paraffin-embedded (FFPE) tumor tissues. DNA was extracted from whole tumor sections and from two to nine different areas microdissected from 32 FFPE UMs. Thirty-one loci on chromosomes 1, 3, 6, and 8 were tested with MLPA for copy number changes. The tumor was considered heterogeneous at a locus if (1) the difference in dosage quotients (DQs) of any two areas was 0.2 or more, and (2) the DQs of the areas belonged to different ranges. Comparison of MLPA data obtained from microdissected areas of the UMs showed heterogeneity in 1 to 26 examined loci in 24 (75%) tumors, with only 25% of the tumors being homogeneous. Intratumor heterogeneity of 3p12.2, 6p21.2, and 8q11.23 was most common, occurring in >30% of the UMs. Gains of chromosome 3 were observed in four UMs, with three of these tumors showing the highest degree of heterogeneity. Copy number variation was associated with differences in tumor cell type, but not with differences in tumor pigmentation or reactive inflammation. UMs with genetic heterogeneity across multiple sample sites showed equivocal MLPA results when the whole tumor section was examined. These results suggest that different clones dilute MLPA results. Heterogeneity of chromosomal abnormalities of chromosomes 1, 3, 6, and 8 is present in most UMs. This heterogeneity causes equivocal MLPA results. One random tumor sample may not be representative of the whole tumor and, therefore, may be insufficient for prognostic testing.

Computational deconvolution of genome wide expression data from Parkinson's and Huntington's disease brain tissues using population-specific expression analysis

PubMed Central

Capurro, Alberto; Bodea, Liviu-Gabriel; Schaefer, Patrick; Luthi-Carter, Ruth; Perreau, Victoria M.

2015-01-01

The characterization of molecular changes in diseased tissues gives insight into pathophysiological mechanisms and is important for therapeutic development. Genome-wide gene expression analysis has proven valuable for identifying biological processes in neurodegenerative diseases using post mortem human brain tissue and numerous datasets are publically available. However, many studies utilize heterogeneous tissue samples consisting of multiple cell types, all of which contribute to global gene expression values, confounding biological interpretation of the data. In particular, changes in numbers of neuronal and glial cells occurring in neurodegeneration confound transcriptomic analyses, particularly in human brain tissues where sample availability and controls are limited. To identify cell specific gene expression changes in neurodegenerative disease, we have applied our recently published computational deconvolution method, population specific expression analysis (PSEA). PSEA estimates cell-type-specific expression values using reference expression measures, which in the case of brain tissue comprises mRNAs with cell-type-specific expression in neurons, astrocytes, oligodendrocytes and microglia. As an exercise in PSEA implementation and hypothesis development regarding neurodegenerative diseases, we applied PSEA to Parkinson's and Huntington's disease (PD, HD) datasets. Genes identified as differentially expressed in substantia nigra pars compacta neurons by PSEA were validated using external laser capture microdissection data. Network analysis and Annotation Clustering (DAVID) identified molecular processes implicated by differential gene expression in specific cell types. The results of these analyses provided new insights into the implementation of PSEA in brain tissues and additional refinement of molecular signatures in human HD and PD. PMID:25620908

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.

Comparison of Different Buffers for Protein Extraction from Formalin-Fixed and Paraffin-Embedded Tissue Specimens

PubMed Central

Shen, Kaini; Sun, Jian; Cao, Xinxin; Zhou, Daobin; Li, Jian

2015-01-01

We determined the best extraction buffer for proteomic investigation using formalin-fixation and paraffin-embedded (FFPE) specimens. A Zwittergent 3–16 based buffer, sodium dodecyl sulfate (SDS)-containing buffer with/without polyethylene glycol 20000 (PEG20000), urea-containing buffer, and FFPE-FASP protein preparation kit were compared for protein extraction from different types of rat FFPE tissues, including the heart, brain, liver, lung, and kidney. All of the samples were divided into two groups of laser microdissected (LMD) and non-LMD specimens. For both kinds of specimens, Zwittergent was the most efficient buffer for identifying peptides and proteins, was broadly applicable to different tissues without impairing the enzymatic digestion, and was well compatible with mass spectrometry analysis. As a high molecular weight carrier substance, PEG20000 improved the identification of peptides and proteins; however, such an advantage is limited to tissues containing submicrograms to micrograms of protein. Considering its low lytic strength, urea-containing buffer would not be the first alternative for protein recovery. In conclusion, Zwittergent 3–16 is an effective buffer for extracting proteins from FFPE specimens for downstream proteomics analysis. PMID:26580073

Impact of Upfront Cellular Enrichment by Laser Capture Microdissection on Protein and Phosphoprotein Drug Target Signaling Activation Measurements in Human Lung Cancer: Implications for Personalized Medicine

PubMed Central

Elisa, Baldelli; B., Haura Eric; Lucio, Crinò; Douglas, Cress W.; Vienna, Ludovini; B., Schabath Matthew; A., Liotta Lance; F., Petricoin Emanuel; Mariaelena, Pierobon

2015-01-01

Purpose The aim of this study was to evaluate whether upfront cellular enrichment via laser capture microdissection is necessary for accurately quantifying predictive biomarkers in non-small cell lung cancer tumors. Experimental design Fifteen snap frozen surgical biopsies were analyzed. Whole tissue lysate and matched highly enriched tumor epithelium via laser capture microdissection (LCM) were obtained for each patient. The expression and activation/phosphorylation levels of 26 proteins were measured by reverse phase protein microarray. Differences in signaling architecture of dissected and undissected matched pairs were visualized using unsupervised clustering analysis, bar graphs, and scatter plots. Results Overall patient matched LCM and undissected material displayed very distinct and differing signaling architectures with 93% of the matched pairs clustering separately. These differences were seen regardless of the amount of starting tumor epithelial content present in the specimen. Conclusions and clinical relevance These results indicate that LCM driven upfront cellular enrichment is necessary to accurately determine the expression/activation levels of predictive protein signaling markers although results should be evaluated in larger clinical settings. Upfront cellular enrichment of the target cell appears to be an important part of the workflow needed for the accurate quantification of predictive protein signaling biomarkers. Larger independent studies are warranted. PMID:25676683

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

DNA methylation profiling of esophageal adenocarcinoma using Methylation Ligation-dependent Macroarray (MLM).

PubMed

Guilleret, Isabelle; Losi, Lorena; Chelbi, Sonia T; Fonda, Sergio; Bougel, Stéphanie; Saponaro, Sara; Gozzi, Gaia; Alberti, Loredana; Braunschweig, Richard; Benhattar, Jean

2016-10-14

Most types of cancer cells are characterized by aberrant methylation of promoter genes. In this study, we described a rapid, reproducible, and relatively inexpensive approach allowing the detection of multiple human methylated promoter genes from many tissue samples, without the need of bisulfite conversion. The Methylation Ligation-dependent Macroarray (MLM), an array-based analysis, was designed in order to measure methylation levels of 58 genes previously described as putative biomarkers of cancer. The performance of the design was proven by screening the methylation profile of DNA from esophageal cell lines, as well as microdissected formalin-fixed and paraffin-embedded (FFPE) tissues from esophageal adenocarcinoma (EAC). Using the MLM approach, we identified 32 (55%) hypermethylated promoters in EAC, and not or rarely methylated in normal tissues. Among them, 21promoters were found aberrantly methylated in more than half of tumors. Moreover, seven of them (ADAMTS18, APC, DKK2, FOXL2, GPX3, TIMP3 and WIF1) were found aberrantly methylated in all or almost all the tumor samples, suggesting an important role for these genes in EAC. In addition, dysregulation of the Wnt pathway with hypermethylation of several Wnt antagonist genes was frequently observed. MLM revealed a homogeneous pattern of methylation for a majority of tumors which were associated with an advanced stage at presentation and a poor prognosis. Interestingly, the few tumors presenting less methylation changes had a lower pathological stage. In conclusion, this study demonstrated the feasibility and accuracy of MLM for DNA methylation profiling of FFPE tissue samples. Copyright © 2016 Elsevier Inc. All rights reserved.

Distribution of toxic alkaloids in tissues from three herbal medicine Aconitum species using laser micro-dissection, UHPLC-QTOF MS and LC-MS/MS techniques.

PubMed

Jaiswal, Yogini; Liang, Zhitao; Ho, Alan; Wong, LaiLai; Yong, Peng; Chen, Hubiao; Zhao, Zhongzhen

2014-11-01

Aconite poisoning continues to be a major type of poisoning caused by herbal drugs in many countries. Nevertheless, despite its toxic characteristics, aconite is used because of its valuable therapeutic benefits. The aim of the present study was to determine the distribution of toxic alkaloids in tissues of aconite roots through chemical profiling. Three species were studied, all being used in traditional Chinese Medicine (TCM) and traditional Indian medicine (Ayurveda), namely: Aconitum carmichaelii, Aconitum kusnezoffii and Aconitum heterophyllum. Laser micro-dissection was used for isolation of target microscopic tissues, such as the metaderm, cortex, xylem, pith, and phloem, with ultra-high performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS) employed for detection of metabolites. Using a multi-targeted approach through auto and targeted LC-MS/MS, 48 known compounds were identified and the presence of aconitine, mesaconitine and hypaconitine that are the biomarkers of this plant was confirmed in the tissues. These results suggest that the three selected toxic alkaloids were exclusively found in A. carmichaelii and A. kusnezoffii. The most toxic components were found in large A. carmichaelii roots with more lateral root projections, and specifically in the metaderm, cork and vascular bundle tissues. The results from metabolite profiling were correlated with morphological features to predict the tissue specific distribution of toxic components and toxicity differences among the selected species. By careful exclusion of tissues having toxic diester diterpenoid alkaloids, the beneficial effects of aconite can still be retained and the frequency of toxicity occurrences can be greatly reduced. Knowledge of tissue-specific metabolite distribution can guide users and herbal drug manufacturers in prudent selection of relatively safer and therapeutically more effective parts of the root. The information provided from this study can contribute towards improved and effective management of therapeutically important, nonetheless, toxic drug such as Aconite. Copyright © 2014 Elsevier Ltd. All rights reserved.

Clinical Implications of Promoter Hypermethylation in RASSF1A and MGMT in Retinoblastoma1

PubMed Central

Choy, Kwong Wai; Lee, Tom C; Cheung, Kin Fai; Fan, Dorothy S P; Lo, Kwok Wai; Beaverson, Katherine L; Abramson, David H; Lam, Dennis S C; Yu, Christopher B O; Pang, Chi Pui

2005-01-01

Abstract We investigated the epigenetic silencing and genetic changes of the RAS-associated domain family 1A (RASSF1A) gene and the O6-methylguanine-DNA methyltransferase (MGMT) gene in retinoblastoma. We extracted DNA from microdissected tumor and normal retina tissues of the same patient in 68 retinoblastoma cases. Promoter methylation in RASSF1A and MGMT was analyzed by methylation-specific PCR, RASSF1A sequence alterations in all coding exons by direct DNA sequencing, and RASSF1A expression by RT-PCR. Cell cycle staging was analyzed by flow cytometry. We detected RASSF1A promoter hypermethylation in 82% of retinoblastoma, in tumor tissues only but not in adjacent normal retinal tissue cells. There was no expression of RASSF1A transcripts in all hypermethylated samples, but RASSF1A transcripts were restored after 5-aza-2′-deoxycytidine treatment with no changes in cell cycle or apoptosis. No mutation in the RASSF1A sequence was found. MGMT hypermethylation was present in 15% of theretinoblastoma samples, and the absence of MGMT hypermethylation was associated (P = .002) with retinoblastoma at advanced Reese-Ellsworth tumor stage. Our results revealed a high RASSF1A hypermethylation frequency in retinoblastoma. The correlation of MGMT inactivation by promoter hypermethylation with lower-stage diseases indicated that MGMT hypermethylation provides useful prognostic information. Epigenetic mechanism plays an important role in the progression of retinoblastoma. PMID:15799820

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.

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...

Isolation and expression of homeobox genes from the embryonic chicken eye.

PubMed

Dhawan, R R; Schoen, T J; Beebe, D C

1997-06-11

To identify homeobox-containing genes that may play a role in the differentiation of ocular tissues. Total RNA was isolated from microdissected chicken embryo eye tissues at 3.5 days of development (embryonic day 3.5; E3.5). An "anchor-oligo-dT primer" was used for the synthesis of cDNA. Degenerate oligonucleotides designed from highly-conserved sequences in the third helix of the homeobox and the "anchor-primer" were used to amplify cDNAs by polymerase chain reaction (PCR). PCR products were cloned and sequenced. The spatial and temporal expression of selected transcripts was mapped by whole-mount in situ hybridization and northern blot analysis. After sequencing eighteen clones we identified a member of the distal-less family (dlx-3) in cDNA from presumptive neural retina and three chicken homologs of the Xenopus "anterior neural fold" (Xanf-1) in cDNA from anterior eye tissue. Dlx transcripts were mapped by in situ hybridization. Expression began at Hamburger and Hamilton stage 14 (E2.5) and was widely distributed in embryonic mesenchyme on E3 and E4. Expression increased in the retina during early development and persisted until after hatching. The one anf clone selected for further study was not detected by in situ or northern blot analysis. It is feasible to isolate homeobox cDNAs directly from microdissected embryonic tissues. Chicken dlx-3 mRNA has a wider distribution in the embryo than expected, based on the expression of the mouse homolog. Dlx-3 may play a role in establishing or maintaining the differentiation of the retina.

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

RANKL/RANK/OPG cytokine receptor system: mRNA expression pattern in BPH, primary and metastatic prostate cancer disease.

PubMed

Christoph, Frank; König, Frank; Lebentrau, Steffen; Jandrig, Burkhard; Krause, Hans; Strenziok, Romy; Schostak, Martin

2018-02-01

The cytokine system RANKL (receptor activator of NF-κB ligand), its receptor RANK and the antagonist OPG (osteoprotegerin) play a critical role in bone turnover. Our investigation was conducted to describe the gene expression at primary tumour site in prostate cancer patients and correlate the results with Gleason Score and PSA level. Seventy-one samples were obtained from prostate cancer patients at the time of radical prostatectomy and palliative prostate resection (n = 71). Patients with benign prostate hyperplasia served as controls (n = 60). We performed real-time RT-PCR after microdissection of the samples. The mRNA expression of RANK was highest in tumour tissue from patients with bone metastases (p < 0.001) as compared to BPH or locally confined tumours, also shown in clinical subgroups distinguished by Gleason Score (< 7 or ≥ 7, p = 0.028) or PSA level (< 10 or ≥ 10 µg/l, p = 0.004). RANKL and OPG mRNA expression was higher in tumour tissue from patients with metastatic compared to local disease. The RANKL/OPG ratio was low in normal prostate tissue and high tumours with bone metastases (p < 0.05). Expression of all three cytokines was high in BPH tissue but did not exceed as much as in the tumour tissue. We demonstrated that RANK, RANKL and OPG are directly expressed by prostate cancer cells at the primary tumour site and showed a clear correlation with Gleason Score, serum PSA level and advanced disease. In BPH, mRNA expression is also detectable, but RANK expression does not exceed as much as compared to tumour tissue.

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.

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.

Quantitative profiling of immune repertoires for minor lymphocyte counts using unique molecular identifiers.

PubMed

Egorov, Evgeny S; Merzlyak, Ekaterina M; Shelenkov, Andrew A; Britanova, Olga V; Sharonov, George V; Staroverov, Dmitriy B; Bolotin, Dmitriy A; Davydov, Alexey N; Barsova, Ekaterina; Lebedev, Yuriy B; Shugay, Mikhail; Chudakov, Dmitriy M

2015-06-15

Emerging high-throughput sequencing methods for the analyses of complex structure of TCR and BCR repertoires give a powerful impulse to adaptive immunity studies. However, there are still essential technical obstacles for performing a truly quantitative analysis. Specifically, it remains challenging to obtain comprehensive information on the clonal composition of small lymphocyte populations, such as Ag-specific, functional, or tissue-resident cell subsets isolated by sorting, microdissection, or fine needle aspirates. In this study, we report a robust approach based on unique molecular identifiers that allows profiling Ag receptors for several hundred to thousand lymphocytes while preserving qualitative and quantitative information on clonal composition of the sample. We also describe several general features regarding the data analysis with unique molecular identifiers that are critical for accurate counting of starting molecules in high-throughput sequencing applications. Copyright © 2015 by The American Association of Immunologists, Inc.

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.

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.

Serial analysis of gene expression identifies connective tissue growth factor expression as a prognostic biomarker in gallbladder cancer.

PubMed

Alvarez, Hector; Corvalan, Alejandro; Roa, Juan C; Argani, Pedram; Murillo, Francisco; Edwards, Jennifer; Beaty, Robert; Feldmann, Georg; Hong, Seung-Mo; Mullendore, Michael; Roa, Ivan; Ibañez, Luis; Pimentel, Fernando; Diaz, Alfonso; Riggins, Gregory J; Maitra, Anirban

2008-05-01

Gallbladder cancer (GBC) is an uncommon neoplasm in the United States, but one with high mortality rates. This malignancy remains largely understudied at the molecular level such that few targeted therapies or predictive biomarkers exist. We built the first series of serial analysis of gene expression (SAGE) libraries from GBC and nonneoplastic gallbladder mucosa, composed of 21-bp long-SAGE tags. SAGE libraries were generated from three stage-matched GBC patients (representing Hispanic/Latino, Native American, and Caucasian ethnicities, respectively) and one histologically alithiasic gallbladder. Real-time quantitative PCR was done on microdissected epithelium from five matched GBC and corresponding nonneoplastic gallbladder mucosa. Immunohistochemical analysis was done on a panel of 182 archival GBC in high-throughput tissue microarray format. SAGE tags corresponding to connective tissue growth factor (CTGF) transcripts were identified as differentially overexpressed in all pairwise comparisons of GBC (P < 0.001). Real-time quantitative PCR confirmed significant overexpression of CTGF transcripts in microdissected primary GBC (P < 0.05), but not in metastatic GBC, compared with nonneoplastic gallbladder epithelium. By immunohistochemistry, 66 of 182 (36%) GBC had high CTGF antigen labeling, which was significantly associated with better survival on univariate analysis (P = 0.0069, log-rank test). An unbiased analysis of the GBC transcriptome by SAGE has identified CTGF expression as a predictive biomarker of favorable prognosis in this malignancy. The SAGE libraries from GBC and nonneoplastic gallbladder mucosa are publicly available at the Cancer Genome Anatomy Project web site and should facilitate much needed research into this lethal neoplasm.

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.

[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.

Factors That Influence the Quality of RNA From the Pancreas of Organ Donors.

PubMed

Philips, Tiffany; Kusmartseva, Irina; Gerling, Ivan C; Campbell-Thompson, Martha; Wasserfall, Clive; Pugliese, Alberto; Longmate, Jeffrey A; Schatz, Desmond A; Atkinson, Mark A; Kaddis, John S

2017-02-01

Attaining high-quality RNA from the tissues or organs of deceased donors used for research can be challenging due to physiological and logistical considerations. In this investigation, METHODS: RNA Integrity Number (RIN) was determined in pancreatic samples from 236 organ donors and used to define high (≥6.5) and low (≤4.5) quality RNAs. Logistic regression was used to evaluate the potential effects of novel or established organ and donor factors on RIN. Univariate analysis revealed donor cause of death (odds ratio [OR], 0.35; 95% confidence interval [CI], 0.15-0.77; P = 0.01), prolonged tissue storage before RNA extraction (OR, 0.65; 95% CI, 0.52-0.79; P < 0.01), pancreas region sampled (multiple comparisons, P < 0.01), and sample type (OR, 0.32; 95% CI, 0.15-0.67; P < 0.01) negatively influenced outcome. Conversely, duration of final hospitalization (OR, 3.95; 95% CI, 1.59-10.37; P < 0.01) and sample collection protocol (OR, 8.48; 95% CI, 3.96-19.30; P < 0.01) positively impacted outcome. Islet RNA obtained via laser capture microdissection improved RIN when compared with total pancreatic RNA from the same donor (ΔRIN = 1.3; 95% CI, 0.6-2.0; P < 0.01). A multivariable model demonstrates that autopsy-free and biopsy-free human pancreata received, processed, and preserved at a single center, using optimized procedures, from organ donors dying of anoxia with normal lipase levels increase the odds of obtaining high-quality RNA.

Factors That Influence the Quality of RNA From the Pancreas of Organ Donors

PubMed Central

Philips, Tiffany; Kusmartseva, Irina; Gerling, Ivan C.; Campbell-Thompson, Martha; Wasserfall, Clive; Pugliese, Alberto; Longmate, Jeffrey A.; Schatz, Desmond A.; Atkinson, Mark A.; Kaddis, John S.

2016-01-01

Objectives Attaining high quality RNA from the tissues or organs of deceased donors used for research can be challenging due to physiological and logistical considerations. In this investigation, Methods RNA Integrity Number (RIN) was determined in pancreatic samples from 236 organ donors and utilized to define high (≥6.5) and low (≤4.5) quality RNA. Logistic regression was used to evaluate the potential effects of novel or established organ and donor factors on RIN. Results Univariate analysis revealed donor cause of death (Odds Ratio [OR]=0.35, 95% Confidence Interval [CI]=0.15–0.77, p=0.01), prolonged tissue storage prior to RNA extraction (OR=0.65, 95%CI 0.52–0.79, p<0.01), pancreas region sampled (multiple comparisons, p<0.01), and sample type (OR=0.32, 95%CI 0.15–0.67, p<0.01) negatively influenced outcome. Conversely, duration of final hospitalization (OR=3.95, 95%CI 1.59–10.37, p<0.01) and sample collection protocol (OR=8.48, 95%CI 3.96–19.30, p<0.01) positively impacted outcome. Islet RNA obtained via laser capture microdissection improved RIN when compared to total pancreatic RNA from the same donor (∆RIN=1.3, 95%CI 0.6–2.0, p<0.01). Conclusions A multivariable model demonstrates that autopsy- and biopsy-free human pancreata received, processed, and preserved at a single center, using optimized procedures, from organ donors dying of anoxia with normal lipase levels increase the odds of obtaining high-quality RNA. PMID:27984510

Roles of Ras Homolog A in Invasive Ductal Breast Carcinoma

PubMed Central

Murakami, Eriko; Nakanishi, Yoko; Hirotani, Yukari; Ohni, Sumie; Tang, Xiaoyan; Masuda, Shinobu; Enomoto, Katsuhisa; Sakurai, Kenichi; Amano, Sadao; Yamada, Tsutomu; Nemoto, Norimichi

2016-01-01

Breast cancer has a poor prognosis owing to tumor cell invasion and metastasis. Although Ras homolog (Rho) A is involved in tumor cell invasion, its role in breast carcinoma is unclear. Here, RhoA expression was examined in invasive ductal carcinoma (IDC), with a focus on its relationships with epidermal-mesenchymal transition (EMT) and collective cell invasion. Forty-four surgical IDC tissue samples and two normal breast tissue samples were obtained. RhoA, E-cadherin, vimentin, and F-actin protein expression were analyzed by immunohistochemistry. RhoA, ROCK, mTOR, AKT1, and PIK3CA mRNA expression were conducted using laser microdissection and semi-nested quantitative reverse transcription-polymerase chain reaction. RhoA expression was stronger on the tumor interface of IDCs than the tumor center (P<0.001). RhoA expression was correlated with ROCK expression only in HER2-subtype IDC (P<0.05). In IDCs co-expressing RhoA and ROCK, F-actin expression was stronger on the tumor interface, particularly at the edges of tumor cells, than it was in ROCK-negative IDCs (P<0.0001). In conclusion, RhoA expression was not correlated with EMT in IDC, but enhanced F-actin expression was localized on the edge of tumor cells that co-expressed ROCK. RhoA/ROCK signaling may be associated with collective cell invasion, particularly in HER2-subtype IDC. PMID:27917007

Roles of Ras Homolog A in Invasive Ductal Breast Carcinoma.

PubMed

Murakami, Eriko; Nakanishi, Yoko; Hirotani, Yukari; Ohni, Sumie; Tang, Xiaoyan; Masuda, Shinobu; Enomoto, Katsuhisa; Sakurai, Kenichi; Amano, Sadao; Yamada, Tsutomu; Nemoto, Norimichi

2016-11-01

Breast cancer has a poor prognosis owing to tumor cell invasion and metastasis. Although Ras homolog (Rho) A is involved in tumor cell invasion, its role in breast carcinoma is unclear. Here, RhoA expression was examined in invasive ductal carcinoma (IDC), with a focus on its relationships with epidermal-mesenchymal transition (EMT) and collective cell invasion. Forty-four surgical IDC tissue samples and two normal breast tissue samples were obtained. RhoA, E-cadherin, vimentin, and F-actin protein expression were analyzed by immunohistochemistry. RhoA , ROCK , mTOR , AKT1 , and PIK3CA mRNA expression were conducted using laser microdissection and semi-nested quantitative reverse transcription-polymerase chain reaction. RhoA expression was stronger on the tumor interface of IDCs than the tumor center ( P <0.001). RhoA expression was correlated with ROCK expression only in HER2-subtype IDC ( P <0.05). In IDCs co-expressing RhoA and ROCK , F-actin expression was stronger on the tumor interface, particularly at the edges of tumor cells, than it was in ROCK -negative IDCs ( P <0.0001). In conclusion, RhoA expression was not correlated with EMT in IDC, but enhanced F-actin expression was localized on the edge of tumor cells that co-expressed ROCK. RhoA/ROCK signaling may be associated with collective cell invasion, particularly in HER2-subtype IDC.

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

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.

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.

Macro-environment of breast carcinoma: frequent genetic alterations in the normal appearing skins of patients with breast cancer.

PubMed

Moinfar, Farid; Beham, Alfred; Friedrich, Gerhard; Deutsch, Alexander; Hrzenjak, Andelko; Luschin, Gero; Tavassoli, Fattaneh A

2008-05-01

Genetic abnormalities in microenvironmental tissues with subsequent alterations of reciprocal interactions between epithelial and mesenchymal cells play a key role in the breast carcinogenesis. Although a few reports have demonstrated abnormal fibroblastic functions in normal-appearing fibroblasts taken from the skins of breast cancer patients, the genetic basis of this phenomenon and its implication for carcinogenesis are unexplored. We analyzed 12 mastectomy specimens showing invasive ductal carcinomas. In each case, morphologically normal epidermis and dermis, carcinoma, normal stroma close to carcinoma, and stroma at a distant from carcinoma were microdissected. Metastatic-free lymphatic tissues from lymph nodes served as a control. Using PCR, DNA extracts were examined with 11 microsatellite markers known for a high frequency of allelic imbalances in breast cancer. Losses of heterozygosity and/or microsatellite instability were detected in 83% of the skin samples occurring either concurrently with or independently from the cancerous tissues. In 80% of these cases at least one microsatellite marker displayed loss of heterozygosity or microsatellite instability in the skin, which was absent in carcinoma. A total of 41% of samples showed alterations of certain loci observed exclusively in the carcinoma but not in the skin compartments. Our study suggests that breast cancer is not just a localized genetic disorder, but rather part of a larger field of genetic alterations/instabilities affecting multiple cell populations in the organ with various cellular elements, ultimately contributing to the manifestation of the more 'localized' carcinoma. These data indicate that more global assessment of tumor micro- and macro-environment is crucial for our understanding of breast carcinogenesis.

MicroRNA-320 family is downregulated in colorectal adenoma and affects tumor proliferation by targeting CDK6.

PubMed

Tadano, Toshihiro; Kakuta, Yoichi; Hamada, Shin; Shimodaira, Yosuke; Kuroha, Masatake; Kawakami, Yoko; Kimura, Tomoya; Shiga, Hisashi; Endo, Katsuya; Masamune, Atsushi; Takahashi, Seiichi; Kinouchi, Yoshitaka; Shimosegawa, Tooru

2016-07-15

To investigate the microRNA (miRNA) expression during histological progression from colorectal normal mucosa through adenoma to carcinoma within a lesion. Using microarray, the sequential changes in miRNA expression profiles were compared in colonic lesions from matched samples; histologically, non-neoplastic mucosa, adenoma, and submucosal invasive carcinoma were microdissected from a tissue sample. Cell proliferation assay was performed to observe the effect of miRNA, and its target genes were predicted using bioinformatics approaches and the expression profile of SW480 transfected with the miRNA mimics. mRNA and protein levels of the target gene in colon cancer cell lines with a mimic control or miRNA mimics were measured using qRT-PCR and Western blotting. The expression levels of miRNA and target gene in colorectal tissue samples were also measured. Microarray analysis identified that the miR-320 family, including miR-320a, miR-320b, miR-320c, miR-320d and miR-320e, were differentially expressed in adenoma and submucosal invasive carcinoma. The miR-320 family, which inhibits cell proliferation, is frequently downregulated in colorectal adenoma and submucosal invasive carcinoma tissues. Seven genes including CDK6 were identified to be common in the results of gene expression array and bioinformatics analyses performed to find the target gene of the miR-320 family. We confirmed that mRNA and protein levels of CDK6 were significantly suppressed in colon cancer cell lines with miR-320 family mimics. CDK6 expression was found to increase from non-neoplastic mucosa through adenoma to submucosal invasive carcinoma tissues and showed an inverse correlation with miR-320 family expression. MiR-320 family affects colorectal tumor proliferation by targeting CDK6, plays important role in its growth, and is considered to be a biomarker for its early detection.

MicroRNA-320 family is downregulated in colorectal adenoma and affects tumor proliferation by targeting CDK6

PubMed Central

Tadano, Toshihiro; Kakuta, Yoichi; Hamada, Shin; Shimodaira, Yosuke; Kuroha, Masatake; Kawakami, Yoko; Kimura, Tomoya; Shiga, Hisashi; Endo, Katsuya; Masamune, Atsushi; Takahashi, Seiichi; Kinouchi, Yoshitaka; Shimosegawa, Tooru

2016-01-01

AIM: To investigate the microRNA (miRNA) expression during histological progression from colorectal normal mucosa through adenoma to carcinoma within a lesion. METHODS: Using microarray, the sequential changes in miRNA expression profiles were compared in colonic lesions from matched samples; histologically, non-neoplastic mucosa, adenoma, and submucosal invasive carcinoma were microdissected from a tissue sample. Cell proliferation assay was performed to observe the effect of miRNA, and its target genes were predicted using bioinformatics approaches and the expression profile of SW480 transfected with the miRNA mimics. mRNA and protein levels of the target gene in colon cancer cell lines with a mimic control or miRNA mimics were measured using qRT-PCR and Western blotting. The expression levels of miRNA and target gene in colorectal tissue samples were also measured. RESULTS: Microarray analysis identified that the miR-320 family, including miR-320a, miR-320b, miR-320c, miR-320d and miR-320e, were differentially expressed in adenoma and submucosal invasive carcinoma. The miR-320 family, which inhibits cell proliferation, is frequently downregulated in colorectal adenoma and submucosal invasive carcinoma tissues. Seven genes including CDK6 were identified to be common in the results of gene expression array and bioinformatics analyses performed to find the target gene of the miR-320 family. We confirmed that mRNA and protein levels of CDK6 were significantly suppressed in colon cancer cell lines with miR-320 family mimics. CDK6 expression was found to increase from non-neoplastic mucosa through adenoma to submucosal invasive carcinoma tissues and showed an inverse correlation with miR-320 family expression. CONCLUSION: MiR-320 family affects colorectal tumor proliferation by targeting CDK6, plays important role in its growth, and is considered to be a biomarker for its early detection. PMID:27559432

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

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

PubMed

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

2015-02-01

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

Global microRNA expression profiling of microdissected tissues identifies miR-135b as a novel biomarker for pancreatic ductal adenocarcinoma.

PubMed

Munding, Johanna B; Adai, Alex T; Maghnouj, Abdelouahid; Urbanik, Aleksandra; Zöllner, Hannah; Liffers, Sven T; Chromik, Ansgar M; Uhl, Waldemar; Szafranska-Schwarzbach, Anna E; Tannapfel, Andrea; Hahn, Stephan A

2012-07-15

Pancreatic ductal adenocarcinoma (PDAC) is known for its poor prognosis resulting from being diagnosed at an advanced stage. Accurate early diagnosis and new therapeutic modalities are therefore urgently needed. MicroRNAs (miRNAs), considered a new class of biomarkers and therapeutic targets, may be able to fulfill those needs. Combining tissue microdissection with global miRNA array analyses, cell type-specific miRNA expression profiles were generated for normal pancreatic ductal cells, acinar cells, PDAC cells derived from xenografts and also from macrodissected chronic pancreatitis (CP) tissues. We identified 78 miRNAs differentially expressed between ND and PDAC cells providing new insights into the miRNA-driven pathophysiological mechanisms involved in PDAC development. Having filtered miRNAs which are upregulated in the three pairwise comparisons of PDAC vs. ND, PDAC vs. AZ and PDAC vs. CP, we identified 15 miRNA biomarker candidates including miR-135b. Using relative qRT-PCR to measure miR-135b normalized to miR-24 in 75 FFPE specimens (42 PDAC and 33 CP) covering a broad range of tumor content, we discriminated CP from PDAC with a sensitivity and specificity of 92.9% [95% CI=(80.5, 98.5)] and 93.4% [95% CI=(79.8, 99.3)], respectively. Furthermore, the area under the curve (AUC) value reached of 0.97 was accompanied by positive and negative predictive values of 95% and 91%, respectively. In conclusion, we report pancreatic cell-specific global miRNA profiles, which offer new candidate miRNAs to be exploited for functional studies in PDAC. Furthermore, we provide evidence that miRNAs are well-suited analytes for development of sensitive and specific aid-in-diagnosis tests for PDAC. Copyright © 2011 UICC.

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

Touch imprint cytology with massively parallel sequencing (TIC-seq): a simple and rapid method to snapshot genetic alterations in tumors.

PubMed

Amemiya, Kenji; Hirotsu, Yosuke; Goto, Taichiro; Nakagomi, Hiroshi; Mochizuki, Hitoshi; Oyama, Toshio; Omata, Masao

2016-12-01

Identifying genetic alterations in tumors is critical for molecular targeting of therapy. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissue is usually employed for genetic analysis. However, DNA extracted from FFPE tissue is often not suitable for analysis because of its low levels and poor quality. Additionally, FFPE sample preparation is time-consuming. To provide early treatment for cancer patients, a more rapid and robust method is required for precision medicine. We present a simple method for genetic analysis, called touch imprint cytology combined with massively paralleled sequencing (touch imprint cytology [TIC]-seq), to detect somatic mutations in tumors. We prepared FFPE tissues and TIC specimens from tumors in nine lung cancer patients and one patient with breast cancer. We found that the quality and quantity of TIC DNA was higher than that of FFPE DNA, which requires microdissection to enrich DNA from target tissues. Targeted sequencing using a next-generation sequencer obtained sufficient sequence data using TIC DNA. Most (92%) somatic mutations in lung primary tumors were found to be consistent between TIC and FFPE DNA. We also applied TIC DNA to primary and metastatic tumor tissues to analyze tumor heterogeneity in a breast cancer patient, and showed that common and distinct mutations among primary and metastatic sites could be classified into two distinct histological subtypes. TIC-seq is an alternative and feasible method to analyze genomic alterations in tumors by simply touching the cut surface of specimens to slides. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

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.

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.

Prognostic impact of a compartment-specific angiogenic marker profile in patients with pancreatic cancer.

PubMed

Kahlert, Christoph; Fiala, Maria; Musso, Gabriel; Halama, Niels; Keim, Sophia; Mazzone, Massimiliano; Lasitschka, Felix; Pecqueux, Mathieu; Klupp, Fee; Schmidt, Thomas; Rahbari, Nuh; Schölch, Sebastian; Pilarsky, Christian; Ulrich, Alexis; Schneider, Martin; Weitz, Juergen; Koch, Moritz

2014-12-30

Pancreatic cancer consists of a heterogenous bulk of tumor cells and stroma cells which contribute to tumor progression by releasing angiogenic factors. Those factors can be detected as circulating serum factors. We performed a compartment-specific analysis of tumor-derived and stroma-derived angiogenic factors to identify biomarkers and molecular targets for the treatment of pancreatic cancer. Kryo-frozen tissue from primary ductal adenocarcinomas (n = 51) was laser-microdissected to isolate tumor and stroma tissue. Expression of 17 angiogenic factors (angiopoietin-2, follistatin, GCSF, HGF, interleukin-8, leptin, PDGF-BB, PECAM-1, VEGF, matrix metalloproteinase -1, -2, -3, -7, -9, -10, -12, and -13) was analyzed using a multiplex elisa assay for tissue-derived proteins and corresponding serum. Our study reveals a compartment-specific expression profile for several angiogenic factors and matrix metalloproteinases. ROC analysis of corresponding serum samples reveals MMP-7 and MMP-12 as strong classifiers for the diagnosis of patients with pancreatic cancer vs. healthy control donors. High expression of tumor-derived PDGF-BB and MMP-1 correlates with prolonged survival in univariate and multivariate analysis. In conclusion, a distinct expression patterns for angiogenic cytokines and MMPs in pancreatic cancer and surrounding stroma may implicate them as novel targets for cancer treatment. Tumor-derived PDGF-BB and MMP-1 are significant and independent prognostic markers for poor survival.

The Recent Developments in Sample Preparation for Mass Spectrometry-Based Metabolomics.

PubMed

Gong, Zhi-Gang; Hu, Jing; Wu, Xi; Xu, Yong-Jiang

2017-07-04

Metabolomics is a critical member in systems biology. Although great progress has been achieved in metabolomics, there are still some problems in sample preparation, data processing and data interpretation. In this review, we intend to explore the roles, challenges and trends in sample preparation for mass spectrometry- (MS-) based metabolomics. The newly emerged sample preparation methods were also critically examined, including laser microdissection, in vivo sampling, dried blood spot, microwave, ultrasound and enzyme-assisted extraction, as well as microextraction techniques. Finally, we provide some conclusions and perspectives for sample preparation in MS-based metabolomics.

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

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

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.

Integrative transcriptome analysis identifies deregulated microRNA-transcription factor networks in lung adenocarcinoma

PubMed Central

Cinegaglia, Naiara C.; Andrade, Sonia Cristina S.; Tokar, Tomas; Pinheiro, Maísa; Severino, Fábio E.; Oliveira, Rogério A.; Hasimoto, Erica N.; Cataneo, Daniele C.; Cataneo, Antônio J.M.; Defaveri, Júlio; Souza, Cristiano P.; Marques, Márcia M.C.; Carvalho, Robson F.; Coutinho, Luiz L.; Gross, Jefferson L.; Rogatto, Silvia R.; Lam, Wan L.; Jurisica, Igor; Reis, Patricia P.

2016-01-01

Herein, we aimed at identifying global transcriptome microRNA (miRNA) changes and miRNA target genes in lung adenocarcinoma. Samples were selected as training (N = 24) and independent validation (N = 34) sets. Tissues were microdissected to obtain >90% tumor or normal lung cells, subjected to miRNA transcriptome sequencing and TaqMan quantitative PCR validation. We further integrated our data with published miRNA and mRNA expression datasets across 1,491 lung adenocarcinoma and 455 normal lung samples. We identified known and novel, significantly over- and under-expressed (p ≤ 0.01 and FDR≤0.1) miRNAs in lung adenocarcinoma compared to normal lung tissue: let-7a, miR-10a, miR-15b, miR-23b, miR-26a, miR-26b, miR-29a, miR-30e, miR-99a, miR-146b, miR-181b, miR-181c, miR-421, miR-181a, miR-574 and miR-1247. Validated miRNAs included let-7a-2, let-7a-3, miR-15b, miR-21, miR-155 and miR-200b; higher levels of miR-21 expression were associated with lower patient survival (p = 0.042). We identified a regulatory network including miR-15b and miR-155, and transcription factors with prognostic value in lung cancer. Our findings may contribute to the development of treatment strategies in lung adenocarcinoma. PMID:27081085

Differential vitamin D 24-hydroxylase/CYP24A1 gene promoter methylation in endothelium from benign and malignant human prostate

PubMed Central

Karpf, Adam R; Omilian, Angela R; Bshara, Wiam; Tian, Lili; Tangrea, Michael A; Morrison, Carl D; Johnson, Candace S

2011-01-01

Epigenetic alterations occur in tumor-associated vessels in the tumor microenvironment. Methylation of the CYP24A1 gene promoter differs in endothelial cells isolated from tumors and non-tumor microenvironments in mice. The epigenetic makeup of endothelial cells of human tumor-associated vasculature is unknown due to difficulty of isolating endothelial cells populations from a heterogeneous tissue microenvironment. To ascertain CYP24A1 promoter methylation in tumor-associated endothelium, we utilized laser microdissection guided by CD31 immunohistochemistry to procure endothelial cells from human prostate tumor specimens. Prostate tissues were obtained following robotic radical prostatectomy from men with clinically localized prostate cancer. Adjacent histologically benign prostate tissues were used to compare endothelium from benign versus tumor microenvironments. Sodium bisulfite sequencing of CYP24A1 promoter region showed that the average CYP24A1 promoter methylation in the endothelium was 20% from the tumor microenvironment compared with 8.2% in the benign microenvironment (p < 0.05). A 2-fold to 17-fold increase in CYP24A1 promoter methylation was observed in the prostate tumor endothelium compared with the matched benign prostate endothelium in four patient samples, while CYP24A1 promoter methylation remained unchanged in two patient samples. In addition, there is no correlation of the level of CYP24A1 promoter methylation in prostate tumor-associated endothelium with that of epithelium/stroma. This study demonstrates that the CYP24A1 promoter is methylated in tumor-associated endothelium, indicating that epigenetic alterations in CYP24A1 may play a role in determining the phenotype of tumor-associated vasculature in the prostate tumor microenvironment. PMID:21725204

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.

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.

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.

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

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.

TET2 mutations in B cells of patients affected by angioimmunoblastic T-cell lymphoma.

PubMed

Schwartz, Friederike H; Cai, Qian; Fellmann, Eva; Hartmann, Sylvia; Mäyränpää, Mikko I; Karjalainen-Lindsberg, Marja-Liisa; Sundström, Christer; Scholtysik, René; Hansmann, Martin-Leo; Küppers, Ralf

2017-06-01

Angioimmunoblastic T-cell lymphomas (AITLs) frequently carry mutations in the TET2 and IDH2 genes. TET2 mutations represent early genetic lesions as they had already been detected in haematopoietic precursor cells of AITL patients. We show by analysis of whole-tissue sections and microdissected PD1 + cells that the frequency of TET2-mutated AITL is presumably even higher than reported (12/13 cases in our collection; 92%). In two-thirds of informative AITLs (6/9), a fraction of B cells was also TET2-mutated. Investigation of four AITLs by TET2 and IGHV gene sequencing of single microdissected B cells showed that between 10% and 60% of polyclonal B cells in AITL lymph nodes harboured the identical TET2 mutations of the respective T-cell lymphoma clone. Thus, TET2-mutated haematopoietic precursor cells in AITL patients not only give rise to the T-cell lymphoma but also generate a large population of mutated mature B cells. Future studies will show whether this is a reason why AITL patients frequently also develop B-cell lymphomas. 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.

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

Glomeruli of Dense Deposit Disease contain components of the alternative and terminal complement pathway

PubMed Central

Sethi, Sanjeev; Gamez, Jeffrey D.; Vrana, Julie A.; Theis, Jason D.; Bergen, H. Robert; Zipfel, Peter F.; Dogan, Ahmet; Smith, Richard J. H.

2009-01-01

Dense Deposit Disease (DDD), or membranoproliferative glomerulonephritis type II, is a rare renal disease characterized by dense deposits in the mesangium and along the glomerular basement membranes that can be seen by electron microscopy. Although these deposits contain complement factor C3, as determined by immunofluorescence microscopy, their precise composition remains unknown. To address this question, we used mass spectrometry to identify the proteins in laser microdissected glomeruli isolated from paraffin-embedded tissue of eight confirmed cases of DDD. Compared to glomeruli from five control patients, we found that all of the glomeruli from patients with DDD contain components of the alternative pathway and terminal complement complex. Factor C9 was uniformly present as well as the two fluid-phase regulators of terminal complement complex clusterin and vitronectin. In contrast, in nine patients with immune complex–mediated membranoproliferative glomerulonephritis, glomerular samples contained mainly immunoglobulins and complement factors C3 and C4. Our study shows that in addition to fluid-phase dysregulation of the alternative pathway, soluble components of the terminal complement complex contribute to glomerular lesions found in DDD. PMID:19177158

Proteomics in investigation of cancer metastasis: functional and clinical consequences and methodological challenges.

PubMed

Maryáš, Josef; Faktor, Jakub; Dvořáková, Monika; Struhárová, Iva; Grell, Peter; Bouchal, Pavel

2014-03-01

Metastases are responsible for most of the cases of death in patients with solid tumors. There is thus an urgent clinical need of better understanding the exact molecular mechanisms and finding novel therapeutics targets and biomarkers of metastatic disease of various tumors. Metastases are formed in a complicated biological process called metastatic cascade. Up to now, proteomics has enabled the identification of number of metastasis-associated proteins and potential biomarkers in cancer tissues, microdissected cells, model systems, and secretomes. Expression profiles and biological role of key proteins were confirmed in verification and functional experiments. This communication reviews these observations and analyses the methodological aspects of the proteomics approaches used. Moreover, it reviews contribution of current proteomics in the field of functional characterization and interactome analysis of proteins involved in various events in metastatic cascade. It is evident that ongoing technical progress will further increase proteome coverage and sample capacity of proteomics technologies, giving complex answers to clinical and functional questions asked. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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.

A method for mass harvesting islets (Brockmann bodies) from teleost fish.

PubMed

Yang, H; Wright, J R

1995-01-01

In certain species of fish, the insulin-producing tissue is uniquely located in separate structures called Brockmann bodies (BBs). Tilapia BBs have been shown to be a simple and inexpensive source of islet cells for xenotransplantation research. Each donor tilapia contains roughly 12-15 BBs, measuring from 0.3 to 5.0 mm in maximum dimension, in a triangular region of adipose tissue bounded by the liver, stomach, and spleen/gallbladder. At present, the larger BBs (usually 2-4) are harvested by microdissecting these "BB regions" using jeweler's forceps and microvascular scissors while being visualized with the aid of a dissecting microscope. It is a simple but time-consuming task that would not be applicable for harvesting massive amounts of BB tissue for large animal studies. Therefore, we have developed an easier and more efficient method of harvesting BBs based on a standard enzymatic method for isolating human adipocytes. BB regions are harvested from donor fish and pooled into a 50 mL plastic tube containing collagenase Type II (3 mg/mL) in Hank's balanced salt solution (HBSS); the tube is then placed in a 37 degrees C waterbath/shaker for roughly 15 min. The exact length of the digestion interval is determined by visual inspection of the tube to determine whether the BBs have been liberated. The digestion is then stopped by adding excess cold HBSS. The adipocytes float while the BBs and residual connective tissue (i.e., a few blood vessels, nerves, and bile ducts) form a pellet. The pellet is washed several times in HBSS and then placed in a culture dish. The BBs are easily handpicked with a siliconized pipette. Based on functional data and DNA content, this new method roughly doubles or triples our yield of BB tissue per donor fish. To determine whether BBs harvested in this manner functioned in a manner similar to those harvested by microdissection, we performed a series of transplants using mass-harvested BBs. Long-term normoglycemia was achieved in streptozotocin-diabetic nude mice and mean graft survival time was not altered in streptozotocin-diabetic euthymic balb/c mice. However, the total weight of donor fish required per recipient was decreased by 50% in both strains.

Deregulation of SYCP2 predicts early stage human papillomavirus-positive oropharyngeal carcinoma: A prospective whole transcriptome analysis.

PubMed

Masterson, Liam; Sorgeloos, Frederic; Winder, David; Lechner, Matt; Marker, Alison; Malhotra, Shalini; Sudhoff, Holger; Jani, Piyush; Goon, Peter; Sterling, Jane

2015-11-01

This study was designed to identify significant differences in gene expression profiles of human papillomavirus (HPV)-positive and HPV-negative oropharyngeal squamous cell carcinomas (OPSCC) and to better understand the functional and biological effects of HPV infection in the premalignant pathway. Twenty-four consecutive patients with locally advanced primary OPSCC were included in a prospective clinical trial. Fresh tissue samples (tumor vs. matched normal epithelium) were subjected to whole transcriptome analysis and the results validated on the same cohort with RT-quantitative real-time PCR. In a separate retrospective cohort of 27 OPSCC patients, laser capture microdissection of formalin-fixed, paraffin-embedded tissue allowed RNA extraction from adjacent regions of normal epithelium, carcinoma in situ (premalignant) and invasive SCC tissue. The majority of patients showed evidence of high-risk HPV16 positivity (80.4%). Predictable fold changes of RNA expression in HPV-associated disease included multiple transcripts within the p53 oncogenic pathway (e.g. CDKN2A/CCND1). Other candidate transcripts found to have altered levels of expression in this study have not previously been established (SFRP1, CRCT1, DLG2, SYCP2, and CRNN). Of these, SYCP2 showed the most consistent fold change from baseline in premalignant tissue; aberrant expression of this protein may contribute to genetic instability during HPV-associated cancer development. If further corroborated, this data may contribute to the development of a non-invasive screening tool. This study is registered with the UK Clinical Research Network (ref.: 11945). © 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

Effects of bone sialoprotein on pancreatic cancer cell growth, invasion and metastasis.

PubMed

Kayed, Hany; Kleeff, Jörg; Keleg, Shereen; Felix, Klaus; Giese, Thomas; Berger, Martin R; Büchler, Markus W; Friess, Helmut

2007-01-08

Bone sialoprotein (BSP) is an acidic glycoprotein that plays an important role in cancer cell growth, migration and invasion. The expression, localization and possible function of BSP in chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC) were analyzed by QRT-PCR, laser capture microdissection, DNA microarray analysis, immunoblotting, radioimmunoassays and immunohistochemistry as well as cell growth, invasion, scattering, and adhesion assays. BSP mRNA was detected in 40.7% of normal, in 80% of CP and in 86.4% of PDAC samples. The median BSP mRNA levels were 6.1 and 0.9copies/microl cDNA in PDAC and CP tissues, respectively, and zero copies/microl cDNA in normal pancreatic tissues. BSP was weakly present in the cytoplasm of islet cells and ductal cells in 20% of normal pancreatic tissues. BSP was localized in the tubular complexes of both CP and PDAC, as well as in pancreatic cancer cells. Five out of 8 pancreatic cancer cell lines expressed BSP mRNA. Recombinant BSP (rBSP) inhibited Capan-1 and SU8686 pancreatic cancer cell growth, with a maximal effect of -46.4+/-12.0% in Capan-1 cells and -45.7+/-14.5% in SU8686 cells. rBSP decreased the invasion of SU8686 cells by -59.1+/-11.2% and of Capan-1 cells by -13.3+/-3.8% (P<0.05), whereas it did not affect scattering or adhesion of both cell lines. In conclusion, endogenous BSP expression levels in pancreatic cancer cells and low to absent BSP expression in the surrounding stromal tissue elements may indirectly act to enhance the proliferation and invasion of pancreatic cancer cells.

Microanalysis of plant cell wall polysaccharides.

PubMed

Obel, Nicolai; Erben, Veronika; Schwarz, Tatjana; Kühnel, Stefan; Fodor, Andrea; Pauly, Markus

2009-09-01

Oligosaccharide Mass Profiling (OLIMP) allows a fast and sensitive assessment of cell wall polymer structure when coupled with Matrix Assisted Laser Desorption Ionisation Time Of Flight Mass Spectrometry (MALDI-TOF MS). The short time required for sample preparation and analysis makes possible the study of a wide range of plant organs, revealing a high degree of heterogeneity in the substitution pattern of wall polymers such as the cross-linking glycan xyloglucan and the pectic polysaccharide homogalacturonan. The high sensitivity of MALDI-TOF allows the use of small amounts of samples, thus making it possible to investigate the wall structure of single cell types when material is collected by such methods as laser micro-dissection. As an example, the analysis of the xyloglucan structure in the leaf cell types outer epidermis layer, entire epidermis cell layer, palisade mesophyll cells, and vascular bundles were investigated. OLIMP is amenable to in situ wall analysis, where wall polymers are analyzed on unprepared plant tissue itself without first isolating cell walls. In addition, OLIMP enables analysis of wall polymers in Golgi-enriched fractions, the location of nascent matrix polysaccharide biosynthesis, enabling separation of the processes of wall biosynthesis versus post-deposition apoplastic metabolism. These new tools will make possible a semi-quantitative analysis of the cell wall at an unprecedented level.

Molecular testing guidelines for lung adenocarcinoma: Utility of cell blocks and concordance between fine-needle aspiration cytology and histology samples

PubMed Central

Heymann, Jonas J.; Bulman, William A.; Maxfield, Roger A.; Powell, Charles A.; Halmos, Balazs; Sonett, Joshua; Beaubier, Nike T.; Crapanzano, John P.; Mansukhani, Mahesh M.; Saqi, Anjali

2014-01-01

Background: Lung cancer is a leading cause of mortality, and patients often present at a late stage. More recently, advances in screening, diagnosing, and treating lung cancer have been made. For instance, greater numbers of minimally invasive procedures are being performed, and identification of lung adenocarcinoma driver mutations has led to the implementation of targeted therapies. Advances in molecular techniques enable use of scant tissue, including cytology specimens. In addition, per recently published consensus guidelines, cytology-derived cell blocks (CBs) are preferred over direct smears. Yet, limited comparison of molecular testing of fine-needle aspiration (FNA) CBs and corresponding histology specimens has been performed. This study aimed to establish concordance of epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma (KRAS) virus homolog testing between FNA CBs and histology samples from the same patients. Materials and Methods: Patients for whom molecular testing for EGFR or KRAS was performed on both FNA CBs and histology samples containing lung adenocarcinoma were identified retrospectively. Following microdissection, when necessary, concordance of EGFR and KRAS molecular testing results between FNA CBs and histology samples was evaluated. Results: EGFR and/or KRAS testing was performed on samples obtained from 26 patients. Concordant results were obtained for all EGFR (22/22) and KRAS (17/17) mutation analyses performed. Conclusions: Identification of mutations in lung adenocarcinomas affects clinical decision-making, and it is important that results from small samples be accurate. This study demonstrates that molecular testing on cytology CBs is as sensitive and specific as that on histology. PMID:24987443

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

Signatures from Tissue-specific MPSS Libraries Identify Transcripts Preferentially Expressed in the Mouse Inner Ear

PubMed Central

Peters, Linda M.; Belyantseva, Inna A.; Lagziel, Ayala; Battey, James F.; Friedman, Thomas B.; Morell, Robert J.

2007-01-01

Specialization in cell function and morphology is influenced by the differential expression of mRNAs, many of which are expressed at low abundance and restricted to certain cell types. Detecting such transcripts in cDNA libraries may require sequencing millions of clones. Massively parallel signature sequencing (MPSS) is well-suited for identifying transcripts that are expressed in discrete cell types and in low abundance. We have made MPSS libraries from microdissections of three inner ear tissues. By comparing these MPSS libraries to those of 87 other tissues included in the Mouse Reference Transcriptome (MRT) online resource, we have identified genes that are highly enriched in, or specific to, the inner ear. We show by RT-PCR and in situ hybridization that signatures unique to the inner ear libraries identify transcripts with highly specific cell-type localizations. These transcripts serve to illustrate the utility of a resource that is available to the research community. Utilization of these resources will increase the number of known transcription units and expand our knowledge of the tissue-specific regulation of the transcriptome. PMID:17049805

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.

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.

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.

Tympanic membrane organ culture using cell culture well inserts engrafted with tympanic membrane tissue explants.

PubMed

Liew, Lawrence J; Day, Richard M; Dilley, Rodney J

2017-03-01

Tissue engineering approaches using growth factors and various materials for repairing chronic perforations of the tympanic membrane are being developed, but there are surprisingly few relevant tissue culture models available to test new treatments. Here, we present a simple three-dimensional model system based on micro-dissecting the rat tympanic membrane umbo and grafting it into the membrane of a cell culture well insert. Cell outgrowth from the graft produced sufficient cells to populate a membrane of similar surface area to the human tympanic membrane within 2 weeks. Tissue grafts from the annulus region also showed cell outgrowth but were not as productive. The umbo organoid supported substantial cell proliferation and migration under the influence of keratinocyte growth medium. Cells from umbo grafts were enzymatically harvested from the polyethylene terephthalate (PET) membrane for expansion in routine culture and cells could be harvested consecutively from the same graft over multiple cycles. We used harvested cells to test cell migration properties and to engraft a porous silk scaffold material as proof-of-principle for tissue engineering applications. This model is simple enough to be widely adopted for tympanic membrane regeneration studies and has promise as a tissue-equivalent model alternative to animal testing.

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

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

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.

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

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.

A Practical Approach to Tumor Heterogeneity in Clinical Research and Diagnostics.

PubMed

Stanta, Giorgio; Bonin, Serena

2018-01-01

This Pathobiology issue tries to better define the complex phenomenon of intratumor heterogeneity (ITH), mostly from a practical point of view. This topic has been chosen because ITH is a central issue in tumor development and has to be investigated directly in patient tissue and immediately applied in the treatment of the presenting patient. Different types of ITH should be considered: clonal genetic and epigenetic evolution, morphological heterogeneity, and tumor sampling, heterogeneity resulting from microenvironmental autocrine and paracrine interaction, and stochastic plasticity related to different functional cell efficiencies. For a higher level of reproducibility in clinical research and diagnostics, it is necessary to establish standardized analytical methods, including microdissection. In situ techniques can be pivotal to explore tumor microenvironment and can be improved with associated digital analysis. Liquid biopsies for plasma DNA analysis are at present the best method to study recurrent tumors with treatment adaptation, and widespread clinical use could be beneficial. The different types of tumor genomic instabilities could have pragmatic applications to rank ITH for clinical applications: treatment approaches differ in patients with a high nucleotide mutation rate and patients with high copy number alterations. © 2017 S. Karger AG, Basel.

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

FOXQ1, a Novel Target of the Wnt Pathway and a New Marker for Activation of Wnt Signaling in Solid Tumors

PubMed Central

Christensen, Jon; Bentz, Susanne; Sengstag, Thierry; Shastri, V. Prasad; Anderle, Pascale

2013-01-01

Background The forkhead box transcription factor FOXQ1 has been shown to be upregulated in colorectal cancer (CRC) and metastatic breast cancer and involved in tumor development, epithelial-mesenchymal transition and chemoresistance. Yet, its transcriptional regulation is still unknown. Methods FOXQ1 mRNA and protein expression were analysed in a panel of CRC cell lines, and laser micro-dissected human biopsy samples by qRT-PCR, microarray GeneChip® U133 Plus 2.0 and western blots. FOXQ1 regulation was assayed by chromatin immunoprecipitation and luciferase reporter assays. Results FOXQ1 was robustly induced in CRC compared to other tumors, but had no predictive value with regards to grade, metastasis and survival in CRC. Prototype-based gene coexpression and gene set enrichment analysis showed a significant association between FOXQ1 and the Wnt pathway in tumors and cancer cell lines from different tissues. In vitro experiments confirmed, on a molecular level, FOXQ1 as a direct Wnt target. Analysis of known Wnt targets identified FOXQ1 as the most suitable marker for canonical Wnt activation across a wide panel of cell lines derived from different tissues. Conclusions Our data show that FOXQ1 is one of the most over-expressed genes in CRC and a direct target of the canonical Wnt pathway. It is a potential new marker for detection of early CRC and Wnt activation in tumors of different origins. PMID:23555880

Tissue-Specific Analysis of Secondary Metabolites Creates a Reliable Morphological Criterion for Quality Grading of Polygoni Multiflori Radix.

PubMed

Liang, Li; Xu, Jun; Liang, Zhi-Tao; Dong, Xiao-Ping; Chen, Hu-Biao; Zhao, Zhong-Zhen

2018-05-08

In commercial herbal markets, Polygoni Multiflori Radix (PMR, the tuberous roots of Polygonum multiflorum Thunb.), a commonly-used Chinese medicinal material, is divided into different grades based on morphological features of size and weight. While more weight and larger size command a higher price, there is no scientific data confirming that the more expensive roots are in fact of better quality. To assess the inherent quality of various grades and of various tissues in PMR and to find reliable morphological indicators of quality, a method combining laser microdissection (LMD) and ultra-performance liquid chromatography triple-quadrupole mass spectrometry (UPLC-QqQ-MS/MS) was applied. Twelve major chemical components were quantitatively determined in both whole material and different tissues of PMR. Determination of the whole material revealed that traditional commercial grades based on size and weight of PRM did not correspond to any significant differences in chemical content. Instead, tissue-specific analysis indicated that the morphological features could be linked with quality in a new way. That is, PMR with broader cork and phloem, as seen in a transverse section, were typically of better quality as these parts are where the bioactive components accumulate. The tissue-specific analysis of secondary metabolites creates a reliable morphological criterion for quality grading of PMR.

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

Purification of cardiac myocytes from human heart biopsies for gene expression analysis.

PubMed

Kosloski, L M; Bales, I K; Allen, K B; Walker, B L; Borkon, A M; Stuart, R S; Pak, A F; Wacker, M J

2009-09-01

The collection of gene expression data from human heart biopsies is important for understanding the cellular mechanisms of arrhythmias and diseases such as cardiac hypertrophy and heart failure. Many clinical and basic research laboratories conduct gene expression analysis using RNA from whole cardiac biopsies. This allows for the analysis of global changes in gene expression in areas of the heart, while eliminating the need for more complex and technically difficult single-cell isolation procedures (such as flow cytometry, laser capture microdissection, etc.) that require expensive equipment and specialized training. The abundance of fibroblasts and other cell types in whole biopsies, however, can complicate gene expression analysis and the interpretation of results. Therefore, we have designed a technique to quickly and easily purify cardiac myocytes from whole cardiac biopsies for RNA extraction. Human heart tissue samples were collected, and our purification method was compared with the standard nonpurification method. Cell imaging using acridine orange staining of the purified sample demonstrated that >98% of total RNA was contained within identifiable cardiac myocytes. Real-time RT-PCR was performed comparing nonpurified and purified samples for the expression of troponin T (myocyte marker), vimentin (fibroblast marker), and alpha-smooth muscle actin (smooth muscle marker). Troponin T expression was significantly increased, and vimentin and alpha-smooth muscle actin were significantly decreased in the purified sample (n = 8; P < 0.05). Extracted RNA was analyzed during each step of the purification, and no significant degradation occurred. These results demonstrate that this isolation method yields a more purified cardiac myocyte RNA sample suitable for downstream applications, such as real-time RT-PCR, and allows for more accurate gene expression changes in cardiac myocytes from heart biopsies.

MicroRNA Expression Profiling to Identify and Validate Reference Genes for the Relative Quantification of microRNA in Rectal Cancer.

PubMed

Eriksen, Anne Haahr Mellergaard; Andersen, Rikke Fredslund; Pallisgaard, Niels; Sørensen, Flemming Brandt; Jakobsen, Anders; Hansen, Torben Frøstrup

2016-01-01

MicroRNAs (miRNAs) play important roles in regulating biological processes at the post-transcriptional level. Deregulation of miRNAs has been observed in cancer, and miRNAs are being investigated as potential biomarkers regarding diagnosis, prognosis and prediction in cancer management. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used, when measuring miRNA expression. Appropriate normalisation of RT-qPCR data is important to ensure reliable results. The aim of the present study was to identify stably expressed miRNAs applicable as normaliser candidates in future studies of miRNA expression in rectal cancer. We performed high-throughput miRNA profiling (OpenArray®) on ten pairs of laser micro-dissected rectal cancer tissue and adjacent stroma. A global mean expression normalisation strategy was applied to identify the most stably expressed miRNAs for subsequent validation. In the first validation experiment, a panel of miRNAs were analysed on 25 pairs of micro dissected rectal cancer tissue and adjacent stroma. Subsequently, the same miRNAs were analysed in 28 pairs of rectal cancer tissue and normal rectal mucosa. From the miRNA profiling experiment, miR-645, miR-193a-5p, miR-27a and let-7g were identified as stably expressed, both in malignant and stromal tissue. In addition, NormFinder confirmed high expression stability for the four miRNAs. In the RT-qPCR based validation experiments, no significant difference between tumour and stroma/normal rectal mucosa was detected for the mean of the normaliser candidates miR-27a, miR-193a-5p and let-7g (first validation P = 0.801, second validation P = 0.321). MiR-645 was excluded from the data analysis, because it was undetected in 35 of 50 samples (first validation) and in 24 of 56 samples (second validation), respectively. Significant difference in expression level of RNU6B was observed between tumour and adjacent stromal (first validation), and between tumour and normal rectal mucosa (second validation). We recommend the mean expression of miR-27a, miR-193a-5p and let-7g as normalisation factor, when performing miRNA expression analyses by RT-qPCR on rectal cancer tissue.

Systematic Identification of Druggable Epithelial-Stromal Crosstalk Signaling Networks in Ovarian Cancer.

PubMed

Yeung, Tsz-Lun; Sheng, Jianting; Leung, Cecilia S; Li, Fuhai; Kim, Jaeyeon; Ho, Samuel Y; Matzuk, Martin M; Lu, Karen H; Wong, Stephen T C; Mok, Samuel C

2018-05-31

Bulk tumor tissue samples are used for generating gene expression profiles in most research studies, making it difficult to decipher the stroma-cancer crosstalk networks. In the present study, we describe the use of microdissected transcriptome profiles for the identification of cancer-stroma crosstalk networks with prognostic value, which presents a unique opportunity for developing new treatment strategies for ovarian cancer. Transcriptome profiles from microdissected ovarian cancer-associated fibroblasts (CAFs) and ovarian cancer cells from patients with high-grade serous ovarian cancer (n = 70) were used as input data for the computational systems biology program CCCExplorer to uncover crosstalk networks between various cell types within the tumor microenvironment. The crosstalk analysis results were subsequently used for discovery of new indications for old drugs in ovarian cancer by computational ranking of candidate agents. Survival analysis was performed on ovarian tumor-bearing Dicer/Pten double-knockout mice treated with calcitriol, a US Food and Drug Administration-approved agent that suppresses the Smad signaling cascade, or vehicle control (9-11 mice per group). All statistical tests were two-sided. Activation of TGF-β-dependent and TGF-β-independent Smad signaling was identified in a particular subtype of CAFs and was associated with poor patient survival (patients with higher levels of Smad-regulated gene expression by CAFs: median overall survival = 15 months, 95% confidence interval [CI] = 12.7 to 17.3 months; vs patients with lower levels of Smad-regulated gene expression: median overall survival = 26 months, 95% CI = 15.9 to 36.1 months, P = .02). In addition, the activated Smad signaling identified in CAFs was found to be targeted by repositioning calcitriol. Calcitriol suppressed Smad signaling in CAFs, inhibited tumor progression in mice, and prolonged the median survival duration of ovarian cancer-bearing mice from 36 to 48 weeks (P = .04). Our findings suggest the feasibility of using novel multicellular systems biology modeling to identify and repurpose known drugs targeting cancer-stroma crosstalk networks, potentially leading to faster and more effective cures for cancers.

MET exon 14 skipping mutation in triple-negative pulmonary adenocarcinomas and pleomorphic carcinomas: An analysis of intratumoral MET status heterogeneity and clinicopathological characteristics.

PubMed

Kwon, Dohee; Koh, Jaemoon; Kim, Sehui; Go, Heounjeong; Kim, Young A; Keam, Bhumsuk; Kim, Tae Min; Kim, Dong-Wan; Jeon, Yoon Kyung; Chung, Doo Hyun

2017-04-01

MET mutations leading to exon 14 skipping rarely occur in non-small cell lung cancer (NSCLC). Recently, small molecule inhibitors targeting MET mutations showed clinical benefit. However, the clinicopathological characteristics of NSCLC harboring MET mutations, and the correlation among mutations, protein expression, and gene copy number of MET in NSCLC remain unclear. Therefore, we address these issues. MET exon 14 skipping mutations were evaluated using real-time quantitative reverse-transcription-PCR (qRT-PCR) in 102 triple-negative (i.e., EGFR mutation (-)/ALK translocation (-)/KRAS mutation (-)) pulmonary adenocarcinomas, and 45 pleomorphic carcinomas. MET mutation and gene copy were also examined in microdissected tissues obtained from tumor areas with heterogeneous MET immunohistochemical expression. MET mutations were detected in 8.8% (9/102) of triple-negative adenocarcinomas and 20% (9/45) of pleomorphic carcinomas of the lung. Patients with MET-mutated adenocarcinomas was significantly older than those without MET mutations (P=0.015). The male to female and ever-to never-smoker ratios were 3:6 and 2:7, respectively, among patients with MET-mutated adenocarcinomas. All (9/9) of the MET-mutated adenocarcinomas showed acinar predominant histology with associated lepidic patterns. In contrast, the male to female and ever- to never-smoker ratios were 8:1 and 7:1, respectively, among patients with MET-mutated pleomorphic carcinomas. The carcinoma component of MET-mutated pleomorphic carcinomas was mostly adenocarcinoma of acinar pattern (8/9). MET mutation was detected by qRT-PCR in all samples with heterogeneous MET expression microdissected from five cases with MET-mutated adenocarcinoma, while MET gene amplification was detected in tumor areas expressing high MET protein levels among MET-mutated adenocarcinomas. MET-mutated NSCLC is characterized by older age in patients with adenocarcinoma and by an acinar histology and variable MET expression in patients with adenocarcinoma and pleomorphic carcinomas. Moreover, MET gene amplification might occur in the tumor cells harboring the MET mutation. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Gene expression profiling of two distinct neuronal populations in the rodent spinal cord.

PubMed

Ryge, Jesper; Westerdahl, Ann-Charlotte; Alstrøm, Preben; Kiehn, Ole

2008-01-01

In the field of neuroscience microarray gene expression profiles on anatomically defined brain structures are being used increasingly to study both normal brain functions as well as pathological states. Fluorescent tracing techniques in brain tissue that identifies distinct neuronal populations can in combination with global gene expression profiling potentially increase the resolution and specificity of such studies to shed new light on neuronal functions at the cellular level. We examine the microarray gene expression profiles of two distinct neuronal populations in the spinal cord of the neonatal rat, the principal motor neurons and specific interneurons involved in motor control. The gene expression profiles of the respective cell populations were obtained from amplified mRNA originating from 50-250 fluorescently identified and laser microdissected cells. In the data analysis we combine a new microarray normalization procedure with a conglomerate measure of significant differential gene expression. Using our methodology we find 32 genes to be more expressed in the interneurons compared to the motor neurons that all except one have not previously been associated with this neuronal population. As a validation of our method we find 17 genes to be more expressed in the motor neurons than in the interneurons and of these only one had not previously been described in this population. We provide an optimized experimental protocol that allows isolation of gene transcripts from fluorescent retrogradely labeled cell populations in fresh tissue, which can be used to generate amplified aRNA for microarray hybridization from as few as 50 laser microdissected cells. Using this optimized experimental protocol in combination with our microarray analysis methodology we find 49 differentially expressed genes between the motor neurons and the interneurons that reflect the functional differences between these two cell populations in generating and transmitting the motor output in the rodent spinal cord.

Gene Expression Profiling of Two Distinct Neuronal Populations in the Rodent Spinal Cord

PubMed Central

Alstrøm, Preben; Kiehn, Ole

2008-01-01

Background In the field of neuroscience microarray gene expression profiles on anatomically defined brain structures are being used increasingly to study both normal brain functions as well as pathological states. Fluorescent tracing techniques in brain tissue that identifies distinct neuronal populations can in combination with global gene expression profiling potentially increase the resolution and specificity of such studies to shed new light on neuronal functions at the cellular level. Methodology/Principal Findings We examine the microarray gene expression profiles of two distinct neuronal populations in the spinal cord of the neonatal rat, the principal motor neurons and specific interneurons involved in motor control. The gene expression profiles of the respective cell populations were obtained from amplified mRNA originating from 50–250 fluorescently identified and laser microdissected cells. In the data analysis we combine a new microarray normalization procedure with a conglomerate measure of significant differential gene expression. Using our methodology we find 32 genes to be more expressed in the interneurons compared to the motor neurons that all except one have not previously been associated with this neuronal population. As a validation of our method we find 17 genes to be more expressed in the motor neurons than in the interneurons and of these only one had not previously been described in this population. Conclusions/Significance We provide an optimized experimental protocol that allows isolation of gene transcripts from fluorescent retrogradely labeled cell populations in fresh tissue, which can be used to generate amplified aRNA for microarray hybridization from as few as 50 laser microdissected cells. Using this optimized experimental protocol in combination with our microarray analysis methodology we find 49 differentially expressed genes between the motor neurons and the interneurons that reflect the functional differences between these two cell populations in generating and transmitting the motor output in the rodent spinal cord. PMID:18923679

Immunohistochemical Markers Distinguishing Cholangiocellular Carcinoma (CCC) from Pancreatic Ductal Adenocarcinoma (PDAC) Discovered by Proteomic Analysis of Microdissected Cells*

PubMed Central

Padden, Juliet; Ahrens, Maike; Kälsch, Julia; Bertram, Stefanie; Megger, Dominik A.; Bracht, Thilo; Eisenacher, Martin; Kocabayoglu, Peri; Meyer, Helmut E.; Sipos, Bence; Baba, Hideo A.; Sitek, Barbara

2016-01-01

Cholangiocellular carcinoma (CCC) and pancreatic ductal adenocarcinoma (PDAC) are two highly aggressive cancer types that arise from epithelial cells of the pancreatobiliary system. Owing to their histological and morphological similarity, differential diagnosis between CCC and metastasis of PDAC located in the liver frequently proves an unsolvable issue for pathologists. The detection of biomarkers with high specificity and sensitivity for the differentiation of these tumor types would therefore be a valuable tool. Here, we address this problem by comparing microdissected CCC and PDAC tumor cells from nine and eleven cancer patients, respectively, in a label-free proteomics approach. The novel biomarker candidates were subsequently verified by immunohistochemical staining of 73 CCC, 78 primary, and 18 metastatic PDAC tissue sections. In the proteome analysis, we found 180 proteins with a significantly differential expression between CCC and PDAC cells (p value < 0.05, absolute fold change > 2). Nine candidate proteins were chosen for an immunohistochemical verification out of which three showed very promising results. These were the annexins ANXA1, ANXA10, and ANXA13. For the correct classification of PDAC, ANXA1 showed a sensitivity of 84% and a specificity of 85% and ANXA10 a sensitivity of 90% at a specificity of 66%. ANXA13 was higher abundant in CCC. It presented a sensitivity of 84% at a specificity of 55%. In metastatic PDAC tissue ANXA1 and ANXA10 showed similar staining behavior as in the primary PDAC tumors (13/18 and 17/18 positive, respectively). ANXA13, however, presented positive staining in eight out of eighteen secondary PDAC tumors and was therefore not suitable for the differentiation of these from CCC. We conclude that ANXA1 and ANXA10 are promising biomarker candidates with high diagnostic values for the differential diagnosis of intrahepatic CCC and metastatic liver tumors deriving from PDAC. PMID:26644413

Immunohistochemical Markers Distinguishing Cholangiocellular Carcinoma (CCC) from Pancreatic Ductal Adenocarcinoma (PDAC) Discovered by Proteomic Analysis of Microdissected Cells.

PubMed

Padden, Juliet; Ahrens, Maike; Kälsch, Julia; Bertram, Stefanie; Megger, Dominik A; Bracht, Thilo; Eisenacher, Martin; Kocabayoglu, Peri; Meyer, Helmut E; Sipos, Bence; Baba, Hideo A; Sitek, Barbara

2016-03-01

Cholangiocellular carcinoma (CCC) and pancreatic ductal adenocarcinoma (PDAC) are two highly aggressive cancer types that arise from epithelial cells of the pancreatobiliary system. Owing to their histological and morphological similarity, differential diagnosis between CCC and metastasis of PDAC located in the liver frequently proves an unsolvable issue for pathologists. The detection of biomarkers with high specificity and sensitivity for the differentiation of these tumor types would therefore be a valuable tool. Here, we address this problem by comparing microdissected CCC and PDAC tumor cells from nine and eleven cancer patients, respectively, in a label-free proteomics approach. The novel biomarker candidates were subsequently verified by immunohistochemical staining of 73 CCC, 78 primary, and 18 metastatic PDAC tissue sections. In the proteome analysis, we found 180 proteins with a significantly differential expression between CCC and PDAC cells (p value < 0.05, absolute fold change > 2). Nine candidate proteins were chosen for an immunohistochemical verification out of which three showed very promising results. These were the annexins ANXA1, ANXA10, and ANXA13. For the correct classification of PDAC, ANXA1 showed a sensitivity of 84% and a specificity of 85% and ANXA10 a sensitivity of 90% at a specificity of 66%. ANXA13 was higher abundant in CCC. It presented a sensitivity of 84% at a specificity of 55%. In metastatic PDAC tissue ANXA1 and ANXA10 showed similar staining behavior as in the primary PDAC tumors (13/18 and 17/18 positive, respectively). ANXA13, however, presented positive staining in eight out of eighteen secondary PDAC tumors and was therefore not suitable for the differentiation of these from CCC. We conclude that ANXA1 and ANXA10 are promising biomarker candidates with high diagnostic values for the differential diagnosis of intrahepatic CCC and metastatic liver tumors deriving from PDAC. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cell-type- and tissue-specific transcriptomes of the white spruce (Picea glauca) bark unmask fine-scale spatial patterns of constitutive and induced conifer defense.

PubMed

Celedon, Jose M; Yuen, Macaire M S; Chiang, Angela; Henderson, Hannah; Reid, Karen E; Bohlmann, Jörg

2017-11-01

Plant defenses often involve specialized cells and tissues. In conifers, specialized cells of the bark are important for defense against insects and pathogens. Using laser microdissection, we characterized the transcriptomes of cortical resin duct cells, phenolic cells and phloem of white spruce (Picea glauca) bark under constitutive and methyl jasmonate (MeJa)-induced conditions, and we compared these transcriptomes with the transcriptome of the bark tissue complex. Overall, ~3700 bark transcripts were differentially expressed in response to MeJa. Approximately 25% of transcripts were expressed in only one cell type, revealing cell specialization at the transcriptome level. MeJa caused cell-type-specific transcriptome responses and changed the overall patterns of cell-type-specific transcript accumulation. Comparison of transcriptomes of the conifer bark tissue complex and specialized cells resolved a masking effect inherent to transcriptome analysis of complex tissues, and showed the actual cell-type-specific transcriptome signatures. Characterization of cell-type-specific transcriptomes is critical to reveal the dynamic patterns of spatial and temporal display of constitutive and induced defense systems in a complex plant tissue or organ. This was demonstrated with the improved resolution of spatially restricted expression of sets of genes of secondary metabolism in the specialized cell types. © 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

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.

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.

Application of laser microdissection to identify the mycorrhizal fungi that establish arbuscules inside root cells

PubMed Central

Berruti, Andrea; Borriello, Roberto; Lumini, Erica; Scariot, Valentina; Bianciotto, Valeria; Balestrini, Raffaella

2013-01-01

Obligate symbiotic fungi that form arbuscular mycorrhizae (AMF; belonging to the Glomeromycota phylum) are some of the most important soil microorganisms. AMFs facilitate mineral nutrient uptake from the soil, in exchange for plant-assimilated carbon, and promote water-stress tolerance and resistance to certain diseases. AMFs colonize the root by producing inter- and intra-cellular hyphae. When the fungus penetrates the inner cortical cells, it produces a complex ramified structure called arbuscule, which is considered the preferential site for nutrient exchange. Direct DNA extraction from the whole root and sequencing of ribosomal gene regions are commonly carried out to investigate intraradical AMF communities. Nevertheless, this protocol cannot discriminate between the AMFs that actively produce arbuscules and those that do not. To solve this issue, the authors have characterized the AMF community of arbusculated cells (AC) through a laser microdissection (LMD) approach, combined with sequencing-based taxa identification. The results were then compared with the AMF community that was found from whole root DNA extraction. The AMF communities originating from the LMD samples and the whole root samples differed remarkably. Five taxa were involved in the production of arbuscules, while two taxa were retrieved inside the root but not in the AC. Unexpectedly, one taxon was found in the AC, but its detection was not possible when extracting from the whole root. Thus, the LMD technique can be considered a powerful tool to obtain more precise knowledge on the symbiotically active intraradical AMF community. PMID:23675380

The genes Scgb1a1, Lpo and Gbp2 characteristically expressed in peri-implant epithelium of rats.

PubMed

Mori, Gentaro; Sasaki, Hodaka; Makabe, Yasushi; Yoshinari, Masao; Yajima, Yasutomo

2016-12-01

The peri-implant epithelium (PIE) plays an important role in the prevention against initial stage of inflammation. To minimize the risk of peri-implantitis, it is necessary to understand the biological characteristics of the PIE. The aim of this study was to investigate the characteristic gene expression profile of PIE as compared to junctional epithelium (JE) using laser microdissection and microarray analysis. Left upper first molars of 4-week-old rat were extracted, and titanium alloy implants were placed. Four weeks after surgery, samples were harvested by laser microdissection, and total RNA samples were isolated. Comprehensive analyses of genes expressed in the JE and PIE were performed using microarray analysis. Confirmation of the differential expression of selected genes was performed by quantitative real-time polymerase chain reaction and immunohistochemistry. The microarray analysis showed that 712 genes were more than twofold change upregulated in the PIE compared with the JE. Genes Scgb1a1 were significantly upregulated more than 19.1-fold, Lpo more than 19.0-fold, and Gbp2 more than 8.9-fold, in the PIE (P < 0.01). Immunohistochemical localization of SCGB1A1, LPO, and GBP2 was observed in PIE. The present results suggested that genes Scgb1a1, Lpo, and Gbp2 are characteristically expressed in the PIE. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A novel method for coral explant culture and micropropagation.

PubMed

Vizel, Maya; Loya, Yossi; Downs, Craig A; Kramarsky-Winter, Esti

2011-06-01

We describe here a method for the micropropagation of coral that creates progeny from tissue explants derived from a single polyp or colonial corals. Coral tissue explants of various sizes (0.5-2.5 mm in diameter) were manually microdissected from the solitary coral Fungia granulosa. Explants could be maintained in an undeveloped state or induced to develop into polyps by manipulating environmental parameters such as light and temperature regimes, as well as substrate type. Fully developed polyps were able to be maintained for a long-term in a closed sea water system. Further, we demonstrate that mature explants are also amenable to this technique with the micropropagation of second-generation explants and their development into mature polyps. We thereby experimentally have established coral clonal lines that maintain their ability to differentiate without the need for chemical induction or genetic manipulation. The versatility of this method is also demonstrated through its application to two other coral species, the colonial corals Oculina patigonica and Favia favus.

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 cannabinoids in laser-microdissected trichomes of medicinal Cannabis sativa using LCMS and cryogenic NMR.

PubMed

Happyana, Nizar; Agnolet, Sara; Muntendam, Remco; Van Dam, Annie; Schneider, Bernd; Kayser, Oliver

2013-03-01

Trichomes, especially the capitate-stalked glandular hairs, are well known as the main sites of cannabinoid and essential oil production of Cannabis sativa. In this study the distribution and density of various types of Cannabis sativa L. trichomes, have been investigated by scanning electron microscopy (SEM). Furthermore, glandular trichomes were isolated over the flowering period (8 weeks) by laser microdissection (LMD) and the cannabinoid profile analyzed by LCMS. Cannabinoids were detected in extracts of 25-143 collected cells of capitate-sessile and capitate stalked trichomes and separately in the gland (head) and the stem of the latter. Δ(9)-Tetrahydrocannabinolic acid [THCA (1)], cannabidiolic acid [CBDA (2)], and cannabigerolic acid [CBGA (3)] were identified as most-abundant compounds in all analyzed samples while their decarboxylated derivatives, Δ(9)-tetrahydrocannabinol [THC (4)], cannabidiol [CBD (5)], and cannabigerol [CBG (6)], co-detected in all samples, were present at significantly lower levels. Cannabichromene [CBC (8)] along with cannabinol (CBN (9)) were identified as minor compounds only in the samples of intact capitate-stalked trichomes and their heads harvested from 8-week old plants. Cryogenic nuclear magnetic resonance spectroscopy (NMR) was used to confirm the occurrence of major cannabinoids, THCA (1) and CBDA (2), in capitate-stalked and capitate-sessile trichomes. Cryogenic NMR enabled the additional identification of cannabichromenic acid [CBCA (7)] in the dissected trichomes, which was not possible by LCMS as standard was not available. The hereby documented detection of metabolites in the stems of capitate-stalked trichomes indicates a complex biosynthesis and localization over the trichome cells forming the glandular secretion unit. Copyright © 2012 Elsevier Ltd. All rights reserved.

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.

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

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.

Morph-X-Select: Morphology-based tissue aptamer selection for ovarian cancer biomarker discovery

PubMed Central

Wang, Hongyu; Li, Xin; Volk, David E.; Lokesh, Ganesh L.-R.; Elizondo-Riojas, Miguel-Angel; Li, Li; Nick, Alpa M.; Sood, Anil K.; Rosenblatt, Kevin P.; Gorenstein, David G.

2016-01-01

High affinity aptamer-based biomarker discovery has the advantage of simultaneously discovering an aptamer affinity reagent and its target biomarker protein. Here, we demonstrate a morphology-based tissue aptamer selection method that enables us to use tissue sections from individual patients and identify high-affinity aptamers and their associated target proteins in a systematic and accurate way. We created a combinatorial DNA aptamer library that has been modified with thiophosphate substitutions of the phosphate ester backbone at selected 5′dA positions for enhanced nuclease resistance and targeting. Based on morphological assessment, we used image-directed laser microdissection (LMD) to dissect regions of interest bound with the thioaptamer (TA) library and further identified target proteins for the selected TAs. We have successfully identified and characterized the lead candidate TA, V5, as a vimentin-specific sequence that has shown specific binding to tumor vasculature of human ovarian tissue and human microvascular endothelial cells. This new Morph-X-Select method allows us to select high-affinity aptamers and their associated target proteins in a specific and accurate way, and could be used for personalized biomarker discovery to improve medical decision-making and to facilitate the development of targeted therapies to achieve more favorable outcomes. PMID:27839510

Histopathologic Diagnosis of Fungal Infections in the 21st Century

PubMed Central

Guarner, Jeannette; Brandt, Mary E.

2011-01-01

Summary: Fungal infections are becoming more frequent because of expansion of at-risk populations and the use of treatment modalities that permit longer survival of these patients. Because histopathologic examination of tissues detects fungal invasion of tissues and vessels as well as the host reaction to the fungus, it is and will remain an important tool to define the diagnostic significance of positive culture isolates or results from PCR testing. However, there are very few instances where the morphological characteristics of fungi are specific. Therefore, histopathologic diagnosis should be primarily descriptive of the fungus and should include the presence or absence of tissue invasion and the host reaction to the infection. The pathology report should also include a comment stating the most frequent fungi associated with that morphology as well as other possible fungi and parasites that should be considered in the differential diagnosis. Alternate techniques have been used to determine the specific agent present in the histopathologic specimen, including immunohistochemistry, in situ hybridization, and PCR. In addition, techniques such as laser microdissection will be useful to detect the now more frequently recognized dual fungal infections and the local environment in which this phenomenon occurs. PMID:21482725

Distinct gene expression profiles characterize the histopathological stages of disease in Helicobacter-induced mucosa-associated lymphoid tissue lymphoma

PubMed Central

Mueller, Anne; O'Rourke, Jani; Grimm, Jan; Guillemin, Karen; Dixon, Michael F.; Lee, Adrian; Falkow, Stanley

2003-01-01

Long-term colonization of humans with Helicobacter pylori can cause the development of gastric B cell mucosa-associated lymphoid tissue lymphoma, yet little is known about the sequence of molecular steps that accompany disease progression. We used microarray analysis and laser microdissection to identify gene expression profiles characteristic and predictive of the various histopathological stages in a mouse model of the disease. The initial step in lymphoma development is marked by infiltration of reactive lymphocytes into the stomach and the launching of a mucosal immune response. Our analysis uncovered molecular markers of both of these processes, including genes coding for the immunoglobulins and the small proline-rich protein Sprr 2A. The subsequent step is characterized histologically by the antigen-driven proliferation and aggregation of B cells and the gradual appearance of lymphoepithelial lesions. In tissues of this stage, we observed increased expression of genes previously associated with malignancy, including the laminin receptor-1 and the multidrug-resistance channel MDR-1. Finally, we found that the transition to destructive lymphoepithelial lesions and malignant lymphoma is marked by an increase in transcription of a single gene encoding calgranulin A/Mrp-8. PMID:12552104

Void space inside the developing seed of Brassica napus and the modelling of its function

PubMed Central

Verboven, Pieter; Herremans, Els; Borisjuk, Ljudmilla; Helfen, Lukas; Ho, Quang Tri; Tschiersch, Henning; Fuchs, Johannes; Nicolaï, Bart M; Rolletschek, Hardy

2013-01-01

The developing seed essentially relies on external oxygen to fuel aerobic respiration, but it is currently unknown how oxygen diffuses into and within the seed, which structural pathways are used and what finally limits gas exchange. By applying synchrotron X-ray computed tomography to developing oilseed rape seeds we uncovered void spaces, and analysed their three-dimensional assembly. Both the testa and the hypocotyl are well endowed with void space, but in the cotyledons, spaces were small and poorly inter-connected. In silico modelling revealed a three orders of magnitude range in oxygen diffusivity from tissue to tissue, and identified major barriers to gas exchange. The oxygen pool stored in the voids is consumed about once per minute. The function of the void space was related to the tissue-specific distribution of storage oils, storage protein and starch, as well as oxygen, water, sugars, amino acids and the level of respiratory activity, analysed using a combination of magnetic resonance imaging, specific oxygen sensors, laser micro-dissection, biochemical and histological methods. We conclude that the size and inter-connectivity of void spaces are major determinants of gas exchange potential, and locally affect the respiratory activity of a developing seed. PMID:23692271

Isolating cells from female/male blood mixtures using florescence in situ hybridization combined with low volume PCR and its application in forensic science.

PubMed

Feng, Lei; Li, Cai-Xia; Han, Jun-Ping; Xu, Cheng; Hu, Lan

2015-11-01

To obtain single-source short tandem repeat (STR) profiles in trace female/male blood mixture samples, we combined florescence in situ hybridization (FISH), laser microdissection, and low volume PCR (LV-PCR) to isolate male/female cells and improve sensitivity. The results showed that isolation of as few as 10 leukocytes was sufficient to yield full STR profiles in fresh female or male blood samples for 32 independent tests with a low additional alleles rate (3.91%) and drop-out alleles rate (5.01%). Moreover, this procedure was tested in two fresh blood mixture series at three ratios (1:5, 1:10, and 1:20), two mock female/male blood mixture casework samples, and one practical casework sample. Male and female STR profiles were successfully detected in all of these samples, showing that this procedure could be used in forensic casework in the future.

Multiomics Analysis of Tumor Microenvironment Reveals Gata2 and miRNA-124-3p as Potential Novel Biomarkers in Ovarian Cancer.

PubMed

Gov, Esra; Kori, Medi; Arga, Kazim Yalcin

2017-10-01

Ovarian cancer is a common and, yet, one of the most deadly human cancers due to its insidious onset and the current lack of robust early diagnostic tests. Tumors are complex tissues comprised of not only malignant cells but also genetically stable stromal cells. Understanding the molecular mechanisms behind epithelial-stromal crosstalk in ovarian cancer is a great challenge in particular. In the present study, we performed comparative analyses of transcriptome data from laser microdissected epithelial, stromal, and ovarian tumor tissues, and identified common and tissue-specific reporter biomolecules-genes, receptors, membrane proteins, transcription factors (TFs), microRNAs (miRNAs), and metabolites-by integration of transcriptome data with genome-scale biomolecular networks. Tissue-specific response maps included common differentially expressed genes (DEGs) and reporter biomolecules were reconstructed and topological analyses were performed. We found that CDK2, EP300, and SRC as receptor-related functions or membrane proteins; Ets1, Ar, Gata2, and Foxp3 as TFs; and miR-16-5p and miR-124-3p as putative biomarkers and warrant further validation research. In addition, we report in this study that Gata2 and miR-124-3p are potential novel reporter biomolecules for ovarian cancer. The study of tissue-specific reporter biomolecules in epithelial cells, stroma, and tumor tissues as exemplified in the present study offers promise in biomarker discovery and diagnostics innovation for common complex human diseases such as ovarian cancer.

Tissue-specific features of the X chromosome and nucleolus spatial dynamics in a malaria mosquito, Anopheles atroparvus

PubMed Central

Bondarenko, Semen M.; Artemov, Gleb N.; Stegniy, Vladimir N.

2017-01-01

Spatial organization of chromosome territories is important for maintenance of genomic stability and regulation of gene expression. Recent studies have shown tissue-specific features of chromosome attachments to the nuclear envelope in various organisms including malaria mosquitoes. However, other spatial characteristics of nucleus organization, like volume and shape of chromosome territories, have not been studied in Anopheles. We conducted a thorough analysis of tissue-specific features of the X chromosome and nucleolus volume and shape in follicular epithelium and nurse cells of the Anopheles atroparvus ovaries using a modern open-source software. DNA of the polytene X chromosome from ovarian nurse cells was obtained by microdissection and was used as a template for amplification with degenerate oligo primers. A fluorescently labeled X chromosome painting probe was hybridized with formaldehyde-fixed ovaries of mosquitoes using a 3D-FISH method. The nucleolus was stained by immunostaining with an anti-fibrillarin antibody. The analysis was conducted with TANGO—a software for a chromosome spatial organization analysis. We show that the volume and position of the X chromosome have tissue-specific characteristics. Unlike nurse cell nuclei, the growth of follicular epithelium nuclei is not accompanied with the proportional growth of the X chromosome. However, the shape of the X chromosome does not differ between the tissues. The dynamics of the X chromosome attachment regions location is tissue-specific and it is correlated with the process of nucleus growth in follicular epithelium and nurse cells. PMID:28158219

Tissue-specific features of the X chromosome and nucleolus spatial dynamics in a malaria mosquito, Anopheles atroparvus.

PubMed

Bondarenko, Semen M; Artemov, Gleb N; Sharakhov, Igor V; Stegniy, Vladimir N

2017-01-01

Spatial organization of chromosome territories is important for maintenance of genomic stability and regulation of gene expression. Recent studies have shown tissue-specific features of chromosome attachments to the nuclear envelope in various organisms including malaria mosquitoes. However, other spatial characteristics of nucleus organization, like volume and shape of chromosome territories, have not been studied in Anopheles. We conducted a thorough analysis of tissue-specific features of the X chromosome and nucleolus volume and shape in follicular epithelium and nurse cells of the Anopheles atroparvus ovaries using a modern open-source software. DNA of the polytene X chromosome from ovarian nurse cells was obtained by microdissection and was used as a template for amplification with degenerate oligo primers. A fluorescently labeled X chromosome painting probe was hybridized with formaldehyde-fixed ovaries of mosquitoes using a 3D-FISH method. The nucleolus was stained by immunostaining with an anti-fibrillarin antibody. The analysis was conducted with TANGO-a software for a chromosome spatial organization analysis. We show that the volume and position of the X chromosome have tissue-specific characteristics. Unlike nurse cell nuclei, the growth of follicular epithelium nuclei is not accompanied with the proportional growth of the X chromosome. However, the shape of the X chromosome does not differ between the tissues. The dynamics of the X chromosome attachment regions location is tissue-specific and it is correlated with the process of nucleus growth in follicular epithelium and nurse cells.

Advanced techniques in placental biology -- workshop report.

PubMed

Nelson, D M; Sadovsky, Y; Robinson, J M; Croy, B A; Rice, G; Kniss, D A

2006-04-01

Major advances in placental biology have been realized as new technologies have been developed and existing methods have been refined in many areas of biological research. Classical anatomy and whole-organ physiology tools once used to analyze placental structure and function have been supplanted by more sophisticated techniques adapted from molecular biology, proteomics, and computational biology and bioinformatics. In addition, significant refinements in morphological study of the placenta and its constituent cell types have improved our ability to assess form and function in highly integrated manner. To offer an overview of modern technologies used by investigators to study the placenta, this workshop: Advanced techniques in placental biology, assembled experts who discussed fundamental principles and real time examples of four separate methodologies. Y. Sadovsky presented the principles of microRNA function as an endogenous mechanism of gene regulation. J. Robinson demonstrated the utility of correlative microscopy in which light-level and transmission electron microscopy are combined to provide cellular and subcellular views of placental cells. A. Croy provided a lecture on the use of microdissection techniques which are invaluable for isolating very small subsets of cell types for molecular analysis. Finally, G. Rice presented an overview methods on profiling of complex protein mixtures within tissue and/or fluid samples that, when refined, will offer databases that will underpin a systems approach to modern trophoblast biology.

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.

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

Differentiation of endosperm transfer cells of barley: a comprehensive analysis at the micro-scale.

PubMed

Thiel, Johannes; Riewe, David; Rutten, Twan; Melzer, Michael; Friedel, Swetlana; Bollenbeck, Felix; Weschke, Winfriede; Weber, Hans

2012-08-01

Barley endosperm cells differentiate into transfer cells (ETCs) opposite the nucellar projection. To comprehensively analyse ETC differentiation, laser microdissection-based transcript and metabolite profiles were obtained from laser microdissected tissues and cell morphology was analysed. Flange-like secondary-wall ingrowths appeared between 5 and 7 days after pollination within the three outermost cell layers. Gene expression analysis indicated that ethylene-signalling pathways initiate ETC morphology. This is accompanied by gene activity related to cell shape control and vesicle transport, with abundant mitochondria and endomembrane structures. Gene expression analyses indicate predominant formation of hemicelluloses, glucuronoxylans and arabinoxylans, and transient formation of callose, together with proline and 4-hydroxyproline biosynthesis. Activation of the methylation cycle is probably required for biosynthesis of phospholipids, pectins and ethylene. Membrane microdomains involving sterols/sphingolipids and remorins are potentially involved in ETC development. The transcriptional activity of assimilate and micronutrient transporters suggests ETCs as the main uptake organs of solutes into the endosperm. Accordingly, the endosperm grows maximally after ETCs are fully developed. Up-regulated gene expression related to amino acid catabolism, C:N balances, carbohydrate oxidation, mitochondrial activity and starch degradation meets high demands for respiratory energy and carbohydrates, required for cell proliferation and wall synthesis. At 10 days after pollination, ETCs undergo further differentiation, potentially initiated by abscisic acid, and metabolism is reprogrammed as shown by activated storage and stress-related processes. Overall, the data provide a comprehensive view of barley ETC differentiation and development, and identify candidate genes and associated pathways. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Aberrant methylation accounts for cell adhesion-related gene silencing during 3-methylcholanthrene and diethylnitrosamine induced multistep rat lung carcinogenesis associated with overexpression of DNA methyltransferases 1 and 3a

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

Liu Wenbin; Cui Zhihong; Ao Lin

To evaluate the significance of alterations in cell adhesion-related genes methylation during lung multistep carcinogenesis induced by the genotoxic carcinogens 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN), tissue samples microdissected from MCA/DEN-induced rat lung carcinogenesis model were subjected to methylation-specific PCR to evaluate the DNA methylation status of CADM1, TIMP3, E-cadherin and N-cadherin. Immunohistochemistry was used to determine protein expression of CADM1, TIMP3, N-cadherin and the DNA methyltransferases (DNMTs) 1, 3a and 3b. E-cadherin hypermethylation was not detected in any tissue. CADM1, TIMP3 and N-cadherin hypermethylation was correlated with the loss of their protein expression during the progression of pathologic lesions. Themore » prevalence of DNA methylation of at least one gene and the average number of methylated genes increased with the histological progression. DNMT1 and DNMT3a protein expression increased progressively during the stages of lung carcinogenesis, whereas DNMT3b overexpression was only found in several samples. Furthermore, DNMT1 protein expression levels were correlated with CADM1 methylation, and DNMT3a protein expression levels were correlated with CADM1, TIMP3 and N-cadherin methylation. The average number of methylated genes during carcinogenesis was significantly correlated with DNMT1 and DNMT3a protein expression levels. Moreover, mRNA expression of CADM1 significantly increased after treatment with DNMT inhibitor 5-aza-2'-deoxycytidine in CADM1-methylated primary tumor cell lines. Our findings suggest that an accumulation of hypermethylation accounts for cell adhesion-related gene silencing is associated with dynamic changes in the progression of MCA/DEN-induced rat lung carcinogenesis. We suggest that DNMT1 and DNMT3a protein overexpression may be responsible for this aberrant DNA methylation.« less

Gene expression abnormalities in histologically normal breast epithelium from patients with luminal type of breast cancer.

PubMed

Zubor, Pavol; Hatok, Jozef; Moricova, Petra; Kajo, Karol; Kapustova, Ivana; Mendelova, Andrea; Racay, Peter; Danko, Jan

2015-05-01

The gene expression profile of breast cancer has been described as a great breakthrough on the way to comprehend differences in cancer origin, behavior and therapy. However, gene expression profile in histologically normal epithelium (HNEpi) which could harbor genetic abnormalities predisposing breast tissue to develop malignancy was minor scope for scientists in the past. Thus, we aimed to analyze gene expressions in HNEpi and breast cancer tissue (BCTis) in order to establish its value as potential diagnostic marker for cancer development. We evaluated a panel of disease-specific genes in luminal type (A/B) of breast cancer and tumor surrounding HNEpi by qRT-PCR Array in 32 microdissected samples. There was 20.2 and 2.4% deregulation rate in genes with at least 2-fold or 5-fold over-expression between luminal (A/B) type breast carcinomas and tumor surrounding HNEpi, respectively. The high-grade luminal carcinomas showed higher number of deregulated genes compared to low-grade cases (50.6 vs. 23.8% with at least 2-fold deregulation rate). The main overexpressed genes in HNEpi were KLK5, SCGB1D2, GSN, EGFR and NGFR. The significant differences in gene expression between BCTis and HNEpi samples were revealed for BAG1, C3, CCNA2, CD44, FGF1, FOSL1, ID2, IL6R, NGFB, NGFR, PAPPA, PLAU, SERPINB5, THBS1 and TP53 gene (p < 0.05) and BCL2L2, CTSB, ITGB4, JUN, KIT, KLF5, SCGB1D2, SCGB2A1, SERPINE1 (p < 0.01), and EGFR, GABRP, GSN, MAP2K7 and THBS2 (p < 0.001), and GSN, KLK5 (p < 0.0001). The ontological gene analyses revealed high deregulations in gene group directly associated with breast cancer prognosis and origin.

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.

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

Identification and characterization of mouse otic sensory lineage genes

PubMed Central

Hartman, Byron H.; Durruthy-Durruthy, Robert; Laske, Roman D.; Losorelli, Steven; Heller, Stefan

2015-01-01

Vertebrate embryogenesis gives rise to all cell types of an organism through the development of many unique lineages derived from the three primordial germ layers. The otic sensory lineage arises from the otic vesicle, a structure formed through invagination of placodal non-neural ectoderm. This developmental lineage possesses unique differentiation potential, giving rise to otic sensory cell populations including hair cells, supporting cells, and ganglion neurons of the auditory and vestibular organs. Here we present a systematic approach to identify transcriptional features that distinguish the otic sensory lineage (from early otic progenitors to otic sensory populations) from other major lineages of vertebrate development. We used a microarray approach to analyze otic sensory lineage populations including microdissected otic vesicles (embryonic day 10.5) as well as isolated neonatal cochlear hair cells and supporting cells at postnatal day 3. Non-otic tissue samples including periotic tissues and whole embryos with otic regions removed were used as reference populations to evaluate otic specificity. Otic populations shared transcriptome-wide correlations in expression profiles that distinguish members of this lineage from non-otic populations. We further analyzed the microarray data using comparative and dimension reduction methods to identify individual genes that are specifically expressed in the otic sensory lineage. This analysis identified and ranked top otic sensory lineage-specific transcripts including Fbxo2, Col9a2, and Oc90, and additional novel otic lineage markers. To validate these results we performed expression analysis on select genes using immunohistochemistry and in situ hybridization. Fbxo2 showed the most striking pattern of specificity to the otic sensory lineage, including robust expression in the early otic vesicle and sustained expression in prosensory progenitors and auditory and vestibular hair cells and supporting cells. PMID:25852475

Proteomics of the human endometrial glandular epithelium and stroma from the proliferative and secretory phases of the menstrual cycle.

PubMed

Hood, Brian L; Liu, Baoquan; Alkhas, Addie; Shoji, Yutaka; Challa, Rusheeswar; Wang, Guisong; Ferguson, Susan; Oliver, Julie; Mitchell, Dave; Bateman, Nicholas W; Zahn, Christopher M; Hamilton, Chad A; Payson, Mark; Lessey, Bruce; Fazleabas, Asgerally T; Maxwell, G Larry; Conrads, Thomas P; Risinger, John I

2015-04-01

Despite its importance in reproductive biology and women's health, a detailed molecular-level understanding of the human endometrium is lacking. Indeed, no comprehensive studies have been undertaken to elucidate the important protein expression differences between the endometrial glandular epithelium and surrounding stroma during the proliferative and midsecretory phases of the menstrual cycle. We utilized laser microdissection to harvest epithelial cells and stromal compartments from proliferative and secretory premenopausal endometrial tissue and performed a global, quantitative mass spectrometry-based proteomics analysis. This analysis identified 1224 total proteins from epithelial cells, among which 318 were differentially abundant between the proliferative and secretory phases (q < 0.05), and 1005 proteins from the stromal compartments, 19 of which were differentially abundant between the phases (q < 0.05). Several proteins were chosen for validation by immunohistochemistry in an independent set of uterine tissues, including carboxypeptidase M, tenascin C, neprilysin, and ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP3). ENPP3, which was elevated in epithelial glandular cells in the secretory phase, was confirmed to be elevated in midsecretory-phase baboon uterine lavage samples and also observed to have an N-linked glycosylated form that was not observed in the proliferative phase. This study provides a detailed view into the global proteomic alterations of the epithelial cells and stromal compartments of the cycling premenopausal endometrium. These proteomic alterations during endometrial remodeling provide a basis for numerous follow-up investigations on the function of these differentially regulated proteins and their role in reproductive biology and endometrial pathologies. © 2015 by the Society for the Study of Reproduction, Inc.

Immunohistochemical detection of the BRAF V600E mutation in papillary thyroid carcinoma. Evaluation against real-time polymerase chain reaction.

PubMed

Paja Fano, Miguel; Ugalde Olano, Aitziber; Fuertes Thomas, Elena; Oleaga Alday, Amelia

2017-02-01

The BRAF V600E mutation is the most common genetic change in papillary thyroid carcinoma and is associated with a poorer clinical course. Usual methods for its study (DNA sequencing or molecular test based on PCR) are expensive and time-consuming. Recently, immunohistochemistry (IHC) for BRAF mutation has been introduced. To compare the results of IHC and real time PCR (RT-PCR) in the detection of BRAF V600E mutation in papillary thyroid carcinoma. Analysis of clinical and pathological differences depending on RT-PCR results is included. A prospective study was performed in 82 consecutive samples, 54 of them taken through a core needle biopsy. IHC was performed on tissue fixed for 24hours with 10% neutral formalin using the anti-BRAF V600E (VE-1) mouse monoclonal primary antibody and was rated as positive or negative. DNA was extracted from formalin-fixed, paraffin-embedded tissues by manual microdissection, and BRAF mutation was detected by RT-PCR using the Cobas® 4800 BRAF V600 mutation test (Roche). Both techniques were concordant in 81 cases, and BRAF was positive in 49. Discordance appeared in a follicular variant showing positive IHC and negative RT-PCR, attributed to histological heterogeneity. Cost of materials for IHC was less than half of the cost for RT-PCR. IHC appears to be a reliable, economical and easily available alternative to molecular biology techniques for routine detection of the BRAF V600E mutation in papillary thyroid carcinoma patients, provided optimal fixation conditions are used. It may be a useful technique in hospitals with no access to molecular biology techniques. Copyright © 2017 SEEN. Publicado por Elsevier España, S.L.U. All rights reserved.

Verification of the anatomy and newly discovered histology of the G-spot complex.

PubMed

Ostrzenski, A; Krajewski, P; Ganjei-Azar, P; Wasiutynski, A J; Scheinberg, M N; Tarka, S; Fudalej, M

2014-10-01

To expand the anatomical investigations of the G-spot and to assess the G-spot's characteristic histological and immunohistochemical features. An observational study. International multicentre. Eight consecutive fresh human female cadavers. Anterior vaginal wall dissections were executed and G-spot microdissections were performed. All specimens were stained with haematoxylin and eosin (H&E). The tissues of two women were selected at random for immunohistochemical staining. The primary outcome measure was to document the anatomy of the G-spot. The secondary outcome measures were to identify the histology of the G-spot and to determine whether histological samples stained with H&E are sufficient to identify the G-spot. The anatomical existence of the G-spot was identified in all women and was in a diagonal plane. In seven (87.5%) and one (12.5%) of the women the G-spot complex was found on the left or right side, respectively. The G-spot was intimately fused with vessels, creating a complex. A large tangled vein-like vascular structure resembled an arteriovenous malformation and there were a few smaller feeding arteries. A band-like structure protruded from the tail of the G-spot. The size of the G-spot varied. Histologically, the G-spot was determined as a neurovascular complex structure. The neural component contained abundant peripheral nerve bundles and a nerve ganglion. The vascular component comprised large vein-like vessels and smaller feeding arteries. Circular and longitudinal muscles covered the G-complex. The anatomy of the G-spot complex was confirmed. The histology of the G-spot presents as neurovascular tissues with a nerve ganglion. H&E staining is sufficient for the identification of the G-spot complex. © 2014 Royal College of Obstetricians and Gynaecologists.

Laser Capture Microdissection in the Genomic and Proteomic Era: Targeting the Genetic Basis of Cancer

PubMed Central

Domazet, Barbara; MacLennan, Gregory T.; Lopez-Beltran, Antonio; Montironi, Rodolfo; Cheng, Liang

2008-01-01

The advent of new technologies has enabled deeper insight into processes atsubcellular levels, which will ultimately improve diagnostic procedures and patient outcome. Thanks to cell enrichment methods, it is now possible to study cells in their native environment. This has greatly contributed to a rapid growth in several areas, such as gene expression analysis, proteomics, and metabolonomics. Laser capture microdissection (LCM) as a method of procuring subpopulations of cells under direct visual inspection is playing an important role in these areas. This review provides an overview of existing LCM technology and its downstream applications in genomics, proteomics, diagnostics and therapy. PMID:18787684

Laser capture microdissection in the genomic and proteomic era: targeting the genetic basis of cancer.

PubMed

Domazet, Barbara; Maclennan, Gregory T; Lopez-Beltran, Antonio; Montironi, Rodolfo; Cheng, Liang

2008-03-15

The advent of new technologies has enabled deeper insight into processes at subcellular levels, which will ultimately improve diagnostic procedures and patient outcome. Thanks to cell enrichment methods, it is now possible to study cells in their native environment. This has greatly contributed to a rapid growth in several areas, such as gene expression analysis, proteomics, and metabolonomics. Laser capture microdissection (LCM) as a method of procuring subpopulations of cells under direct visual inspection is playing an important role in these areas. This review provides an overview of existing LCM technology and its downstream applications in genomics, proteomics, diagnostics and therapy.

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.

Adventitial Tertiary Lymphoid Organs as Potential Source of MicroRNA Biomarkers for Abdominal Aortic Aneurysm

PubMed Central

Spear, Rafaelle; Boytard, Ludovic; Blervaque, Renaud; Chwastyniak, Maggy; Hot, David; Vanhoutte, Jonathan; Staels, Bart; Lemoine, Yves; Lamblin, Nicolas; Pruvot, François-René; Haulon, Stephan; Amouyel, Philippe; Pinet, Florence

2015-01-01

Abdominal aortic aneurysm (AAA) is an inflammatory disease associated with marked changes in the cellular composition of the aortic wall. This study aims to identify microRNA (miRNA) expression in aneurysmal inflammatory cells isolated by laser microdissection from human tissue samples. The distribution of inflammatory cells (neutrophils, B and T lymphocytes, mast cells) was evaluated in human AAA biopsies. We observed in half of the samples that adventitial tertiary lymphoid organs (ATLOs) with a thickness from 0.5 to 2 mm were located exclusively in the adventitia. Out of the 850 miRNA that were screened by microarray in isolated ATLOs (n = 2), 164 miRNAs were detected in ATLOs. The three miRNAs (miR-15a-3p, miR-30a-5p and miR-489-3p) with the highest expression levels were chosen and their expression quantified by RT-PCR in isolated ATLOs (n = 4), M1 (n = 2) and M2 macrophages (n = 2) and entire aneurysmal biopsies (n = 3). Except for the miR-30a-5p, a similar modulation was found in ATLOs and the two subtypes of macrophages. The modulated miRNAs were then evaluated in the plasma of AAA patients for their potential as AAA biomarkers. Our data emphasize the potential of miR-15a-3p and miR-30a-5p as biomarkers of AAA but also as triggers of ATLO evolution. Further investigations will be required to evaluate their targets in order to better understand AAA pathophysiology. PMID:25993295

The GDNF System Is Altered in Diverticular Disease – Implications for Pathogenesis

PubMed Central

Böttner, Martina; Barrenschee, Martina; Hellwig, Ines; Harde, Jonas; Egberts, Jan-Hendrik; Becker, Thomas; Zorenkov, Dimitri; Schäfer, Karl-Herbert; Wedel, Thilo

2013-01-01

Background & Aims Absence of glial cell line-derived neurotrophic factor (GDNF) leads to intestinal aganglionosis. We recently demonstrated that patients with diverticular disease (DD) exhibit hypoganglionosis suggesting neurotrophic factor deprivation. Thus, we screened mRNA expression pattern of the GDNF system in DD and examined the effects of GDNF on cultured enteric neurons. Methods Colonic specimens obtained from patients with DD (n = 21) and controls (n = 20) were assessed for mRNA expression levels of the GDNF system (GDNF, GDNF receptors GFRα1 and RET). To identify the tissue source of GDNF and its receptors, laser-microdissected (LMD) samples of human myenteric ganglia and intestinal muscle layers were analyzed separately by qPCR. Furthermore, the effects of GDNF treatment on cultured enteric neurons (receptor expression, neuronal differentiation and plasticity) were monitored. Results mRNA expression of GDNF and its receptors was significantly down-regulated in the muscularis propria of patients with DD. LMD samples revealed high expression of GDNF in circular and longitudinal muscle layers, whereas GDNF receptors were also expressed in myenteric ganglia. GDNF treatment of cultured enteric neurons increased mRNA expression of its receptors and promoted neuronal differentiation and plasticity revealed by synaptophysin mRNA and protein expression. Conclusions Our results suggest that the GDNF system is compromised in DD. In vitro studies demonstrate that GDNF enhances expression of its receptors and promotes enteric neuronal differentiation and plasticity. Since patients with DD exhibit hypoganglionosis, we propose that the observed enteric neuronal loss in DD may be due to lacking neurotrophic support mediated by the GDNF system. PMID:23805210

Inferring Viral Dynamics in Chronically HCV Infected Patients from the Spatial Distribution of Infected Hepatocytes

DOE PAGES

Graw, Frederik; Balagopal, Ashwin; Kandathil, Abraham J.; ...

2014-11-13

Chronic liver infection by hepatitis C virus (HCV) is a major public health concern. Despite partly successful treatment options, several aspects of intrahepatic HCV infection dynamics are still poorly understood, including the preferred mode of viral propagation, as well as the proportion of infected hepatocytes. Answers to these questions have important implications for the development of therapeutic interventions. In this study, we present methods to analyze the spatial distribution of infected hepatocytes obtained by single cell laser capture microdissection from liver biopsy samples of patients chronically infected with HCV. By characterizing the internal structure of clusters of infected cells, wemore » are able to evaluate hypotheses about intrahepatic infection dynamics. We found that individual clusters on biopsy samples range in size from 4-50 infected cells. In addition, the HCV RNA content in a cluster declines from the cell that presumably founded the cluster to cells at the maximal cluster extension. These observations support the idea that HCV infection in the liver is seeded randomly (e.g. from the blood) and then spreads locally. Assuming that the amount of intracellular HCV RNA is a proxy for how long a cell has been infected, we estimate based on models of intracellular HCV RNA replication and accumulation that cells in clusters have been infected on average for less than a week. Further, we do not find a relationship between the cluster size and the estimated cluster expansion time. Lastly, our method represents a novel approach to make inferences about infection dynamics in solid tissues from static spatial data.« less

Inferring Viral Dynamics in Chronically HCV Infected Patients from the Spatial Distribution of Infected Hepatocytes

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

Graw, Frederik; Balagopal, Ashwin; Kandathil, Abraham J.

Chronic liver infection by hepatitis C virus (HCV) is a major public health concern. Despite partly successful treatment options, several aspects of intrahepatic HCV infection dynamics are still poorly understood, including the preferred mode of viral propagation, as well as the proportion of infected hepatocytes. Answers to these questions have important implications for the development of therapeutic interventions. In this study, we present methods to analyze the spatial distribution of infected hepatocytes obtained by single cell laser capture microdissection from liver biopsy samples of patients chronically infected with HCV. By characterizing the internal structure of clusters of infected cells, wemore » are able to evaluate hypotheses about intrahepatic infection dynamics. We found that individual clusters on biopsy samples range in size from 4-50 infected cells. In addition, the HCV RNA content in a cluster declines from the cell that presumably founded the cluster to cells at the maximal cluster extension. These observations support the idea that HCV infection in the liver is seeded randomly (e.g. from the blood) and then spreads locally. Assuming that the amount of intracellular HCV RNA is a proxy for how long a cell has been infected, we estimate based on models of intracellular HCV RNA replication and accumulation that cells in clusters have been infected on average for less than a week. Further, we do not find a relationship between the cluster size and the estimated cluster expansion time. Lastly, our method represents a novel approach to make inferences about infection dynamics in solid tissues from static spatial data.« less

Angioimmunoblastic T-cell lymphoma: more than a disease of T follicular helper cells.

PubMed

Lemonnier, François; Mak, Tak W

2017-08-01

Angioimmunoblastic T-cell lymphoma (AITL) is one of the most frequent entities of peripheral T-cell lymphoma. An AITL has two components: the AITL tumour cells, which have a T follicular helper (TFH) cell phenotype, and a surrounding and extensive tumour microenvironment that is populated with various reactive cell types, including B cells. Recurrent TET2 mutations have been described in 50-80% of AITLs, possibly occurring in a haematopoietic progenitor cell. An article published recently in the Journal of Pathology describes the use of microdissection to isolate PD1 + AITL tumour cells and CD20 + B cells from the AITL microenvironment, and to show that TET2 mutations are actually more frequent in these diseases than previously thought. Whereas TET2 mutations were detected in only six of 13 AITLs, 12 of 13 samples of microdissected PD1 + AITL tumour cells possessed this mutation. Moreover, TET2 mutations were detected in CD20 + B cells from the AITL microenvironment in six of nine informative cases. These results confirm that TET2 mutation is an early event in the majority of AITL cases, and that the driving molecular anomalies are not restricted to the T lineage tumour cells. 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.

Macrophage Infiltration Is a Causative Factor for Ligamentum Flavum Hypertrophy through the Activation of Collagen Production in Fibroblasts.

PubMed

Saito, Takeyuki; Hara, Masamitsu; Kumamaru, Hiromi; Kobayakawa, Kazu; Yokota, Kazuya; Kijima, Ken; Yoshizaki, Shingo; Harimaya, Katsumi; Matsumoto, Yoshihiro; Kawaguchi, Kenichi; Hayashida, Mitsumasa; Inagaki, Yutaka; Shiba, Keiichiro; Nakashima, Yasuharu; Okada, Seiji

2017-12-01

Ligamentum flavum (LF) hypertrophy causes lumbar spinal canal stenosis, leading to leg pain and disability in activities of daily living in elderly individuals. Although previous studies have been performed on LF hypertrophy, its pathomechanisms have not been fully elucidated. In this study, we demonstrated that infiltrating macrophages were a causative factor for LF hypertrophy. Induction of macrophages into the mouse LF by applying a microinjury resulted in LF hypertrophy along with collagen accumulation and fibroblasts proliferation at the injured site, which were very similar to the characteristics observed in the severely hypertrophied LF of human. However, we found that macrophage depletion by injecting clodronate-containing liposomes counteracted LF hypertrophy even with microinjury. For identification of fibroblasts in the LF, we used collagen type I α 2 linked to green fluorescent protein transgenic mice and selectively isolated green fluorescent protein-positive fibroblasts from the microinjured LF using laser microdissection. A quantitative RT-PCR on laser microdissection samples revealed that the gene expression of collagen markedly increased in the fibroblasts at the injured site with infiltrating macrophages compared with the uninjured location. These results suggested that macrophage infiltration was crucial for LF hypertrophy by stimulating collagen production in fibroblasts, providing better understanding of the pathophysiology of LF hypertrophy. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Nanoproteomic analysis of extracellular receptor kinase-1/2 post-translational activation in microdissected human hyperplastic colon lesions.

PubMed

Drew, David A; Devers, Thomas; Horelik, Nicole; Yang, Shi; O'Brien, Michael; Wu, Rong; Rosenberg, Daniel W

2013-05-01

Oncogenic activation resulting in hyperproliferative lesions within the colonic mucosa has been identified in putative precancerous lesions, aberrant crypt foci (ACF). KRAS and BRAF mutation status was determined in 172 ACF identified in the colorectum of screening subjects by in situ high-definition, magnifying chromoendoscopy. Lesions were stratified according to histology (serrated vs. distended). Due to their limiting size, however, it was not technically feasible to examine downstream signaling consequences of these oncogenic mutations. We have combined ultraviolet-infrared (UV/IR) microdissection with an ultrasensitive nanofluidic proteomic immunoassay (NIA) to enable accurate quantification of posttranslational modifications to mitogen-activated protein kinase (MAPK) in total protein lysates isolated from hyperproliferative crypts and adjacent normal mucosa. Using this approach, levels of singly and dually (activated) phosphorylated isoforms of extracellular receptor kinase(ERK)-1 and ERK-2 were quantified in samples containing as little as 16 ng of total protein recovered from <200 cells. ERK activation is responsible for observed hyperplasia found in these early lesions, but is not directly dependent on KRAS and/or BRAF mutation status. This study describes the novel use of a sensitive nanofluidic platform to measure oncogene-driven proteomic changes in diminutive lesions and highlights the advantage of this approach over classical immunohistochemistry-based analyses. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Whole Transcriptome Analysis of Pre-invasive and Invasive Early Squamous Lung Carcinoma in Archival Laser Microdissected Samples.

PubMed

Koper, Andre; Zeef, Leo A H; Joseph, Leena; Kerr, Keith; Gosney, John; Lindsay, Mark A; Booton, Richard

2017-01-10

Preinvasive squamous cell cancer (PSCC) are local transformations of bronchial epithelia that are frequently observed in current or former smokers. Their different grades and sizes suggest a continuum of dysplastic change with increasing severity, which may culminate in invasive squamous cell carcinoma (ISCC). As a consequence of the difficulty in isolating cancerous cells from biopsies, the molecular pathology that underlies their histological variability remains largely unknown. To address this issue, we have employed microdissection to isolate normal bronchial epithelia and cancerous cells from low- and high-grade PSCC and ISCC, from paraffin embedded (FFPE) biopsies and determined gene expression using Affymetric Human Exon 1.0 ST arrays. Tests for differential gene expression were performed using the Bioconductor package limma followed by functional analyses of differentially expressed genes in IPA. Examination of differential gene expression showed small differences between low- and high-grade PSCC but substantial changes between PSCC and ISCC samples (184 vs 1200 p-value <0.05, fc ±1.75). However, the majority of the differentially expressed PSCC genes (142 genes: 77%) were shared with those in ISCC samples. Pathway analysis showed that these shared genes are associated with DNA damage response, DNA/RNA metabolism and inflammation as major biological themes. Cluster analysis identified 12 distinct patterns of gene expression including progressive up or down-regulation across PSCC and ISCC. Pathway analysis of incrementally up-regulated genes revealed again significant enrichment of terms related to DNA damage response, DNA/RNA metabolism, inflammation, survival and proliferation. Altered expression of selected genes was confirmed using RT-PCR, as well as immunohistochemistry in an independent set of 45 ISCCs. Gene expression profiles in PSCC and ISCC differ greatly in terms of numbers of genes with altered transcriptional activity. However, altered gene expression in PSCC affects canonical pathways and cellular and biological processes, such as inflammation and DNA damage response, which are highly consistent with hallmarks of cancer.

Renal Amyloidosis: Origin and Clinicopathologic Correlations of 474 Recent Cases

PubMed Central

Said, Samar M.; Sethi, Sanjeev; Valeri, Anthony M.; Leung, Nelson; Cornell, Lynn D.; Fidler, Mary E.; Herrera Hernandez, Loren; Vrana, Julie A.; Theis, Jason D.; Quint, Patrick S.; Dogan, Ahmet

2013-01-01

Summary Background and objectives The kidney is the organ most commonly involved in systemic amyloidosis. This study reports the largest clinicopathologic series of renal amyloidosis. Design, setting, participants, & measurements This study provides characteristics of 474 renal amyloidosis cases evaluated at the Mayo Clinic Renal Pathology Laboratory from 2007 to 2011, including age, sex, serum creatinine, proteinuria, type of amyloid, and tissue distribution according to type. Results The type of amyloid was Ig amyloidosis in 407 patients (85.9%), AA amyloidosis in 33 (7.0%), leukocyte chemotactic factor 2 amyloidosis in 13 (2.7%), fibrinogen A α chain amyloidosis in 6 (1.3%), Apo AI, Apo AII, or Apo AIV amyloidosis in 3 (0.6%), combined AA amyloidosis/Ig heavy and light chain amyloidosis in 1 (0.2%), and unclassified in 11 (2.3%). Laser microdissection/mass spectrometry, performed in 147 cases, was needed to determine the origin of amyloid in 74 of the 474 cases (16%), whereas immunofluorescence failed to diagnose 28 of 384 light chain amyloidosis cases (7.3%). Leukocyte chemotactic factor 2 amyloidosis and Apo AI, Apo AII, or Apo AIV amyloidosis were characterized by diffuse interstitial deposition, whereas fibrinogen A α chain amyloidosis showed obliterative glomerular involvement. Compared with other types, Ig amyloidosis was associated with lower serum creatinine, higher degree of proteinuria, and amyloid spicules. Conclusions In the authors’ experience, the vast majority of renal amyloidosis cases are Ig derived. The newly identified leukocyte chemotactic factor 2 amyloidosis form was the most common of the rarer causes of renal amyloidosis. With the advent of laser microdissection/mass spectrometry for amyloid typing, the origin of renal amyloidosis can be determined in >97% of cases. PMID:23704299

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.

High resolution chromosome 3p, 8p, 9q and 22q allelotyping analysis in the pathogenesis of gallbladder carcinoma

PubMed Central

Wistuba, I I; Maitra, A; Carrasco, R; Tang, M; Troncoso, P; Minna, J D; Gazdar, A F

2002-01-01

Our recent genome-wide allelotyping analysis of gallbladder carcinoma identified 3p, 8p, 9q and 22q as chromosomal regions with frequent loss of heterozygosity. The present study was undertaken to more precisely identify the presence and location of regions of frequent allele loss involving those chromosomes in gallbladder carcinoma. Microdissected tissue from 24 gallbladder carcinoma were analysed for PCR-based loss of heterozygosity using 81 microsatellite markers spanning chromosome 3p (n=26), 8p (n=14), 9q (n=29) and 22q (n=12) regions. We also studied the role of those allele losses in gallbladder carcinoma pathogenesis by examining 45 microdissected normal and dysplastic gallbladder epithelia accompanying gallbladder carcinoma, using 17 microsatellite markers. Overall frequencies of loss of heterozygosity at 3p (100%), 8p (100%), 9q (88%), and 22q (92%) sites were very high in gallbladder carcinoma, and we identified 13 distinct regions undergoing frequent loss of heterozygosity in tumours. Allele losses were frequently detected in normal and dysplastic gallbladder epithelia. There was a progressive increase of the overall loss of heterozygosity frequency with increasing severity of histopathological changes. Allele losses were not random and followed a sequence. This study refines several distinct chromosome 3p, 8p, 9q and 22q regions undergoing frequent allele loss in gallbladder carcinoma that will aid in the positional identification of tumour suppressor genes involved in gallbladder carcinoma pathogenesis. British Journal of Cancer (2002) 87, 432–440. doi:10.1038/sj.bjc.6600490 www.bjcancer.com © 2002 Cancer Research UK PMID:12177780

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.

Monoclonal origin of peritoneal implants and lymph node deposits in serous borderline ovarian tumors (s-BOT) with high intratumoral homogeneity.

PubMed

Horn, Lars-Christian; Höhn, Anne K; Einenkel, Jens; Siebolts, Udo

2014-11-01

Molecular studies have shown that the most prevalent mutations in serous ovarian borderline tumors (s-BOT) are BRAF and/or KRAS alterations. About one third of s-BOT represent peritoneal implants and/or lymph node involvement. These extraovarian deposits may be monoclonal or polyclonal in origin. To test both the hypotheses, mutational analyses using pyrosequencing for BRAF codon 600 and KRAS codon 12/13 and 61 of microdissected tissue was performed in 15 s-BOT and their invasive and noninvasive peritoneal implants. Two to 6 implants from different peritoneal sites were examined in 13 cases. Lymph node deposits were available for the analysis in 3 cases. Six s-BOT showed mutation in exon 2 codon 12 of the KRAS proto-oncogen. Five additional cases showed BRAF p.V600E mutation representing an overall mutation rate of 73.3%. Multiple (2-6) peritoneal implants were analyzed after microdissection in 13 of 15 cases. All showed identical mutational results when compared with the ovarian site of the disease. All lymph node deposits, including those with multiple deposits in different nodes, showed identical results, suggesting high intratumoral mutational homogeneity. The evidence presented in this study and the majority of data reported in the literature support the hypothesis that s-BOT with their peritoneal implants and lymph node deposits show identical mutational status of BRAF and KRAS suggesting a monoclonal rather than a polyclonal disease regarding these both tested genetic loci. In addition, a high intratumoral genetic homogeneity can be suggested. In conclusion, the results of the present study support the monoclonal origin of s-BOT and their peritoneal implants and lymph node deposits.

LOH at 16p13 is a novel chromosomal alteration detected in benign and malignant microdissected papillary neoplasms of the breast.

PubMed

Lininger, R A; Park, W S; Man, Y G; Pham, T; MacGrogan, G; Zhuang, Z; Tavassoli, F A

1998-10-01

Papillary carcinoma of the breast is a variant of predominantly intraductal carcinoma characterized by a papillary growth pattern with fibrovascular support. Loss of heterozygosity (LOH) was evaluated at multiple chromosomal loci (including loci reported to show frequent genetic alterations in breast cancer) to determine the frequency of genetic mutations in these tumors and their precursors. Thirty-three papillary lesions of the breast (6 papillary carcinomas, 12 carcinomas arising in a papilloma, and 15 intraductal papillomas with florid epithelial hyperplasia) were retrieved from the files of the Armed Forces Institute of Pathology (AFIP). Tumor cells and normal tissue were microdissected in each case and screened for LOH at INT-2 and p53 as well as several loci on chromosome 16p13 in the TSC2/PKD1 gene region (D16S423, D16S663, D16S665). LOH on chromosome 16p13 was present in 10 of 16 (63%) informative cases of either papillary carcinoma or carcinoma arising in a papilloma as well as in 6 of 10 (60%) informative cases of intraductal papilloma with florid epithelial hyperplasia (IDH). One case showed simultaneous LOH in both the florid IDH and carcinoma components of a papilloma. LOH was not observed at either INT-2 or p53 in any of the papillary carcinomas or papillomas with florid IDH. In conclusion, a high frequency of LOH at chromosome 16p13 (the TSC2/PKD1 gene region) is in both papillary carcinomas of the breast as well as in papillomas with florid IDH, including a case with LOH present simultaneously in both components. These findings suggest that chromosome 16p contains a tumor suppressor gene that frequently is mutated early in papillary neoplasia.

Identification and functional validation of HPV-mediated hypermethylation in head and neck squamous cell carcinoma

PubMed Central

2013-01-01

Background Human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) represents a distinct clinical and epidemiological condition compared with HPV-negative (HPV-) HNSCC. To test the possible involvement of epigenetic modulation by HPV in HNSCC, we conducted a genome-wide DNA-methylation analysis. Methods Using laser-capture microdissection of 42 formalin-fixed paraffin wax-embedded (FFPE) HNSCCs, we generated DNA-methylation profiles of 18 HPV+ and 14 HPV- samples, using Infinium 450 k BeadArray technology. Methylation data were validated in two sets of independent HPV+/HPV- HNSCC samples (fresh-frozen samples and cell lines) using two independent methods (Infinium 450 k and whole-genome methylated DNA immunoprecipitation sequencing (MeDIP-seq)). For the functional analysis, an HPV- HNSCC cell line was transduced with lentiviral constructs containing the two HPV oncogenes (E6 and E7), and effects on methylation were assayed using the Infinium 450 k technology. Results and discussion Unsupervised clustering over the methylation variable positions (MVPs) with greatest variation showed that samples segregated in accordance with HPV status, but also that HPV+ tumors are heterogeneous. MVPs were significantly enriched at transcriptional start sites, leading to the identification of a candidate CpG island methylator phenotype in a sub-group of the HPV+ tumors. Supervised analysis identified a strong preponderance (87%) of MVPs towards hypermethylation in HPV+ HNSCC. Meta-analysis of our HNSCC and publicly available methylation data in cervical and lung cancers confirmed the observed DNA-methylation signature to be HPV-specific and tissue-independent. Grouping of MVPs into functionally more significant differentially methylated regions identified 43 hypermethylated promoter DMRs, including for three cadherins of the Polycomb group target genes. Integration with independent expression data showed strong negative correlation, especially for the cadherin gene-family members. Combinatorial ectopic expression of the two HPV oncogenes (E6 and E7) in an HPV- HNSCC cell line partially phenocopied the hypermethylation signature seen in HPV+ HNSCC tumors, and established E6 as the main viral effector gene. Conclusions Our data establish that archival FFPE tissue is very suitable for this type of methylome analysis, and suggest that HPV modulates the HNSCC epigenome through hypermethylation of Polycomb repressive complex 2 target genes such as cadherins, which are implicated in tumor progression and metastasis. PMID:23419152

Plasma and brain angiotensin concentrations associated with water response behavior in the desert anuran, Scaphiopus couchii under natural conditions in the field.

PubMed

Johnson, William E; Hillyard, Stanley D; Propper, Catherine R

2010-12-01

Terrestrial amphibians obtain water by absorption across a specialized region of the ventral skin and exhibit a behavior, the water absorption response (WR) to place that region in contact with moist surfaces. Spadefoot toads (Scaphiopus couchii) spend dry months of the year in burrows, then emerge during brief periods of summer rainfall and seek water sources for rehydration and reproduction. We tested the hypothesis that these toads have changes in plasma and/or central angiotensin concentrations that are associated with seasonal emergence and WR behavior. Immunoreactive concentrations of combined angiotensin II and III (ir-ANG) were measured in plasma samples and microdissected regions of brain tissue taken from toads moving across the road or toads showing WR behavior in shallow puddles on the road. Plasma ir-ANG concentrations were not significantly different between these groups, but were significantly higher in the periventricular region of the hypothalamus in toads showing WR behavior. Concentrations in other brain regions, while highly variable among individuals, were not different between groups. Within the context of the natural history of a specialized desert toad, these results support the hypothesis that ir-ANG is associated with WR behavior in spadefoot toads in a manner analogous to oral drinking exhibited by other vertebrate clades. Copyright © 2010 Elsevier Inc. All rights reserved.

Lysyl oxidase expression is decreased in the developing diaphragm and lungs of nitrofen-induced congenital diaphragmatic hernia.

PubMed

Takahashi, Toshiaki; Friedmacher, Florian; Takahashi, Hiromizu; Daniel Hofmann, Alejandro; Puri, Prem

2015-02-01

Malformation of the nonmuscular tissue components in congenital diaphragmatic hernia (CDH) is thought to underlie the diaphragmatic defect, causing intrathoracic herniation of abdominal viscera and thus disturbing normal lung development. It has been shown that diaphragmatic and pulmonary morphogeneses require the structural integrity of connective tissue, and developmental mutations that inhibit the formation of extracellular matrix (ECM) result in CDH with hypoplastic lungs. Lysyl oxidase (lox), an extracellular enzyme that catalyzes the cross-linking of ECM proteins, plays an essential role during diaphragmatic and pulmonary development by controlling the formation of connective tissue. Furthermore, lox (-/-) knockouts exhibit abnormal connective tissue with diaphragmatic defects and impaired airway morphogenesis. We designed this study to investigate the hypothesis that diaphragmatic and pulmonary lox expression is decreased in the nitrofen-induced CDH model. Timed-pregnant Sprague-Dawley rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on selected time points D15 and D18. The micro-dissected fetal diaphragms (n=48) and lungs (n=48) were divided into two groups: control and nitrofen-exposed samples (n=12 per specimen and time point, respectively). Diaphragmatic and pulmonary gene expression levels of lox were analyzed by quantitative real-time polymerase chain reaction. Immunohistochemical staining was performed to evaluate lox protein expression in diaphragms and lungs. Relative mRNA expression of lox was significantly reduced in diaphragms and lungs of nitrofen-exposed fetuses on D15 (0.29 ± 0.08 vs. 0.12 ± 0.05; p<0.05 and 0.52 ± 0.44 vs. 0.20 ± 0.04; p<0.05) and D18 (0.90 ± 0.25 vs. 0.57 ± 0.23; p<0.05 and 0.59 ± 0.26 vs. 0.35 ± 0.09; p<0.05) compared with controls. Diaphragmatic and pulmonary immunoreactivity of lox was markedly decreased in nitrofen-exposed fetuses on D15 and D18 compared with controls. Decreased lox expression during diaphragmatic development and lung branching morphogenesis may interfere with normal cross-linking of ECM proteins, disrupting the integrity of connective tissue, and contributing to the diaphragmatic defect and impaired airway formation in the nitrofen-induced CDH model. Georg Thieme Verlag KG Stuttgart · New York.

Spectroscopic imaging system for high-throughput viability assessment of ovarian spheroids or microdissected tumor tissues (MDTs) in a microfluidic chip

NASA Astrophysics Data System (ADS)

St-Georges-Robillard, A.; Masse, M.; Kendall-Dupont, J.; Strupler, M.; Patra, B.; Jermyn, M.; Mes-Masson, A.-M.; Leblond, F.; Gervais, T.

2016-02-01

There is a growing effort in the biomicrosystems community to develop a personalized treatment response assay for cancer patients using primary cells, patient-derived spheroids, or live tissues on-chip. Recently, our group has developed a technique to cut tumors in 350 μm diameter microtissues and keep them alive on-chip, enabling multiplexed in vitro drug assays on primary tumor tissue. Two-photon microscopy, confocal microscopy and flow cytometry are the current standard to assay tissue chemosensitivity on-chip. While these techniques provide microscopic and molecular information, they are not adapted for high-throughput analysis of microtissues. We present a spectroscopic imaging system that allows rapid quantitative measurements of multiple fluorescent viability markers simultaneously by using a liquid crystal tunable filter to record fluorescence and transmittance spectra. As a proof of concept, 24 spheroids composed of ovarian cancer cell line OV90 were formed in a microfluidic chip, stained with two live cell markers (CellTrackerTM Green and Orange), and imaged. Fluorescence images acquired were normalized to the acquisition time and gain of the camera, dark noise was removed, spectral calibration was applied, and spatial uniformity was corrected. Spectral un-mixing was applied to separate each fluorophore's contribution. We have demonstrated that rapid and simultaneous viability measurements on multiple spheroids can be achieved, which will have a significant impact on the prediction of a tumor's response to multiple treatment options. This technique may be applied as well in drug discovery to assess the potential of a drug candidate directly on human primary tissue.

Full field optical coherence tomography can identify spermatogenesis in a rodent sertoli-cell only model.

PubMed

Ramasamy, Ranjith; Sterling, Joshua; Manzoor, Maryem; Salamoon, Bekheit; Jain, Manu; Fisher, Erik; Li, Phillip S; Schlegel, Peter N; Mukherjee, Sushmita

2012-01-01

Microdissection testicular sperm extraction (micro-TESE) has replaced conventional testis biopsies as a method of choice for obtaining sperm for in vitro fertilization for men with nonobstructive azoospermia. A technical challenge of micro-TESE is that the low magnification inspection of the tubules with a surgical microscope is insufficient to definitively identify sperm-containing tubules, necessitating tissue removal and cytologic assessment. Full field optical coherence tomography (FFOCT) uses white light interference microscopy to generate quick high-resolution tomographic images of fresh (unprocessed and unstained) tissue. Furthermore, by using a nonlaser safe light source (150 W halogen lamp) for tissue illumination, it ensures that the sperm extracted for in vitro fertilization are not photo-damaged or mutagenized. A focal Sertoli-cell only rodent model was created with busulfan injection in adult rats. Ex vivo testicular tissues from both normal and busulfan-treated rats were imaged with a commercial modified FFOCT system, Light-CT™, and the images were correlated with gold standard hematoxylin and eosin staining. Light-CT™ identified spermatogenesis within the seminiferous tubules in freshly excised testicular tissue, without the use of exogenous contrast or fixation. Normal adult rats exhibited tubules with uniform size and shape (diameter 328 ±11 μm). The busulfan-treated animals showed marked heterogeneity in tubular size and shape (diameter 178 ± 35 μm) and only 10% contained sperm within the lumen. FFOCT has the potential to facilitate real-time visualization of spermatogenesis in humans, and aid in micro-TESE for men with infertility.

Evidence that molecular changes in cells occur before morphological alterations during the progression of breast ductal carcinoma

PubMed Central

Castro, Nadia P; Osório, Cynthia ABT; Torres, César; Bastos, Elen P; Mourão-Neto, Mário; Soares, Fernando A; Brentani, Helena P; Carraro, Dirce M

2008-01-01

Introduction Ductal carcinoma in situ (DCIS) of the breast includes a heterogeneous group of preinvasive tumors with uncertain evolution. Definition of the molecular factors necessary for progression to invasive disease is crucial to determining which lesions are likely to become invasive. To obtain insight into the molecular basis of DCIS, we compared the gene expression pattern of cells from the following samples: non-neoplastic, pure DCIS, in situ component of lesions with co-existing invasive ductal carcinoma, and invasive ductal carcinoma. Methods Forty-one samples were evaluated: four non-neoplastic, five pure DCIS, 22 in situ component of lesions with co-existing invasive ductal carcinoma, and 10 invasive ductal carcinoma. Pure cell populations were isolated using laser microdissection. Total RNA was purified, DNase treated, and amplified using the T7-based method. Microarray analysis was conducted using a customized cDNA platform. The concept of molecular divergence was applied to classify the sample groups using analysis of variance followed by Tukey's test. Results Among the tumor sample groups, cells from pure DCIS exhibited the most divergent molecular profile, consequently identifying cells from in situ component of lesions with co-existing invasive ductal carcinoma as very similar to cells from invasive lesions. Additionally, we identified 147 genes that were differentially expressed between pure DCIS and in situ component of lesions with co-existing invasive ductal carcinoma, which can discriminate samples representative of in situ component of lesions with co-existing invasive ductal carcinoma from 60% of pure DCIS samples. A gene subset was evaluated using quantitative RT-PCR, which confirmed differential expression for 62.5% and 60.0% of them using initial and partial independent sample groups, respectively. Among these genes, LOX and SULF-1 exhibited features that identify them as potential participants in the malignant process of DCIS. Conclusions We identified new genes that are potentially involved in the malignant transformation of DCIS, and our findings strongly suggest that cells from the in situ component of lesions with co-existing invasive ductal carcinoma exhibit molecular alterations that enable them to invade the surrounding tissue before morphological changes in the lesion become apparent. PMID:18928525

Evidence that molecular changes in cells occur before morphological alterations during the progression of breast ductal carcinoma.

PubMed

Castro, Nadia P; Osório, Cynthia A B T; Torres, César; Bastos, Elen P; Mourão-Neto, Mário; Soares, Fernando A; Brentani, Helena P; Carraro, Dirce M

2008-01-01

Ductal carcinoma in situ (DCIS) of the breast includes a heterogeneous group of preinvasive tumors with uncertain evolution. Definition of the molecular factors necessary for progression to invasive disease is crucial to determining which lesions are likely to become invasive. To obtain insight into the molecular basis of DCIS, we compared the gene expression pattern of cells from the following samples: non-neoplastic, pure DCIS, in situ component of lesions with co-existing invasive ductal carcinoma, and invasive ductal carcinoma. Forty-one samples were evaluated: four non-neoplastic, five pure DCIS, 22 in situ component of lesions with co-existing invasive ductal carcinoma, and 10 invasive ductal carcinoma. Pure cell populations were isolated using laser microdissection. Total RNA was purified, DNase treated, and amplified using the T7-based method. Microarray analysis was conducted using a customized cDNA platform. The concept of molecular divergence was applied to classify the sample groups using analysis of variance followed by Tukey's test. Among the tumor sample groups, cells from pure DCIS exhibited the most divergent molecular profile, consequently identifying cells from in situ component of lesions with co-existing invasive ductal carcinoma as very similar to cells from invasive lesions. Additionally, we identified 147 genes that were differentially expressed between pure DCIS and in situ component of lesions with co-existing invasive ductal carcinoma, which can discriminate samples representative of in situ component of lesions with co-existing invasive ductal carcinoma from 60% of pure DCIS samples. A gene subset was evaluated using quantitative RT-PCR, which confirmed differential expression for 62.5% and 60.0% of them using initial and partial independent sample groups, respectively. Among these genes, LOX and SULF-1 exhibited features that identify them as potential participants in the malignant process of DCIS. We identified new genes that are potentially involved in the malignant transformation of DCIS, and our findings strongly suggest that cells from the in situ component of lesions with co-existing invasive ductal carcinoma exhibit molecular alterations that enable them to invade the surrounding tissue before morphological changes in the lesion become apparent.

A Role for the Stele in Intertissue Signaling in the Initiation of Abscission in Bean Leaves (Phaseolus vulgaris L.).

PubMed Central

Thompson, D. S.; Osborne, D. J.

1994-01-01

A combination of microdissection and viscometric endo-[beta]-1,4-glucanhydrolase assays was used to investigate if the early appearance of the abscission-related isoelectric point-9.5 endo-[beta]-1,4-glucanhydrolase in the stele of the pulvinus and abscission zone of the foliar abscission zone of Phaseolus vulgaris L. prior to cell separation (reported by E. del Campillo, P.D. Reid, R. Sexton, L.N.Lewis [1990] Plant Cell 2: 245-254) indicates that the vascular tissue of this region has a specific role in abscission. We find that no endo-[beta]-1,4-glucanhydrolase activity or cell separation is detectable in the abscission zone cortex if the abscission zone cortex is separated from the stele tissue. If the stele is separated from the abscission zone cortex after a lag period but again before any endo-[beta]-1,4-glucanhydrolase activity is present in the abscission zone cortex, then the enzyme is produced in the cortex and abscission ensues. We conclude that the cortex of the abscission zone is able to abscind independently of the vascular tissue only after the vascular tissue has begun to respond to abscission-promoting signals. We suggest that ethylene promotes formation of an abscission-permitting signal in the stele of the abscission zone and pulvinus, and that this signal is an essential elicitor for the synthesis of cell separation enzymes in the target cells of the abscission zone cortex. PMID:12232206

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

Whole-genome multiple displacement amplification from single cells.

PubMed

Spits, Claudia; Le Caignec, Cédric; De Rycke, Martine; Van Haute, Lindsey; Van Steirteghem, André; Liebaers, Inge; Sermon, Karen

2006-01-01

Multiple displacement amplification (MDA) is a recently described method of whole-genome amplification (WGA) that has proven efficient in the amplification of small amounts of DNA, including DNA from single cells. Compared with PCR-based WGA methods, MDA generates DNA with a higher molecular weight and shows better genome coverage. This protocol was developed for preimplantation genetic diagnosis, and details a method for performing single-cell MDA using the phi29 DNA polymerase. It can also be useful for the amplification of other minute quantities of DNA, such as from forensic material or microdissected tissue. The protocol includes the collection and lysis of single cells, and all materials and steps involved in the MDA reaction. The whole procedure takes 3 h and generates 1-2 microg of DNA from a single cell, which is suitable for multiple downstream applications, such as sequencing, short tandem repeat analysis or array comparative genomic hybridization.

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

Massive expression of germ cell-specific genes is a hallmark of cancer and a potential target for novel treatment development.

PubMed

Bruggeman, Jan Willem; Koster, Jan; Lodder, Paul; Repping, Sjoerd; Hamer, Geert

2018-06-15

Cancer cells have been found to frequently express genes that are normally restricted to the testis, often referred to as cancer/testis (CT) antigens or genes. Because germ cell-specific antigens are not recognized as "self" by the innate immune system, CT-genes have previously been suggested as ideal candidate targets for cancer therapy. The use of CT-genes in cancer therapy has thus far been unsuccessful, most likely because their identification has relied on gene expression in whole testis, including the testicular somatic cells, precluding the detection of true germ cell-specific genes. By comparing the transcriptomes of micro-dissected germ cell subtypes, representing the main developmental stages of human spermatogenesis, with the publicly accessible transcriptomes of 2617 samples from 49 different healthy somatic tissues and 9232 samples from 33 tumor types, we here discover hundreds of true germ cell-specific cancer expressed genes. Strikingly, we found these germ cell cancer genes (GC-genes) to be widely expressed in all analyzed tumors. Many GC-genes appeared to be involved in processes that are likely to actively promote tumor viability, proliferation and metastasis. Targeting these true GC-genes thus has the potential to inhibit tumor growth with infertility being the only possible side effect. Moreover, we identified a subset of GC-genes that are not expressed in spermatogonial stem cells. Targeting of this GC-gene subset is predicted to only lead to temporary infertility, as untargeted spermatogonial stem cells can recover spermatogenesis after treatment. Our GC-gene dataset enables improved understanding of tumor biology and provides multiple novel targets for cancer treatment.

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.

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.

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

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

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.

Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens.

PubMed

Sho, Shonan; Court, Colin M; Kim, Stephen; Braxton, David R; Hou, Shuang; Muthusamy, V Raman; Watson, Rabindra R; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S

2017-01-01

Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare mutation subtypes representing tumor heterogeneity.

Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens

PubMed Central

Court, Colin M.; Kim, Stephen; Braxton, David R.; Hou, Shuang; Muthusamy, V. Raman; Watson, Rabindra R.; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S.

2017-01-01

Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare mutation subtypes representing tumor heterogeneity. PMID:28125707

Molecular Genetic Evidence for a Common Clonal Origin of Urinary Bladder Small Cell Carcinoma and Coexisting Urothelial Carcinoma

PubMed Central

Cheng, Liang; Jones, Timothy D.; McCarthy, Ryan P.; Eble, John N.; Wang, Mingsheng; MacLennan, Gregory T.; Lopez-Beltran, Antonio; Yang, Ximing J.; Koch, Michael O.; Zhang, Shaobo; Pan, Chong-Xian; Baldridge, Lee Ann

2005-01-01

In most cases, small-cell carcinoma of the urinary bladder is admixed with other histological types of bladder carcinoma. To understand the pathogenetic relationship between the two tumor types, we analyzed histologically distinct tumor cell populations from the same patient for loss of heterozygosity (LOH) and X chromosome inactivation (in female patients). We examined five polymorphic microsatellite markers located on chromosome 3p25-26 (D3S3050), chromosome 9p21 (IFNA and D9S171), chromosome 9q32-33 (D9S177), and chromosome 17p13 (TP53) in 20 patients with small-cell carcinoma of the urinary bladder and concurrent urothelial carcinoma. DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser-assisted microdissection. A nearly identical pattern of allelic loss was observed in the two tumor types in all cases, with an overall frequency of allelic loss of 90% (18 of 20 cases). Three patients showed different allelic loss patterns in the two tumor types at a single locus; however, the LOH patterns at the remaining loci were identical. Similarly, the same pattern of nonrandom X chromosome inactivation was present in both carcinoma components in the four cases analyzed. Concordant genetic alterations and X chromosome inactivation between small-cell carcinoma and coexisting urothelial carcinoma suggest that both tumor components originate from the same cells in the urothelium. PMID:15855652

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

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.

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.

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

Loss of Stromal Caveolin-1 Expression: A Novel Tumor Microenvironment Biomarker That Can Predict Poor Clinical Outcomes for Pancreatic Cancer

PubMed Central

Shan, Tao; Lu, Hongwei; Ji, Hong; Li, Yiming; Guo, Jian; Chen, Xi; Wu, Tao

2014-01-01

Aims Cancer development and progression is not only associated with the tumor cell proliferation but also depends on the interaction between tumor cells and the stromal microenvironment. A new understanding of the role of the tumor microenvironment suggests that the loss of stromal caveolin-1 (Cav-1) as a key regulator may become a potential therapy target. This study aims to elucidate whether stromal Cav-1 expression in pancreatic cancer can be a strong prognosis biomarker. Methods Tissue samples from 45 pancreatic cancer patients were studied. Parenchyma and stroma were separated and purified using laser capture microdissection. Stromal Cav-1 expression was measured from pancreatic cancer, paraneoplastic, and normal tissue using immunohistochemistry. We analyzed the correlation of stromal Cav-1 expression with clinicopathologic features and prognostic indicators, such as tumor marker HER-2/neu gene. Results Specimens from six patients (13.3%) showed high levels of stromal Cav-1 staining, those from eight patients (17.8%) showed a lower, intermediate level of staining, whereas those from 31 patients (68.9%) showed an absence of staining. Cav-1 expression in cancer-associated fibroblasts was lower than that in paracancer-associated and in normal fibroblasts. Stromal Cav-1 loss was associated with TNM stage (P = 0.018), lymph node metastasis (P = 0.014), distant metastasis (P = 0.027), and HER-2/neu amplification (P = 0.007). The relationships of age, sex, histological grade, and tumor size with stromal Cav-1 expression were not significant (P>0.05). A negative correlation was found between circulating tumor cells and stromal Cav-1 expression (P<0.05). Conclusion The loss of stromal Cav-1 in pancreatic cancer was an independent prognostic indicator, thus suggesting that stromal Cav-1 may be an effective therapeutic target for patients with pancreatic cancer. PMID:24949874

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.

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

Amyloidosis: A cancer-derived paraproteinemia and kidney involvement.

PubMed

Małyszko, Jolanta; Kozłowska, Klaudia; Małyszko, Jacek Stanisław

2017-03-01

Amyloidosis is the general term describing the extracellular tissue deposition of fibrils composed of low molecular weight subunits of a variety of proteins. There are multiple different human protein precursors of amyloid fibrils. Amyloid deposits are stained using Congo Red and show typical apple-green birefringence in polarized microscopy. Nowadays, a novel technique LMD/MS technique or laser microdissection combined with mass spectrometry help to diagnose amyloidosis. Amyloidosis of the kidney is typically classified as being either one of two types: AL or AA. Less common is the hereditary amyloidosis. Clinical manifestations are usually determined by the type of precursor protein, the tissue distribution, and the amount of amyloid deposition. Renal manifestation is usually present as asymptomatic proteinuria or clinically apparent nephrotic syndrome. In some patients clinical presentation include impaired kidney function with no or mild proteinuria. Patients with renal amyloidosis who progress to end-stage renal disease (ESRD) can be treated with either dialysis or renal transplantation. Diagnosis of amyloidosis is prerequisite to consider treatment options to avoid unnecessary chemotherapy. Treatment of amyloidosis is aimed at decreasing the precursors of fibrillary proteins and/or decrease in synthesis/deposition of amyloid fibrils. It depends upon the type of amyloidosis and cause of excess fibril production. Copyright © 2016 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.

Ambient Mass Spectrometry Imaging Using Direct Liquid Extraction Techniques

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

Laskin, Julia; Lanekoff, Ingela

2015-11-13

Mass spectrometry imaging (MSI) is a powerful analytical technique that enables label-free spatial localization and identification of molecules in complex samples.1-4 MSI applications range from forensics5 to clinical research6 and from understanding microbial communication7-8 to imaging biomolecules in tissues.1, 9-10 Recently, MSI protocols have been reviewed.11 Ambient ionization techniques enable direct analysis of complex samples under atmospheric pressure without special sample pretreatment.3, 12-16 In fact, in ambient ionization mass spectrometry, sample processing (e.g., extraction, dilution, preconcentration, or desorption) occurs during the analysis.17 This substantially speeds up analysis and eliminates any possible effects of sample preparation on the localization of moleculesmore » in the sample.3, 8, 12-14, 18-20 Venter and co-workers have classified ambient ionization techniques into three major categories based on the sample processing steps involved: 1) liquid extraction techniques, in which analyte molecules are removed from the sample and extracted into a solvent prior to ionization; 2) desorption techniques capable of generating free ions directly from substrates; and 3) desorption techniques that produce larger particles subsequently captured by an electrospray plume and ionized.17 This review focuses on localized analysis and ambient imaging of complex samples using a subset of ambient ionization methods broadly defined as “liquid extraction techniques” based on the classification introduced by Venter and co-workers.17 Specifically, we include techniques where analyte molecules are desorbed from solid or liquid samples using charged droplet bombardment, liquid extraction, physisorption, chemisorption, mechanical force, laser ablation, or laser capture microdissection. Analyte extraction is followed by soft ionization that generates ions corresponding to intact species. Some of the key advantages of liquid extraction techniques include the ease of operation, ability to analyze samples in their native environments, speed of analysis, and ability to tune the extraction solvent composition to a problem at hand. For example, solvent composition may be optimized for efficient extraction of different classes of analytes from the sample or for quantification or online derivatization through reactive analysis. In this review, we will: 1) introduce individual liquid extraction techniques capable of localized analysis and imaging, 2) describe approaches for quantitative MSI experiments free of matrix effects, 3) discuss advantages of reactive analysis for MSI experiments, and 4) highlight selected applications (published between 2012 and 2015) that focus on imaging and spatial profiling of molecules in complex biological and environmental samples.« less

Scanning electron microscopic appearance of rat otocyst of the twelfth postcoital day: elaboration of a method.

PubMed

Marovitz, W F; Khan, K M

1977-01-01

A method for removal, fixation, microdissection, and drying of early rat otocyst for examination by the scanning electron microscope is elaborated. Tissues were dissected, fixed as for conventional transmission electron microscopy and dried by critical point evaporation using amylacetate as the transitional fluid and carbon dioxide as the pressure head. Otocysts were either dissected at the time of initial fixation, or subsequent to drying. The otocyst of the 12th postcoital day was used as a model system in this preliminary report. Critical point drying retained the overall configuration and the fine ultrastructural detail of the otocyst. The interior otocystic surface was visualized and cilia bearing cells of the luminal surface were identified. Most if not all of these cells had a comspicuous, but short kinocillum which terminated in an ovoid bulb. The scanning electron microscopic appearance was correlated to the transmission electron microscopic image seen in the second paper in this Supplement.

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

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.

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

Serum biomarkers of Burkholderia mallei infection elucidated by proteomic imaging of skin and lung abscesses.

PubMed

Glaros, Trevor G; Blancett, Candace D; Bell, Todd M; Natesan, Mohan; Ulrich, Robert G

2015-01-01

The bacterium Burkholderia mallei is the etiological agent of glanders, a highly contagious, often fatal zoonotic infectious disease that is also a biodefense concern. Clinical laboratory assays that analyze blood or other biological fluids are the highest priority because these specimens can be collected with minimal risk to the patient. However, progress in developing sensitive assays for monitoring B. mallei infection is hampered by a shortage of useful biomarkers. Reasoning that there should be a strong correlation between the proteomes of infected tissues and circulating serum, we employed imaging mass spectrometry (IMS) of thin-sectioned tissues from Chlorocebus aethiops (African green) monkeys infected with B. mallei to localize host and pathogen proteins that were associated with abscesses. Using laser-capture microdissection of specific regions identified by IMS and histology within the tissue sections, a more extensive proteomic analysis was performed by a technique that combined the physical separation capabilities of liquid chromatography (LC) with the sensitive mass analysis capabilities of mass spectrometry (LC-MS/MS). By examining standard formalin-fixed, paraffin-embedded tissue sections, this strategy resulted in the identification of several proteins that were associated with lung and skin abscesses, including the host protein calprotectin and the pathogen protein GroEL. Elevated levels of calprotectin detected by ELISA and antibody responses to GroEL, measured by a microarray of the bacterial proteome, were subsequently detected in the sera of C. aethiops, Macaca mulatta, and Macaca fascicularis primates infected with B. mallei. Our results demonstrate that a combination of multidimensional MS analysis of traditional histology specimens with high-content protein microarrays can be used to discover lead pairs of host-pathogen biomarkers of infection that are identifiable in biological fluids.

miR-125b-1 and miR-378a are predictive biomarkers for the efficacy of vaccine treatment against colorectal cancer.

PubMed

Tanaka, Hironori; Hazama, Shoichi; Iida, Michihisa; Tsunedomi, Ryouichi; Takenouchi, Hiroko; Nakajima, Masao; Tokumitsu, Yukio; Kanekiyo, Shinsuke; Shindo, Yoshitaro; Tomochika, Shinobu; Tokuhisa, Yoshihiro; Sakamoto, Kazuhiko; Suzuki, Nobuaki; Takeda, Shigeru; Yamamoto, Shigeru; Yoshino, Shigefumi; Ueno, Tomio; Hamamoto, Yoshihiko; Fujita, Yusuke; Tanaka, Hiroaki; Tahara, Ko; Shimizu, Ryoichi; Okuno, Kiyotaka; Fujita, Koji; Kuroda, Masahiko; Nakamura, Yusuke; Nagano, Hiroaki

2017-11-01

Many clinical trials of peptide vaccines have been conducted. However, these vaccines have provided clinical benefits in only a small fraction of patients. The purpose of the present study was to explore microRNAs (miRNAs) as novel predictive biomarkers for the efficacy of vaccine treatment against colorectal cancer. First, we carried out microarray analysis of pretreatment cancer tissues in a phase I study, in which peptide vaccines alone were given. Candidate miRNAs were selected by comparison of the better prognosis group with the poorer prognosis group. Next, we conducted microarray analysis of cancer tissues in a phase II study, in which peptide vaccines combined with chemotherapy were given. Candidate miRNAs were further selected by a similar comparison of prognosis. Subsequently, we carried out reverse-transcription PCR analysis of phase II cases, separating cancer tissues into cancer cells and stromal tissue using laser capture microdissection. Treatment effect in relation to overall survival (OS) and miRNA expression was analyzed. Three miRNA predictors were negatively associated with OS: miR-125b-1 in cancer cells (P = 0.040), and miR-378a in both cancer cells (P = 0.009) and stromal cells (P < 0.001). Multivariate analysis showed that expression of miR-378a in stromal cells was the best among the three predictors (HR, 2.730; 95% CI, 1.027-7.585; P = 0.044). In conclusion, miR-125b-1 and miR-378a expression might be considered as novel biomarkers to predict the efficacy of vaccine treatment against colorectal cancer. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Evaluation of adenoassociated virus 2 and human papilloma virus 16 and 18 infection in cervical cancer biopsies.

PubMed

Ahn, Woong Shick; Bae, Su Mi; Chung, Jae Eun; Lee, Hyun Kyung; Kim, Byung Kee; Lee, Joon Mo; Namkoong, Sung Eun; Kim, Chong Kook; Sin, Jeong- Im

2003-04-01

Protective roles of adenoassociated virus (AAV) 2 in cervical tumorigenesis are controversial. In an effort to clarify this issue, we tested prevalence of AAV 2 and human papillomavirus (HPV) infection in cervical lesions and adjacent normal tissues. Tissues of cervical intraepithelial neoplasm (CIN) I (20 patients), CIN II (24 patients), CIN III (25 patients), and invasive cancer (23 patients) were investigated by microdissection and PCR using HPV-16-, HVP-18-, and AAV-2-specific primers. AAV 2 was detected in 11 out of 20 CIN I (55%), 21 out of 24 CIN II (84.5%), 13 out of 25 CIN III (52%), and 12 out of 23 invasive cancer cases (52.2%). However, HPV 16 was detected in none out of 20 CIN I, 2 out of 24 CIN II (8.3%), 6 out of 25 CIN III (24%), and 6 out of 23 invasive cancer cases (26.1%). HPV 18 was detected in 1 case in CIN II (4.2%) and 2 cases in CIN III (8%). In 92 perilesional normal tissues, AAV 2 was detected in 53 cases (57.6%), displaying 25% of CIN I, 83.3% of CIN II, 52% of CIN III, and 65.2% of invasive cancer. The differences in AAV 2 prevalence are not significant between CIN and normal tissues. However, differences in HPV 16 are significant in CIN III and invasive cancer, as compared to CIN I, CIN II, and normal, suggesting no significant correlation between AAV 2 and cervical cancer. Thus, these results support the notion that AAV 2 is not associated with cervical tumorigenesis.

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

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.

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

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.

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.

FGFR3, PIK3CA and RAS mutations in benign lichenoid keratosis.

PubMed

Groesser, L; Herschberger, E; Landthaler, M; Hafner, C

2012-04-01

Benign lichenoid keratoses (BLKs) are solitary skin lesions which have been proposed to represent a regressive form of pre-existent epidermal tumours such as solar lentigo or seborrhoeic keratosis. However, the genetic basis of BLK is unknown. FGFR3, PIK3CA and RAS mutations have been shown to be involved in the pathogenesis of seborrhoeic keratosis and solar lentigo. We thus investigated whether these mutations are also present in BLK. After manual microdissection and DNA isolation, 52 BLKs were screened for FGFR3, PIK3CA and RAS hotspot mutations using SNaPshot(®) multiplex assays. We identified 6/52 (12%) FGFR3 mutations, 10/52 (19%) PIK3CA mutations, 6/52 (12%) HRAS mutations and 2/52 (4%) KRAS mutations. FGFR3 and RAS mutations were mutually exclusive. One BLK showed a simultaneous PIK3CA and HRAS mutation. In nine BLKs with a mutation, nonlesional control tissue from the epidermal margin and the dermal lymphocytic infiltrate were wild-type, indicating that these mutations are somatic. To demonstrate that these findings are specific, 10 samples of lichen planus were analysed without evidence for FGFR3, PIK3CA or RAS mutations. Our results indicate that FGFR3, PIK3CA and RAS mutations are present in approximately 50% of BLKs. These findings support the concept on the molecular genetic level that at least a proportion of BLKs represents regressive variants resulting from former benign epidermal tumours such as seborrhoeic keratosis and solar lentigo. © 2011 The Authors. BJD © 2011 British Association of Dermatologists 2011.

Biomineralization, life-time of odontogenic cells and differential expression of the two homeobox genes MSX-1 and DLX-2 in transgenic mice.

PubMed

Lézot, F; Thomas, B; Hotton, D; Forest, N; Orestes-Cardoso, S; Robert, B; Sharpe, P; Berdal, A

2000-03-01

Msx and Dlx homeobox genes encode for transcription factors that control early morphogenesis. More specifically, Msx-1, Msx-2, and Dlx-2 homeobox genes contribute to the initial patterning of the dentition. The present study is devoted to the potential role of those homeobox genes during the late formation of mineralized tissues, using the rodent incisor as an experimental system. The continuously erupting mandibular incisor allows (1) the coinvestigation of the whole sequences of amelogenesis and dentinogenesis, aligned along the main dental axis in a single sample in situ and (2) the differential characterization of transcripts generated by epithelial and ectomesenchymal odontogenic cells. Northern blot experiments on microdissected cells showed the continuing expression of Msx-2 and Dlx-2 in the later stages of dental biomineralization, differentially in epithelial and ectomesenchymal compartments. Transgenic mice produced with LacZ reporter constructs for Dlx-2 and Msx-1 were used to detect different components of the gene expression patterns with the sensitive beta-galactosidase histoenzymology. The results show a prominent epithelial involvement of Dlx-2, with stage-specific variations in the cells involved in enamel formation. Quantitative analyses identified specific modulations of Dlx-2 expression in ameloblasts depending on the anatomical sites of the incisor, showing more specifically an inverse linear relationship between the Dlx-2 promoter activity level and enamel thickness. This investigation extends the role of homeoproteins to postmitotic stages, which would control secretory cell activity, in a site-specific manner as shown here for Dlx-2.

Otoconial complexes as ion reservoirs in endolymph

NASA Technical Reports Server (NTRS)

Ross, M. D.; Williams, T. J.

1982-01-01

Scintillation spectrometry was employed to examine the Ca-45(2+) uptake and exchange by otoconial complexes in the sensory region endolymph medium, and a comparison was made with bone mineral deposition. CaCl was injected intraperitoneally into 222 rats and blood samples were collected at set intervals during the subsequent 15 min-l mo life durations of the animals. The animals were eventually sacrificed and saccular and utricular otoconial complexes were microdissected while bone chips from the otic bone and femur were gathered by scraping. Ca-45 was present in the saccular otoconial complexes within 15 min of injection, an uptake similar to the bone deposition, while slower rates were observed with the utricular complexes. Utricular uptake, however, accelerated 5-6 hr postinjection, and total otoconial content was always lower than proportional bone absorption.

Distribution of erlotinib in rash and normal skin in cancer patients receiving erlotinib visualized by matrix assisted laser desorption/ionization mass spectrometry imaging.

PubMed

Nishimura, Meiko; Hayashi, Mitsuhiro; Mizutani, Yu; Takenaka, Kei; Imamura, Yoshinori; Chayahara, Naoko; Toyoda, Masanori; Kiyota, Naomi; Mukohara, Toru; Aikawa, Hiroaki; Fujiwara, Yasuhiro; Hamada, Akinobu; Minami, Hironobu

2018-04-06

The development of skin rashes is the most common adverse event observed in cancer patients treated with epidermal growth factor receptor-tyrosine kinase inhibitors such as erlotinib. However, the pharmacological evidence has not been fully revealed. Erlotinib distribution in the rashes was more heterogeneous than that in the normal skin, and the rashes contained statistically higher concentrations of erlotinib than adjacent normal skin in the superficial skin layer (229 ± 192 vs. 120 ± 103 ions/mm 2 ; P = 0.009 in paired t -test). LC-MS/MS confirmed that the concentration of erlotinib in the skin rashes was higher than that in normal skin in the superficial skin layer (1946 ± 1258 vs. 1174 ± 662 ng/cm 3 ; P = 0.028 in paired t -test). The results of MALDI-MSI and LC-MS/MS were well correlated (coefficient of correlation 0.879, P < 0.0001). Focal distribution of erlotinib in the skin tissue was visualized using non-labeled MALDI-MSI. Erlotinib concentration in the superficial layer of the skin rashes was higher than that in the adjacent normal skin. We examined patients with advanced pancreatic cancer who developed skin rashes after treatment with erlotinib and gemcitabine. We biopsied both the rash and adjacent normal skin tissues, and visualized and compared the distribution of erlotinib within the skin using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The tissue concentration of erlotinib was also measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with laser microdissection.

Etiological role of human papillomavirus infection for inverted papilloma of the bladder.

PubMed

Shigehara, Kazuyoshi; Sasagawa, Toshiyuki; Doorbar, John; Kawaguchi, Shohei; Kobori, Yoshitomo; Nakashima, Takao; Shimamura, Masayoshi; Maeda, Yuji; Miyagi, Tohru; Kitagawa, Yasuhide; Kadono, Yoshifumi; Konaka, Hiroyuki; Mizokami, Atsushi; Koh, Eitetsu; Namiki, Mikio

2011-02-01

The status of human papillomavirus (HPV) infection in urothelial inverted papilloma was examined in the present study. Formalin-fixed and paraffin-embedded tissues from eight cases of inverted papilloma of the bladder were studied. The presence of HPV-DNA was examined by modified GP5/6+PCR using archival tissue sections by microdissection. HPV genotype was determined with a Hybri-Max HPV genotyping kit. Immunohistochemical analysis for p16-INK4a, mcm7, HPV-E4, and L1, and in situ hybridization for the HPV genome were performed. HPV was detected in seven of eight cases (87.5%) of inverted papilloma. Three cases were diagnosed as inverted papilloma with atypia, while the remaining five were typical cases. HPV-18 was detected in two cases, including one inverted papilloma with atypia, and HPV-16 was detected in four cases, including one inverted papilloma with atypia. Multiple HPV type infection was detected in one typical case and one atypical case. High-risk HPV was present in all HPV-positive cases. Cellular proteins, p16-INK4a and mcm7, which are surrogate markers for HPV-E7 expression, were detected in all HPV-positive cases, and their levels were higher in inverted papilloma with atypia than in typical cases. In contrast, HPV-E4 and L1, which are markers for HPV propagation, were observed in some parts of the typical inverted papilloma tissue. High-risk HPV infection may be one of the causes of urothelial inverted papilloma, and inverted papilloma with atypia may have malignant potential. 2010 Wiley-Liss, Inc.

Distribution of erlotinib in rash and normal skin in cancer patients receiving erlotinib visualized by matrix assisted laser desorption/ionization mass spectrometry imaging

PubMed Central

Mizutani, Yu; Takenaka, Kei; Imamura, Yoshinori; Chayahara, Naoko; Toyoda, Masanori; Kiyota, Naomi; Mukohara, Toru; Aikawa, Hiroaki; Fujiwara, Yasuhiro; Hamada, Akinobu; Minami, Hironobu

2018-01-01

Background The development of skin rashes is the most common adverse event observed in cancer patients treated with epidermal growth factor receptor-tyrosine kinase inhibitors such as erlotinib. However, the pharmacological evidence has not been fully revealed. Results Erlotinib distribution in the rashes was more heterogeneous than that in the normal skin, and the rashes contained statistically higher concentrations of erlotinib than adjacent normal skin in the superficial skin layer (229 ± 192 vs. 120 ± 103 ions/mm2; P = 0.009 in paired t-test). LC-MS/MS confirmed that the concentration of erlotinib in the skin rashes was higher than that in normal skin in the superficial skin layer (1946 ± 1258 vs. 1174 ± 662 ng/cm3; P = 0.028 in paired t-test). The results of MALDI-MSI and LC-MS/MS were well correlated (coefficient of correlation 0.879, P < 0.0001). Conclusions Focal distribution of erlotinib in the skin tissue was visualized using non-labeled MALDI-MSI. Erlotinib concentration in the superficial layer of the skin rashes was higher than that in the adjacent normal skin. Methods We examined patients with advanced pancreatic cancer who developed skin rashes after treatment with erlotinib and gemcitabine. We biopsied both the rash and adjacent normal skin tissues, and visualized and compared the distribution of erlotinib within the skin using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). The tissue concentration of erlotinib was also measured by liquid chromatography-tandem mass spectrometry (LC–MS/MS) with laser microdissection. PMID:29719624

Development of endosperm transfer cells in barley.

PubMed

Thiel, Johannes

2014-01-01

Endosperm transfer cells (ETCs) are positioned at the intersection of maternal and filial tissues in seeds of cereals and represent a bottleneck for apoplasmic transport of assimilates into the endosperm. Endosperm cellularization starts at the maternal-filial boundary and generates the highly specialized ETCs. During differentiation barley ETCs develop characteristic flange-like wall ingrowths to facilitate effective nutrient transfer. A comprehensive morphological analysis depicted distinct developmental time points in establishment of transfer cell (TC) morphology and revealed intracellular changes possibly associated with cell wall metabolism. Embedded inside the grain, ETCs are barely accessible by manual preparation. To get tissue-specific information about ETC specification and differentiation, laser microdissection (LM)-based methods were used for transcript and metabolite profiling. Transcriptome analysis of ETCs at different developmental stages by microarrays indicated activated gene expression programs related to control of cell proliferation and cell shape, cell wall and carbohydrate metabolism reflecting the morphological changes during early ETC development. Transporter genes reveal distinct expression patterns suggesting a switch from active to passive modes of nutrient uptake with the onset of grain filling. Tissue-specific RNA-seq of the differentiating ETC region from the syncytial stage until functionality in nutrient transfer identified a high number of novel transcripts putatively involved in ETC differentiation. An essential role for two-component signaling (TCS) pathways in ETC development of barley emerged from this analysis. Correlative data provide evidence for abscisic acid and ethylene influences on ETC differentiation and hint at a crosstalk between hormone signal transduction and TCS phosphorelays. Collectively, the data expose a comprehensive view on ETC development, associated pathways and identified candidate genes for ETC specification.

Development of endosperm transfer cells in barley

PubMed Central

Thiel, Johannes

2014-01-01

Endosperm transfer cells (ETCs) are positioned at the intersection of maternal and filial tissues in seeds of cereals and represent a bottleneck for apoplasmic transport of assimilates into the endosperm. Endosperm cellularization starts at the maternal-filial boundary and generates the highly specialized ETCs. During differentiation barley ETCs develop characteristic flange-like wall ingrowths to facilitate effective nutrient transfer. A comprehensive morphological analysis depicted distinct developmental time points in establishment of transfer cell (TC) morphology and revealed intracellular changes possibly associated with cell wall metabolism. Embedded inside the grain, ETCs are barely accessible by manual preparation. To get tissue-specific information about ETC specification and differentiation, laser microdissection (LM)-based methods were used for transcript and metabolite profiling. Transcriptome analysis of ETCs at different developmental stages by microarrays indicated activated gene expression programs related to control of cell proliferation and cell shape, cell wall and carbohydrate metabolism reflecting the morphological changes during early ETC development. Transporter genes reveal distinct expression patterns suggesting a switch from active to passive modes of nutrient uptake with the onset of grain filling. Tissue-specific RNA-seq of the differentiating ETC region from the syncytial stage until functionality in nutrient transfer identified a high number of novel transcripts putatively involved in ETC differentiation. An essential role for two-component signaling (TCS) pathways in ETC development of barley emerged from this analysis. Correlative data provide evidence for abscisic acid and ethylene influences on ETC differentiation and hint at a crosstalk between hormone signal transduction and TCS phosphorelays. Collectively, the data expose a comprehensive view on ETC development, associated pathways and identified candidate genes for ETC specification. PMID:24723929

Enabling high-precision nonlinear three-dimensional photoprocessing of premeditated designs on a conventional multiphoton imaging system

NASA Astrophysics Data System (ADS)

Garsha, Karl E.

2004-06-01

There is an increasing amount of interest in functionalized microstructural, microphotonic and microelectromechanical systems (MEMS) for use in biological applications. By scanning a tightly focused ultra-short pulsed laser beam inside a wide variety of commercially available polymer systems, the flexibility of the multiphoton microscope can be extended to include routine manufacturing of micro-devices with feature sizes well below the diffraction limit. Compared with lithography, two-photon polymerization has the unique ability to additively realize designs with high resolution in three dimensions; this permits the construction of cross-linked components and structures with hollow cavities. In light of the increasing availability of multiphoton imaging systems at research facilities, femtosecond laser manufacturing becomes particularly attractive in that the modality provides a readily accessible, rapid and high-accuracy 3-D processing capability to biological investigators interested in culture scaffolds and biomimetic tissue engineering, bio-MEMS, biomicrophotonics and microfluidics applications. This manuscript overviews recent efforts towards to enabling user accessible 3-D micro-manufacturing capabilities on a conventional proprietary-based imaging system. Software which permits the off-line design of microstructures and leverages the extensibility of proprietary LCSM image acquisition software to realize designs is introduced. The requirements for multiphoton photo-disruption (ablation) are in some ways analogous to those for multiphoton polymerization. Hence, "beam-steering" also facilitates precision photo-disruption of biological tissues with 3-D resolution, and applications involving tissue microdissection and intracellular microsurgery or three-dimensionally resolved fluorescence recovery after photobleaching (FRAP) studies can benefit from this work as well.

CD90/THY1 is over-expressed in prostate cancer-associated fibroblasts and could serve as a cancer biomarker

PubMed Central

True, Lawrence D; Zhang, Hui; Ye, Mingliang; Huang, Chung-Ying; Nelson, Peter S; von Haller, Priska D; Tjoelker, Larry W; Kim, Jong-Seo; Qian, Wei-Jun; Smith, Richard D; Ellis, William J; Liebeskind, Emily S; Liu, Alvin Y

2010-01-01

A by-product in the processing of prostate tissue for cell sorting by collagenase digestion is the media supernatant that remains after the cells are harvested. These supernatants contain proteins made by the cells within the tissue. Quantitative proteomic analysis of N-glycosylated proteins detected an increased amount of CD90/THY1 in cancer supernatants compared to non-cancer supernatants. Immunohistochemistry showed that in all carcinomas, regardless of Gleason grade, a layer of CD90-positive stromal fibroblastic cells, approximately 5-to-10 cells deep, was localized to tumor glands. In contrast, a no more than 1-cell wide girth of CD90-positive stromal cells was found around benign glands. The increased number of CD90-positive stromal cells in cancer correlated with overexpression of CD90 mRNA detected by gene expression analysis of stromal cells obtained by laser-capture microdissection. There is increasing evidence that cancer-associated stroma plays a role in both tumor progression and carcinogenesis. Most experiments to identify cancer biomarkers have focused on the cancer cells. CD90, being a marker for prostate cancer-associated stroma, might be a potential biomarker for this cancer. A non-invasive test could be provided by a urine test. Proteomic analysis of urine from patients with prostate cancer identified CD90; conversely, CD90 was not detected in the urine of post-prostatectomy patients. Furthermore, this urinary CD90 protein was a variant CD90 protein not known to be expressed by such cells as lymphocytes that express CD90. These CD90 results were obtained from ∼90 cases consisting of proteomic analysis of tissue and urine, immunohistochemistry, Western blot analysis of tissue media, flow cytometry of cells from digested tissue, and reverse transcriptase polymerase chain reaction analysis of isolated stromal cells. PMID:20562849

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

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.

The clonal origin and clonal evolution of epithelial tumours

PubMed Central

Garcia, Sergio Britto; Novelli, Marco; Wright, Nicholas A

2000-01-01

While the origin of tumours, whether from one cell or many, has been a source of fascination for experimental oncologists for some time, in recent years there has been a veritable explosion of information about the clonal architecture of tumours and their antecedents, stimulated, in the main, by the ready accessibility of new molecular techniques. While most of these new results have apparently confirmed the monoclonal origin of human epithelial (and other) tumours, there are a significant number of studies in which this conclusion just cannot be made. Moreover, analysis of many articles show that the potential impact of such considerations as patch size and clonal evolution on determinations of clonality have largely been ignored, with the result that a number of these studies are confounded. However, the clonal architecture of preneoplastic lesions provide some interesting insights — many lesions which might have been hitherto regarded as hyperplasias are apparently clonal in derivation. If this is indeed true, it calls into some question our hopeful corollary that a monoclonal origin presages a neoplastic habitus. Finally, it is clear, for many reasons, that methods of analysis which involve the disaggregation of tissues, albeit microdissected, are far from ideal and we should be putting more effort into techniques where the clonal architecture of normal tissues, preneoplastic and preinvasive lesions and their derivative tumours can be directly visualized in situ. PMID:10762440

The Successful Diagnosis and Typing of Systemic Amyloidosis Using A Microwave-Assisted Filter-Aided Fast Sample Preparation Method and LC/MS/MS Analysis

PubMed Central

Zou, Lili; Shen, Kaini; Zhong, Dingrong; Zhou, Daobin; Sun, Wei; Li, Jian

2015-01-01

Laser microdissection followed by mass spectrometry has been successfully used for amyloid typing. However, sample contamination can interfere with proteomic analysis, and overnight digestion limits the analytical throughput. Moreover, current quantitative analysis methods are based on the spectrum count, which ignores differences in protein length and may lead to misdiagnoses. Here, we developed a microwave-assisted filter-aided sample preparation (maFASP) method that can efficiently remove contaminants with a 10-kDa cutoff ultrafiltration unit and can accelerate the digestion process with the assistance of a microwave. Additionally, two parameters (P- and D-scores) based on the exponentially modified protein abundance index were developed to define the existence of amyloid deposits and those causative proteins with the greatest abundance. Using our protocol, twenty cases of systemic amyloidosis that were well-typed according to clinical diagnostic standards (training group) and another twenty-four cases without subtype diagnoses (validation group) were analyzed. Using this approach, sample preparation could be completed within four hours. We successfully subtyped 100% of the cases in the training group, and the diagnostic success rate in the validation group was 91.7%. This maFASP-aided proteomic protocol represents an efficient approach for amyloid diagnosis and subtyping, particularly for serum-contaminated samples. PMID:25984759

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...

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.

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.

Ovine secondary follicles vitrified out the ovarian tissue grow and develop in vitro better than those vitrified into the ovarian fragments.

PubMed

Lunardi, Franciele Osmarini; de Aguiar, Francisco Leo Nascimento; Duarte, Ana Beatriz Graça; Araújo, Valdevane Rocha; de Lima, Laritza Ferreira; Ribeiro de Sá, Naiza Arcângela; Vieira Correia, Hudson Henrique; Domingues, Sheyla Farhayldes Souza; Campello, Cláudio Cabral; Smitz, Johan; de Figueiredo, José Ricardo; Ribeiro Rodrigues, Ana Paula

2016-04-15

Cryopreservation of preantral follicles is a promising technique to preserve female fertility. The aim of this study was to evaluate the effect of vitrification on the development of secondary follicles included in ovarian tissue or isolated after microdissection. An important end point included is the capacity of grown oocytes to resume meiosis. Sheep ovarian cortexes were cut into fragments and split into three different groups: (1) fresh (control): secondary follicles isolated without any previous vitrification; (2) follicle-vitrification (follicle-vit): secondary follicles vitrified in isolated form; and (3) tissue-vitrification (tissue-vit): secondary follicles vitrified within fragments of ovarian tissue (in situ former) and subsequently subjected to isolation. From the three groups, isolated secondary follicles were submitted to IVC for 18 days. After IVC, cumulus-oocyte complexes (COCs) were harvested from follicles. As an additional control group, in vivo grown, in vivo-grown COCs were collected from antral ovarian follicles. All, recovered COCs were matured and the chromatin configuration was evaluated. Data were analyzed by ANOVA, and the means were compared by Student-Newman-Keuls test, and by chi-square. Differences were considered to be significant when P < 0.05. Isolated preantral follicles from all treatments had normal morphology, antrum formation, and low follicle degeneration after IVC. The growth rate between control and follicle-vit did not differ (P > 0.05), and was higher (P < 0.05) than for tissue-vit. The percentage of follicles that decreased diameter during IVC was significantly higher in tissue-vit than the in follicle-vit. Recovery rate of oocytes from normal follicles was higher in follicle-vit than in tissue-vit. Furthermore, oocyte viability was lower in tissue-vit than other treatments, and follicle-vit did not differ from control and in vivo grown. The percentage of oocytes meiosis resuming was not different between treatments except for in vivo grown. After vitrification, only follicle-vit showed metaphase I oocyte. We conclude that secondary follicles vitrified after isolation displayed a better follicular growth rate, oocyte viability, percentage of oocytes reaching the metaphase I stage, and fewer follicles with decreased diameter after IVC. Copyright © 2016 Elsevier Inc. All rights reserved.

[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.

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

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.

Acute EBV infection masquerading as "In-situ Follicular Lymphoma": a pitfall in the differential diagnosis of this entity.

PubMed

Gru, Alejandro A; Kreisel, Friederike; Duncavage, Eric; Nguyen, TuDung T; Hassan, Anjum; Frater, John L

2013-06-19

We present the case of a 30 year-old man who was referred for evaluation of diffuse lymphadenopathy. Six weeks prior, he noticed darkening of his urine associated with pale stools, nausea and an eventual 30 lb weight loss within a month. The initial laboratory findings showed elevation of the liver enzymes. A CT scan showed mesenteric and periaortic lymphadenopathy with the largest lymph node measuring 2.8 cm. Other laboratory results were otherwise unremarkable (including a normal LDH) with the exception of positive serum antibodies against Epstein-Barr virus (EBV) associated antigens (IgM+ and IgG+). An excisional biopsy of 4 of the small neck lymph nodes showed a normal architecture with prominent follicles and an intact capsule. But, by immunohistochemistry two of the follicles showed aberrant coexpression of BCL-2, in addition to CD10 and BCL-6. In-situ hybridization for early Epstein-Barr virus mRNA (EBER) and immunohistochemistry for latent membrane protein-1 (LMP-1) stained both scattered positive cells, as well as BCL-2 positive B-cells. Although an original diagnosis of in-situ follicular lymphoma was favored at an outside facility, additional interphase fluorescence in situ hybridization (FISH) studies for t(14;18);(IGH-BCL2) rearrangement (performed on the BCL-2 + follicles microdissected from the tissue block; Abott probe dual colour fusion) and molecular studies (IGH gene rearrangement by PCR, also performed on the microdissected follicles) were negative. Serologic studies (positive EBV antibodies) and immunostains in conjunction with the molecular studies confirmed the reactive nature of the changes. Our case also shows direct immunopathogenic evidence of BCL-2 expression among the EBV-infected cells, which has to our knowledge not been previously documented in vivo. A diagnosis of EBV infection should, therefore, be considered when confronted with BCL-2 expression in germinal centers, particularly in younger individuals, as the diagnosis of FLIS may lead to extensive and invasive haematologic work-ups. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1323656318940068.

Laryngeal mucosa elasticity and viscosity in high and low relative air humidity.

PubMed

Hemler, R J; Wieneke, G H; Lebacq, J; Dejonckere, P H

2001-03-01

In earlier work we showed that low relative humidity (RH) of inhaled air causes acoustic voice parameters such as jitter and shimmer to deteriorate. Other authors have shown negative effects on vocal efficiency. To explain these changes in the mechanical properties of the vocal folds, the effects of changes in RH of the air passing over microdissected mucosa of sheep larynges were studied. The dissected surface of the tissue specimen just touched Ringer solution and air of varying RH was blown over the specimen. The mucosa specimen was subjected to sinusoidal oscillations of length (strain) and the resulting force (stress) was measured. The gain and phase angle between the imposed strain and resulting stress were measured, and elasticity and viscosity were calculated. Two different air conditions were tested: air with high RH (100%) vs air with low RH (0%). Viscosity and stiffness increased significantly in both ambient conditions (P < 0.01). Dry dehydrating air resulted in a stiffer and more viscous cover than humid air (P < 0.001). These changes in mechanical characteristics may contribute to the effects on voice parameters described in earlier work.

Primary Glomerulonephritis with Unique C4d Deposition and Concurrent Non-infectious Intermediate Uveitis: a Case Report and Literature Review

PubMed Central

2018-01-01

C4 glomerulopathy is a recently introduced entity that presents with bright C4d staining and minimal or absent immunoglobulin and C3 staining. We report a case of a 62-year-old man with C4 glomerulonephritis (GN) and uveitis. He presented to the nephrology department with proteinuria and hematuria. The patient also had intermediate uveitis along with proteinuria and hematuria. A kidney biopsy that was performed in light of continuing proteinuria and hematuria showed a focal proliferative, focal sclerotic glomerulopathy pattern on light microscopy, absent staining for immunoglobulin or C3 by immunofluorescence microscopy, with bright staining for C4d on immunohistochemistry, and electron-dense deposits on electron microscopy. Consequently, C4 GN was suggested as the pathologic diagnosis. Although laser microdissection and mass spectrometry for glomerular deposit and pathologic evaluation of the retinal tissue were not performed, this is the first report of C4 GN in Korea and the first case of coexisting C4 GN and uveitis in the English literature. PMID:29713256

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

Spatio-Temporal Dynamics of Fructan Metabolism in Developing Barley Grains[W

PubMed Central

Peukert, Manuela; Thiel, Johannes; Peshev, Darin; Weschke, Winfriede; Van den Ende, Wim; Mock, Hans-Peter; Matros, Andrea

2014-01-01

Barley (Hordeum vulgare) grain development follows a series of defined morphological and physiological stages and depends on the supply of assimilates (mainly sucrose) from the mother plant. Here, spatio-temporal patterns of sugar distributions were investigated by mass spectrometric imaging, targeted metabolite analyses, and transcript profiling of microdissected grain tissues. Distinct spatio-temporal sugar balances were observed, which may relate to differentiation and grain filling processes. Notably, various types of oligofructans showed specific distribution patterns. Levan- and graminan-type oligofructans were synthesized in the cellularized endosperm prior to the commencement of starch biosynthesis, while during the storage phase, inulin-type oligofructans accumulated to a high concentration in and around the nascent endosperm cavity. In the shrunken endosperm mutant seg8, with a decreased sucrose flux toward the endosperm, fructan accumulation was impaired. The tight partitioning of oligofructan biosynthesis hints at distinct functions of the various fructan types in the young endosperm prior to starch accumulation and in the endosperm transfer cells that accomplish the assimilate supply toward the endosperm at the storage phase. PMID:25271242

Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target.

PubMed

Apps, John R; Carreno, Gabriela; Gonzalez-Meljem, Jose Mario; Haston, Scott; Guiho, Romain; Cooper, Julie E; Manshaei, Saba; Jani, Nital; Hölsken, Annett; Pettorini, Benedetta; Beynon, Robert J; Simpson, Deborah M; Fraser, Helen C; Hong, Ying; Hallang, Shirleen; Stone, Thomas J; Virasami, Alex; Donson, Andrew M; Jones, David; Aquilina, Kristian; Spoudeas, Helen; Joshi, Abhijit R; Grundy, Richard; Storer, Lisa C D; Korbonits, Márta; Hilton, David A; Tossell, Kyoko; Thavaraj, Selvam; Ungless, Mark A; Gil, Jesus; Buslei, Rolf; Hankinson, Todd; Hargrave, Darren; Goding, Colin; Andoniadou, Cynthia L; Brogan, Paul; Jacques, Thomas S; Williams, Hywel J; Martinez-Barbera, Juan Pedro

2018-05-01

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. β-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.

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.

Congestive Heart Failure Leads to Prolongation of the PR Interval and Atrioventricular Junction Enlargement and Ion Channel Remodelling in the Rabbit

PubMed Central

Nikolaidou, Theodora; Cai, Xue J.; Stephenson, Robert S.; Yanni, Joseph; Lowe, Tristan; Atkinson, Andrew J.; Jones, Caroline B.; Sardar, Rida; Corno, Antonio F.; Dobrzynski, Halina; Withers, Philip J.; Jarvis, Jonathan C.; Hart, George; Boyett, Mark R.

2015-01-01

Heart failure is a major killer worldwide. Atrioventricular conduction block is common in heart failure; it is associated with worse outcomes and can lead to syncope and bradycardic death. We examine the effect of heart failure on anatomical and ion channel remodelling in the rabbit atrioventricular junction (AVJ). Heart failure was induced in New Zealand rabbits by disruption of the aortic valve and banding of the abdominal aorta resulting in volume and pressure overload. Laser micro-dissection and real-time polymerase chain reaction (RT-PCR) were employed to investigate the effects of heart failure on ion channel remodelling in four regions of the rabbit AVJ and in septal tissues. Investigation of the AVJ anatomy was performed using micro-computed tomography (micro-CT). Heart failure animals developed first degree heart block. Heart failure caused ventricular myocardial volume increase with a 35% elongation of the AVJ. There was downregulation of HCN1 and Cx43 mRNA transcripts across all regions and downregulation of Cav1.3 in the transitional tissue. Cx40 mRNA was significantly downregulated in the atrial septum and AVJ tissues but not in the ventricular septum. mRNA abundance for ANP, CLCN2 and Navβ1 was increased with heart failure; Nav1.1 was increased in the inferior nodal extension/compact node area. Heart failure in the rabbit leads to prolongation of the PR interval and this is accompanied by downregulation of HCN1, Cav1.3, Cx40 and Cx43 mRNAs and anatomical enlargement of the entire heart and AVJ. PMID:26509807

Elevated osteopontin and thrombospondin expression identifies malignant human breast carcinoma but is not indicative of metastatic status

PubMed Central

Wang-Rodriguez, Jessica; Urquidi, Virginia; Rivard, Amber; Goodison, Steve

2003-01-01

Background Our previous characterization of a human breast tumor metastasis model identified several candidate metastasis genes. The expression of osteopontin (OPN) correlated with the metastatic phenotype, whereas thrombospondin-1 (TSP-1) and tyrosinase-related protein-1 (TYRP-1) correlated with the nonmetastatic phenotype of independent MDA-MB-435 cell lines implanted orthotopically into athymic mice. The aim of the present study was to examine the cellular distribution of these molecules in human breast tissue and to determine whether the relative expression level of these three genes is associated with human breast tumor metastasis. Methods Sixty-eight fresh, frozen specimens including 31 primary infiltrating ductal carcinomas, 22 nodal metastases, 10 fibroadenomas, and five normal breast tissues were evaluated for OPN expression, TSP-1 expression and TYRP-1 expression. Immunohistochemistry was performed to monitor the cellular distribution and to qualitatively assess expression. Quantitative analysis was achieved by enrichment of breast epithelial cells using laser-capture microdissection and subsequent real-time, quantitative PCR. Results The epithelial components of the breast tissue were the source of OPN and TSP-1 expression, whereas TYRP-1 was present in both the epithelial and stromal components. Both OPN and TSP-1 expression were significantly higher in malignant epithelial sources over normal and benign epithelial sources, but no difference in expression levels was evident between primary tumors with or without metastases, nor between primary and metastatic carcinomas. Conclusion Elevated expression of OPN and TSP-1 may play a role in the pathogenesis of breast cancer. The multiplex analysis of these molecules may enhance our ability to diagnose and/or prognosticate human breast malignancy. PMID:12927044

Functional, Anatomical, and Molecular Investigation of the Cardiac Conduction System and Arrhythmogenic Atrioventricular Ring Tissue in the Rat Heart

PubMed Central

Atkinson, Andrew J.; Logantha, Sunil Jit R. J.; Hao, Guoliang; Yanni, Joseph; Fedorenko, Olga; Sinha, Aditi; Gilbert, Stephen H.; Benson, Alan P.; Buckley, David L.; Anderson, Robert H.; Boyett, Mark R.; Dobrzynski, Halina

2013-01-01

Background The cardiac conduction system consists of the sinus node, nodal extensions, atrioventricular (AV) node, penetrating bundle, bundle branches, and Purkinje fibers. Node‐like AV ring tissue also exists at the AV junctions, and the right and left rings unite at the retroaortic node. The study aims were to (1) construct a 3‐dimensional anatomical model of the AV rings and retroaortic node, (2) map electrical activation in the right ring and study its action potential characteristics, and (3) examine gene expression in the right ring and retroaortic node. Methods and Results Three‐dimensional reconstruction (based on magnetic resonance imaging, histology, and immunohistochemistry) showed the extent and organization of the specialized tissues (eg, how the AV rings form the right and left nodal extensions into the AV node). Multiextracellular electrode array and microelectrode mapping of isolated right ring preparations revealed robust spontaneous activity with characteristic diastolic depolarization. Using laser microdissection gene expression measured at the mRNA level (using quantitative PCR) and protein level (using immunohistochemistry and Western blotting) showed that the right ring and retroaortic node, like the sinus node and AV node but, unlike ventricular muscle, had statistically significant higher expression of key transcription factors (including Tbx3, Msx2, and Id2) and ion channels (including HCN4, Cav3.1, Cav3.2, Kv1.5, SK1, Kir3.1, and Kir3.4) and lower expression of other key ion channels (Nav1.5 and Kir2.1). Conclusions The AV rings and retroaortic node possess gene expression profiles similar to that of the AV node. Ion channel expression and electrophysiological recordings show the AV rings could act as ectopic pacemakers and a source of atrial tachycardia. PMID:24356527

Maternal smoke exposure decreases mesenchymal proliferation and modulates Rho-GTPase-dependent actin cytoskeletal signaling in fetal lungs.

PubMed

Unachukwu, Uchenna; Trischler, Jordis; Goldklang, Monica; Xiao, Rui; D'Armiento, Jeanine

2017-06-01

The present study tested the hypothesis that maternal smoke exposure results in fetal lung growth retardation due to dysregulation in various signaling pathways, including the Wnt (wingless-related integration site)/β-catenin pathway. Pregnant female C57BL/6J mice were exposed to cigarette smoke (100-150 mg/m 3 ) or room air, and offspring were humanely killed on 12.5, 14.5, 16.5, and 18.5 d post coitum (dpc). We assessed lung stereology with Cavalieri estimation; apoptosis with proliferating cell nuclear antigen, TUNEL, and caspase assays; and gene expression with quantitative PCR (qPCR) and RNA sequencing on lung epithelium and mesenchyme retrieved by laser capture microdissection. Results demonstrated a significant decrease in body weight and lung volume of smoke-exposed embryos. At 16.5 dpc, the reduction in lung volume was due to loss of lung mesenchymal tissue correlating with a decrease in cell proliferation ( n = 10; air: 61.65% vs. smoke: 44.21%, P < 0.05). RNA sequence analysis demonstrated an alteration in the Wnt pathway, and qPCR confirmed an increased expression of secreted frizzled-related protein 1 (sFRP-1) [ n = 12; relative quantification (RQ) 1 vs. 2.33, P < 0.05] and down-regulation of Cyclin D1 ( n = 7; RQ 1 vs. 0.61, P < 0.05) in mesenchymal tissue. Furthermore, genome expression studies revealed a smoke-induced up-regulation of Rho-GTPase-dependent actin cytoskeletal signaling that can lead to loss of tissue integrity.-Unachukwu, U., Trischler, J., Goldklang, M., Xiao, R., D'Armiento, J. Maternal smoke exposure decreases mesenchymal proliferation and modulates Rho-GTPase-dependent actin cytoskeletal signaling in fetal lungs. © FASEB.

Bathing in carbon dioxide-enriched water alters protein expression in keratinocytes of skin tissue in rats.

PubMed

Kälsch, Julia; Pott, Leona L; Takeda, Atsushi; Kumamoto, Hideo; Möllmann, Dorothe; Canbay, Ali; Sitek, Barbara; Baba, Hideo A

2017-04-01

Beneficial effects of balneotherapy using naturally occurring carbonated water (CO 2 enriched) have been known since the Middle Ages. Although this therapy is clinically applied for peripheral artery disease and skin disorder, the underlying mechanisms are not fully elucidated.Under controlled conditions, rats were bathed in either CO 2 -enriched water (CO 2 content 1200 mg/L) or tap water, both at 37 °C, for 10 min daily over 4 weeks. Proliferation activity was assessed by Ki67 immunohistochemistry of the epidermis of the abdomen. The capillary density was assessed by immunodetection of isolectin-positive cells. Using cryo-fixed abdominal skin epidermis, follicle cells and stroma tissue containing capillaries were separately isolated by means of laser microdissection and subjected to proteomic analysis using label-free technique. Differentially expressed proteins were validated by immunohistochemistry.Proliferation activity of keratinocytes was not significantly different in the epidermis after bathing in CO 2 -enriched water, and also, capillary density did not change. Proteomic analysis revealed up to 36 significantly regulated proteins in the analyzed tissue. Based on the best expression profiles, ten proteins were selected for immunohistochemical validation. Only one protein, far upstream element binding protein 2 (FUBP2), was similarly downregulated in the epidermis after bathing in CO 2 -enriched water with both techniques. Low FUBP2 expression was associated with low c-Myc immune-expression in keratinocytes.Long-term bathing in CO 2 -enriched water showed a cellular protein response of epithelial cells in the epidermis which was detectable by two different methods. However, differences in proliferation activity or capillary density were not detected in the normal skin.

The potential role of myocardial serotonin receptor 2B expression in canine dilated cardiomyopathy.

PubMed

Fonfara, Sonja; Hetzel, Udo; Oyama, Mark A; Kipar, Anja

2014-03-01

Serotonin signalling in the heart is mediated by receptor subtype 2B (5-HTR2B). A contribution of serotonin to valvular disease has been reported, but myocardial expression of 5-HTR2B and its role in canine dilated cardiomyopathy (DCM) is not known. The aim of the present study was to investigate myocardial 5-HTR2B mRNA expression in dogs with DCM and to correlate results with expression of markers for inflammation and remodelling. Myocardial samples from eight healthy dogs, four dogs with DCM, five with cardiac diseases other than DCM and six with systemic non-cardiac diseases were investigated for 5-HTR2B mRNA expression using quantitative PCR (qPCR). The results were compared to mRNA expression of selected cytokines, matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinase (TIMP). Laser microdissection with subsequent qPCR and immunohistochemistry were employed to identify the cells expressing 5-HTR2B. The myocardium of control dogs showed constitutive 5-HTR2B mRNA expression. In dogs with DCM, 5-HTR2B mRNA values were significantly greater than in all other groups, with highest levels of expression in the left ventricle and right atrium. Myocytes were identified as the source of 5-HTR2B mRNA and protein. A significant positive correlation of 5-HTR2B mRNA with expression of several cytokines, MMPs and TIMPs was observed. The findings suggest that serotonin might play a role in normal cardiac structure and function and could contribute to myocardial remodelling and functional impairment in dogs with DCM. Copyright © 2013 Elsevier Ltd. All rights reserved.

Ovarian mucinous tumors arising from mature cystic teratomas--a molecular genetic approach for understanding the cellular origin.

PubMed

Fujii, Kaho; Yamashita, Yoriko; Yamamoto, Toshimichi; Takahashi, Koji; Hashimoto, Katsunori; Miyata, Tomoko; Kawai, Kumi; Kikkawa, Fumitaka; Toyokuni, Shinya; Nagasaka, Tetsuro

2014-04-01

Mucinous tumors of the ovary are frequently associated with mature cystic teratomas, and it has been speculated that the mucinous tumors arise from teratoma components. The cellular origins of mature cystic teratomas are believed to be post-meiotic ovarian germ cells, and the analysis of microsatellite markers such as short tandem repeats is suitable for determining the cellular origin of tumors. In this study, we analyzed 3 ovarian mature cystic teratomas, all of which were associated with simultaneous ovarian mucinous tumors within the same ovary. Two of the 3 mucinous tumors were intestinal-type and the other was endocervical type. A laser capture microdissection technique was used to separate the epithelial component of the mucinous tumor, the components of the mature cystic teratoma, and control ovarian somatic tissue. Using short tandem repeat analysis based on 6 markers (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, and D17S1290), we could distinguish the germ cell (homozygous) or somatic (heterozygous) origin of a given component in each sample. The epithelial components of the intestinal-type mucinous tumors in cases 1 and 2 were homozygous, and the epithelial component in case 3 (endocervical type) was heterozygous. All teratomatous components were homozygous, and the control components were heterozygous. In addition, we analyzed 3 mature cystic teratomas without mucinous tumors, and all 3 were homozygous in the tumor component. Our data suggest that the origin of mucinous tumors in the ovary may differ among histological subtypes, and intestinal-type mucinous tumors may arise from mature cystic teratomas, although endocervical-type mucinous tumors may not. Copyright © 2014 Elsevier Inc. All rights reserved.

Somatic GNAS Mutation Causes Widespread and Diffuse Pituitary Disease in Acromegalic Patients with McCune-Albright Syndrome

PubMed Central

Vortmeyer, Alexander O.; Gläsker, Sven; Mehta, Gautam U.; Abu-Asab, Mones S.; Smith, Jonathan H.; Zhuang, Zhengping; Collins, Michael T.

2012-01-01

Context: McCune-Albright syndrome (MAS) is caused by sporadic mutations of the GNAS. Patients exhibit features of acromegaly. In most patients, GH-secreting pituitary adenomas have been held responsible for this presentation. However, surgical adenomectomy rarely eliminates excess GH production. Objective: The aim of this study was to elucidate pituitary pathology in patients with MAS and to explain the basis of failure of adenomectomy to eliminate GH hypersecretion. Design and Setting: We conducted a case series at the National Institutes of Health. Intervention(s): Interventions included medical therapy and transsphenoidal surgery. Patients and Main Outcome Measures: We studied clinical and imaging features and the histology and molecular features of the pituitary of four acromegalic MAS patients. Results: We identified widespread and diffuse pituitary gland disease. The primary pathological changes were characterized by hyperplastic and neoplastic change, associated with overrepresentation of somatotroph cells in structurally intact tissue areas. Genetic analysis of multiple microdissected samples of any type of histological area consistently revealed identical GNAS mutations in individual patients. The only patient with remission after surgery received complete hypophysectomy in addition to removal of multiple GH-secreting tumors. Conclusions: These findings indicate developmental effects of GNAS mutation on the entire anterior pituitary gland. The pituitary of individual cases contains a spectrum of changes with regions of normal appearing gland, hyperplasia, and areas of fully developed adenoma formation, as well as transitional stages between these entities. The primary change underlying acromegaly in MAS patients is somatotroph hyperplasia involving the entire pituitary gland, with or without development of somatotroph adenoma. Thus, successful clinical management, whether it is medical, surgical, or via irradiation, must target the entire pituitary, not just the adenomas evident on imaging. PMID:22564667

Transcriptional response to hypoxic stress in melanoma and prognostic potential of GBE1 and BNIP3.

PubMed

Buart, Stéphanie; Terry, Stéphane; Noman, Muhammad Z; Lanoy, Emilie; Boutros, Céline; Fogel, Paul; Dessen, Philippe; Meurice, Guillaume; Gaston-Mathé, Yann; Vielh, Philippe; Roy, Séverine; Routier, Emilie; Marty, Virginie; Ferlicot, Sophie; Legrès, Luc; Bouchtaoui, Morad El; Kamsu-Kom, Nyam; Muret, Jane; Deutsch, Eric; Eggermont, Alexander; Soria, Jean-Charles; Robert, Caroline; Chouaib, Salem

2017-12-12

Gradients of hypoxia occur in most solid tumors and cells found in hypoxic regions are associated with the most aggressive and therapy-resistant fractions of the tumor. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia in melanoma. Using microarray technology, whole genome gene expression profiling was first performed on established melanoma cell lines. From gene set enrichment analyses, we derived a robust 35 probes signature (hypomel for HYPOxia MELanoma) associated with hypoxia-response pathways, including 26 genes up regulated, and 9 genes down regulated. The microarray data were validated by RT-qPCR for the 35 transcripts. We then validated the signature in hypoxic zones from 8 patient specimens using laser microdissection or macrodissection of Formalin fixed-paraffin-embedded (FFPE) material, followed with RT-qPCR. Moreover, a similar hypoxia-associated gene expression profile was observed using NanoString technology to analyze RNAs from FFPE melanoma tissues of a cohort of 19 patients treated with anti-PD1. Analysis of NanoString data from validation sets using Non-Negative Matrix Factorization (NMF) analysis (26 genes up regulated in hypoxia) and dual clustering (samples and genes) further revealed that the increased level of BNIP3 (Bcl-2 adenovirus E1B 19 kDa-interacting protein 3)/GBE1 (glycogen branching enzyme1) differential pair correlates with the lack of response of melanoma patients to anti-PD1 (pembrolizumab) immunotherapy. These studies suggest that through elevated glycogenic flux and induction of autophagy, hypoxia is a critical molecular program that could be considered as a prognostic factor for melanoma.

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...

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 ...

BRAF mutation is unusual in chronic lymphocytic thyroiditis-associated papillary thyroid carcinomas and absent in non-neoplastic nuclear atypia of thyroiditis.

PubMed

Sargent, Rachel; LiVolsi, Virginia; Murphy, Justin; Mantha, Geeta; Hunt, Jennifer L

2006-01-01

Chronic lymphocytic thyroiditis (CLT) has an epidemiological relationship to papillary thyroid carcinoma (PTC). The follicular epithelium in CLT can be markedly atypical, with cytologic changes ranging from oncocytic morphology to clearing and overlapping. At the molecular level, the association between CLT and PTC is more controversial. In order to further characterize the molecular changes in CLT, this study examined the BRAF gene in 27 patient samples with CLT and without carcinoma and 28 samples with CLT and carcinoma (12 conventional papillary carcinomas, 13 follicular variants, and 3 tall cell variants). Microdissection, PCR, and sequencing of exon 15 of the BRAF gene were performed. BRAF mutations were uncommon in the cases studied with only two microscopic and two clinically sized PTCs had BRAF mutations (14%). There was no evidence of BRAF mutation in any of the areas with atypical follicular epithelium in CLT. These data suggest that BRAF is a less frequent mechanism of tumorigenesis in a background of CLT and that BRAF mutation is not present in the atypical follicular epithelium of CLT.

Identification of serum angiopoietin-2 as a biomarker for clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy.

PubMed

Goede, V; Coutelle, O; Neuneier, J; Reinacher-Schick, A; Schnell, R; Koslowsky, T C; Weihrauch, M R; Cremer, B; Kashkar, H; Odenthal, M; Augustin, H G; Schmiegel, W; Hallek, M; Hacker, U T

2010-10-26

The combination of chemotherapy with the vascular endothelial growth factor (VEGF) antibody bevacizumab is a standard of care in advanced colorectal cancer (CRC). However, biomarkers predicting outcome of bevacizumab-containing treatment are lacking. As angiopoietin-2 (Ang-2) is a key regulator of vascular remodelling in concert with VEGF, we investigated its role as a biomarker in metastatic CRC. Serum Ang-2 levels were measured in 33 healthy volunteers and 90 patients with CRC. Of these, 34 had metastatic disease and received bevacizumab-containing therapy. To determine the tissue of origin of Ang-2, quantitative real-time PCR was performed on microdissected cryosections of human CRC and in a murine xenograft model of CRC using species-specific amplification. Ang-2 originated from the stromal compartment of CRC tissues. Serum Ang-2 levels were significantly elevated in patients with metastatic CRC compared with healthy controls. Amongst patients receiving bevacizumab-containing treatment, low pre-therapeutic serum Ang-2 levels were associated with a significant better response rate (82 vs 31%; P<0.01), a prolonged median progression-free survival (14.1 vs 8.5 months; P<0.01) and a reduction of 91% in the hazard of death (P<0.05). Serum Ang-2 is a candidate biomarker for outcome of patients with metastatic CRC treated with bevacizumab-containing therapy, and it should be further validated to customise combined chemotherapeutic and anti-angiogenic treatment.

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.

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 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

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

IL-10 -1082 SNP and IL-10 in primary CNS and vitreoretinal lymphomas.

PubMed

Ramkumar, Hema L; Shen, De Fen; Tuo, Jingsheng; Braziel, Rita M; Coupland, Sarah E; Smith, Justine R; Chan, Chi-Chao

2012-10-01

Most primary central nervous system lymphomas (PCNSLs) and primary vitreoretinal lymphomas (PVRLs) are B-cell lymphomas that produce high levels of interleukin (IL)-10, which is linked to rapid disease progression. The IL-10 (-1082) G → A polymorphism (IL-10 SNP) is associated with improved survival in certain non-CNS lymphoma patients. PDCD4 is a tumor suppressor gene and upstream regulator of IL-10. This study examined the correlation between the IL-10 SNP, PDCD4 mRNA expression, and IL-10 expression (at transcript and protein levels) in these lymphoma cells. Single-nucleotide polymorphism (SNP)-typing at IL-10 (-1082) was performed after microdissecting cytospun PVRL cells from 26 specimens. Vitreal IL-10 and IL-6 levels were measured by ELISA. PCNSL cells from 52 paraffin-embedded sections were microdissected and SNP typed on genomic DNA. RT-PCR was performed to analyze expression of IL-10 and PDCD4 mRNA. IL-10 (-1082) SNP typing was performed on blood samples of 96 healthy controls. We measured IL-10 (-1082) SNP expression in 26 PVRLs and 52 PCNSLs and examined its relationship with IL-10 protein and gene expression, respectively. More PVRL patients expressed one copy of the IL-10 ( -1082 )  G → A SNP with the GA genotype compared to controls. The frequencies of the three genotypes (AA, AG, GG) significantly differed in PVRL versus controls and in PCNSL versus controls. In PVRLs, the vitreal IL-10/IL-6 ratio was higher in IL-10 (-1082) AG and IL-10 (-1082) AA patients, compared to IL-10 (-1082) GG patients. IL-10 mRNA expression was higher in IL-10 (-1082) AG and IL-10 (-1082) AA PCNSLs, compared to IL-10 (-1082) GG PCNSLs. No correlation was found between IL-10 and PDCD4 expression levels in 37 PCNSL samples. PVRL and PCNSL patients had similar IL-10 (-1082) A allele frequencies, but genotype distributions differed from healthy controls. The findings suggest that the IL-10 (-1082) A allele is a risk factor for higher IL-10 levels in PVRLs and PCNSLs. Higher IL-10 levels have been correlated with more aggressive disease in both PVRLs and PCNSLs, making this finding an important and potentially clinically significant observation.

IL-10 -1082 SNP and IL-10 in primary CNS and vitreoretinal lymphomas

PubMed Central

Ramkumar, Hema L.; Shen, De Fen; Tuo, Jingsheng; Braziel, Rita M.; Coupland, Sarah E.; Smith, Justine R.

2012-01-01

Objectives Most primary central nervous system lymphomas (PCNSLs) and primary vitreoretinal lymphomas (PVRLs) are B-cell lymphomas that produce high levels of interleukin (IL)-10, which is linked to rapid disease progression. The IL-10-1082G→A polymorphism (IL-10 SNP) is associated with improved survival in certain non-CNS lymphoma patients. PDCD4 is a tumor suppressor gene and upstream regulator of IL-10. This study examined the correlation between the IL-10 SNP, PDCD4 mRNA expression, and IL-10 expression (at transcript and protein levels) in these lymphoma cells. Materials and methods Single-nucleotide polymorphism (SNP)-typing at IL-10-1082 was performed after micro-dissecting cytospun PVRL cells from 26 specimens. Vitreal IL-10 and IL-6 levels were measured by ELISA. PCNSL cells from 52 paraffin-embedded sections were microdissected and SNP typed on genomic DNA. RT-PCR was performed to analyze expression of IL-10 and PDCD4 mRNA. IL-10-1082 SNP typing was performed on blood samples of 96 healthy controls. We measured IL-10-1082 SNP expression in 26 PVRLs and 52 PCNSLs and examined its relationship with IL-10 protein and gene expression, respectively. Results More PVRL patients expressed one copy of the IL-10-1082G→A SNP with the GA genotype compared to controls. The frequencies of the three genotypes (AA, AG, GG) significantly differed in PVRL versus controls and in PCNSL versus controls. In PVRLs, the vitreal IL-10/IL-6 ratio was higher in IL-10-1082 AG and IL-10-1082 AA patients, compared to IL-10-1082 GG patients. IL-10 mRNA expression was higher in IL-10-1082 AG and IL-10-1082 AA PCNSLs, compared to IL-10-1082 GG PCNSLs. No correlation was found between IL-10 and PDCD4 expression levels in 37 PCNSL samples. Conclusions PVRL and PCNSL patients had similar IL-10-1082 A allele frequencies, but genotype distributions differed from healthy controls. The findings suggest that the IL-10-1082 A allele is a risk factor for higher IL-10 levels in PVRLs and PCNSLs. Higher IL-10 levels have been correlated with more aggressive disease in both PVRLs and PCNSLs, making this finding an important and potentially clinically significant observation. PMID:22628023

TS and ERCC-1 mRNA expressions and clinical outcome in patients with metastatic colon cancer in CONFIRM-1 and -2 clinical trials.

PubMed

Grimminger, P P; Shi, M; Barrett, C; Lebwohl, D; Danenberg, K D; Brabender, J; Vigen, C L P; Danenberg, P V; Winder, T; Lenz, H-J

2012-10-01

To validate established cutoff levels of thymidylate synthase (TS) and excision repair cross-complementing (ERCC-1) intratumoral mRNA expressions in tumor samples from metastatic colorectal cancer (mCRC) patients treated with PTK787/ZK222584 (PTK/ZK). From 122 samples of patients with mCRC enrolled in CONFIRM-1 (Colorectal Oral Novel Therapy for the Inhibition of Angiogenesis and Retarding of Metastases) or CONFIRM-2, mRNA was isolated of microdissected formalin-fixed paraffin-embedded samples and quantitated using TaqMan-based technology. Existing TS and ERCC-1 cutoff levels were tested for their prognostic value in first-line and second-line therapy. TS expression was associated with overall survival (OS) in first-line, but not second-line therapy. ERCC-1 was associated with OS in patients treated with first-line and second-line FOLFOX4. In first-line FOLFOX4, combination of high TS and/or high ERCC-1 was associated with shorter OS. A correlation was observed between ERCC-1 expression and benefit from PTK/ZK+FOLFOX4 treatment. TS and ERCC-1 expression is associated with clinical outcome in mCRC. Baseline TS and ERCC-1 levels may allow the selection of patients who benefit from FOLFOX4 chemotherapy.

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...

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...

An epigenetically derived monoclonal origin for recurrent respiratory papillomatosis.

PubMed

Stephen, Josena Kunjoonju; Vaught, Lori E; Chen, Kang Mei; Shah, Veena; Schweitzer, Vanessa G; Gardner, Glendon; Benninger, Michael S; Worsham, Maria J

2007-07-01

To investigate the contribution of promoter methylation-mediated epigenetic events in recurrent respiratory papillomatosis tumorigenesis. Archival tissue DNA, extracted from microdissected papilloma lesions, was interrogated for methylation status by means of the novel, multigene methylation-specific multiplex ligation-dependent probe amplification assay. Fifteen subjects with recurrent respiratory papillomatosis, 3 females and 12 males, all with adult onset of illness (age range, 23-73 years) except for 1 female patient with juvenile onset (1 year old). Promoter hypermethylation was recorded in 14 of 15 cases, and 19 of 22 unique methylation-prone cancer genes in the multigene panel had altered DNA methylation in at least 1 laryngeal papilloma biopsy specimen. Identical abnormally methylated genes were found in 5 of 15 recurrent cases, of which the CDKN2B gene was hypermethylated in all 5 cases. Dissimilar epigenetic events were noted in the remaining cases. A clonal origin was derived for 5 of 15 recurrent respiratory papillomatosis biopsy specimens based on identical epigenetic events. The high frequency of epigenetic events, characterized by consistent promoter hypermethylation of multiple tumor suppressor genes, points to the use of gene silencing mechanisms in the pathogenesis of recurrent respiratory papillomatosis.

Dilatational band formation in bone

PubMed Central

Poundarik, Atharva A.; Diab, Tamim; Sroga, Grazyna E.; Ural, Ani; Boskey, Adele L.; Gundberg, Caren M.; Vashishth, Deepak

2012-01-01

Toughening in hierarchically structured materials like bone arises from the arrangement of constituent material elements and their interactions. Unlike microcracking, which entails micrometer-level separation, there is no known evidence of fracture at the level of bone’s nanostructure. Here, we show that the initiation of fracture occurs in bone at the nanometer scale by dilatational bands. Through fatigue and indentation tests and laser confocal, scanning electron, and atomic force microscopies on human and bovine bone specimens, we established that dilatational bands of the order of 100 nm form as ellipsoidal voids in between fused mineral aggregates and two adjacent proteins, osteocalcin (OC) and osteopontin (OPN). Laser microdissection and ELISA of bone microdamage support our claim that OC and OPN colocalize with dilatational bands. Fracture tests on bones from OC and/or OPN knockout mice (OC−/−, OPN−/−, OC-OPN−/−;−/−) confirm that these two proteins regulate dilatational band formation and bone matrix toughness. On the basis of these observations, we propose molecular deformation and fracture mechanics models, illustrating the role of OC and OPN in dilatational band formation, and predict that the nanometer scale of tissue organization, associated with dilatational bands, affects fracture at higher scales and determines fracture toughness of bone. PMID:23129653

Isolation and characterization of 21 novel expressed DNA sequences from the distal region of human chromosome 4p

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

Ishida, Yoshikazu; Hadano, Shinji; Nagayama, Tomiko

1994-07-15

The authors have established an approach to the isolation of expressed DNA sequences from a defined region of the human chromosome. The method relies on the direct screening of cDNA libraries using pooled single-copy microclones generated by a laser chromosome microdissection in conjunction with a single unique primer polymerase chain reaction (SUP-PCR) procedure. They applied this method to the distal region of human chromosome 4p (4p15-4pter), which contains the Huntington disease (HD) and the Wolf-Hirschhorn syndrome (WHS) loci. Twenty-one nonoverlapping and region-specific cDNA clones encoding novel genes were isolated in this manner. Ten of 21 clones were subregionally assigned tomore » 4p16.1-4pter, and the remainder mapped to the region proximal to 4p16.1. Northern blot and reverse transcription followed by the PCR (RT-PCR) analysis revealed that 16 of these 21 clones detected transcripts in total RNA from human tissues. The method is applicable to other chromosomal regions and is a powerful approach to the isolation of region-specific cDNA clones. 44 refs., 3 figs., 3 tabs.« less

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

Coexpression of an unusual form of the EWS-WT1 fusion transcript and interleukin 2/15 receptor betamRNA in a desmoplastic small round cell tumour.

PubMed

Nakanishi, Y; Oinuma, T; Sano, M; Fuchinoue, F; Komatsu, K; Seki, T; Obana, Y; Tabata, M; Kikuchi, K; Shimamura, M; Ohmori, K; Nemoto, N

2006-10-01

The beta chain of the interleukin 2/15 receptor (IL-2/15Rbeta) is induced by the expression of the EWS-WT1. A case of desmoplastic small round cell tumour (DSRCT) expressing only an unusual EWS-WT1 treated by us is reported here. To characterise an unusual form of EWS-WT1. Frozen tissue sections of the axillary tumour were examined using a laser-assisted microdissection technique and reverse transcriptase polymerase chain reaction. The novel fusion of exon 8 of EWS and the defective exon 10 of WT1 (-KTS) was detected. Although it was an unusual form, the coexpression of the present EWS-WT1, IL-2/15Rbeta and Janus kinase (JAK1) mRNA was detected in the tumour cells. IL-2 and signal transducers and activators of transcription (STAT5) mRNA were detected in both tumour and stromal cells. The induction of the IL-2/15 receptor signalling pathway may contribute to tumorigenesis in DSRCT through a paracrine or an autocrine system, even though the EWS-WT1 was an unusual form.

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.

Single cell subtractive transcriptomics for identification of cell-specifically expressed candidate genes of pyrrolizidine alkaloid biosynthesis.

PubMed

Sievert, Christian; Beuerle, Till; Hollmann, Julien; Ober, Dietrich

2015-09-01

Progress has recently been made in the elucidation of pathways of secondary metabolism. However, because of its diversity, genetic information concerning biosynthetic details is still missing for many natural products. This is also the case for the biosynthesis of pyrrolizidine alkaloids. To close this gap, we tested strategies using tissues that express this pathway in comparison to tissues in which this pathway is not expressed. As many pathways of secondary metabolism are known to be induced by jasmonates, the pyrrolizidine alkaloid-producing species Heliotropium indicum, Symphytum officinale, and Cynoglossum officinale of the Boraginales order were treated with methyl jasmonate. An effect on pyrrolizidine alkaloid levels and on transcript levels of homospermidine synthase, the first specific enzyme of pyrrolizidine alkaloid biosynthesis, was not detectable. Therefore, a method was developed by making use of the often observed cell-specific production of secondary compounds. H. indicum produces pyrrolizidine alkaloids exclusively in the shoot. Homospermidine synthase is expressed only in the cells of the lower leaf epidermis and the epidermis of the stem. Suggesting that the whole pathway of pyrrolizidine alkaloid biosynthesis might be localized in these cells, we have isolated single cells of the upper and lower epidermis by laser-capture microdissection. The resulting cDNA preparations have been used in a subtractive transcriptomic approach. Quantitative real-time polymerase chain reaction has shown that the resulting library is significantly enriched for homospermidine-synthase-coding transcripts providing a valuable source for the identification of further genes involved in pyrrolizidine alkaloid biosynthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of nitrofen-induced congenital diaphragmatic hernia.

PubMed

Takahashi, Toshiaki; Zimmer, Julia; Friedmacher, Florian; Puri, Prem

2016-12-01

Pleuroperitoneal folds (PPFs) are the source of the primordial diaphragm's muscle connective tissue (MCT), and developmental mutations have been shown to result in congenital diaphragmatic hernia (CDH). The protein paired-related homeobox 1 (Prx1) labels migrating PPF cells and stimulates expression of transcription factor 4 (Tcf4), a novel MCT marker that controls morphogenesis of the fetal diaphragm. We hypothesized that diaphragmatic Prx1 and Tcf4 expression is decreased in the nitrofen-induced CDH model. Time-mated rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms were microdissected on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Gene expression levels of Prx1 and Tcf4 were analyzed by qRT-PCR. Immunofluorescence double staining for Prx1 and Tcf4 was performed to evaluate protein expression and localization. Relative mRNA expression of Prx1 and Tcf4 was significantly downregulated in PPFs (D13), developing diaphragms (D15) and fully muscularized diaphragms (D18) of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy revealed markedly diminished Prx1 and Tcf4 expression in diaphragmatic MCT of nitrofen-exposed fetuses on D13, D15, and D18 compared to controls. Decreased expression of Prx1 and Tcf4 in the fetal diaphragm may cause defects in the PPF-derived MCT, leading to development of CDH in the nitrofen model. Level 2c (Centre for Evidence-Based Medicine, Oxford). Copyright © 2016 Elsevier Inc. All rights reserved.

Restructuring the vocal fold lamina propria with endoscopic microdissection.

PubMed

Bartlett, Rebecca S; Hoffman, Henry T; Dailey, Seth H; Bock, Jonathan M; Klemuk, Sarah A; Askeland, Ryan W; Ahlrichs-Hanson, Jan S; Heaford, Andrew C; Thibeault, Susan L

2013-11-01

The purposes of this preclinical study were to investigate histologic and rheologic outcomes of Microendoscopy of Reinke's space (MERS)-guided minithyrotomy and to assess its instrumentation. Human cadaveric and in vivo animal study. Three human cadaveric larynges were treated with MERS-guided placement of Radiesse VoiceGel and immediately evaluated histologically for biomaterial location. In the second part of this investigation, two scarred porcine larynges were treated with MERS-guided placement of HyStem-VF and rheologically evaluated 6 weeks later. Student t tests determined differences in viscoelastic properties of treated/untreated vocal folds. Sialendoscopes and microendoscopes were subjectively compared for their visualization capacity. MERS imaged the subepithelial area and vocal ligament, guiding both tissue dissection and biomaterial positioning. Sialendoscopes provided adequate visualization and feature incorporated working channels. Enhanced image clarity was created in a gas-filled rather than saline-filled environment, per rater judgment. Histological analysis revealed desirable biomaterial positioning with MERS. Per rheological analysis, viscoelastic properties of the MERS-treated porcine vocal folds compared to uninjured vocal folds 6 weeks following treatment did not statistically differ. MERS-guided laryngoplasty using sialendoscopes yielded satisfactory biomaterial positioning in the short-term and normalized rheologic tissue properties in the long-term, contributing to proof of concept for MERS in the treatment of scarring. Strengths of MERS include direct, real-time visualization of Reinke's space and an ability to manipulate surgical instruments parallel to the vocal fold edge while maintaining an intact epithelium. Future work will explore the clinical utility of MERS for addressing scarring, sulcus vocalis, and other intracordal processes. Copyright © 2013 The American Laryngological, Rhinological and Otological Society, Inc.

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

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

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.

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.

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

MicroRNA-196a-5p is a potential prognostic marker of delayed lymph node metastasis in early-stage tongue squamous cell carcinoma

PubMed Central

Maruyama, Tessho; Nishihara, Kazuhide; Umikawa, Masato; Arasaki, Akira; Nakasone, Toshiyuki; Nimura, Fumikazu; Matayoshi, Akira; Takei, Kimiko; Nakachi, Saori; Kariya, Ken-Ichi; Yoshimi, Naoki

2018-01-01

MicroRNAs (miRs) are expected to serve as prognostic tools for cancer. However, many miRs have been reported as prognostic markers of recurrence or metastasis in oral squamous cell carcinoma patients. We aimed to determine the prognostic markers in early-stage tongue squamous cell carcinoma (TSCC). Based on previous studies, we hypothesized that miR-10a, 10b, 196a-5p, 196a-3p, and 196b were prognostic markers and we retrospectively performed miR expression analyses using formalin-fixed paraffin-embedded sections of surgical specimens. Total RNA was isolated from cancer tissues and adjacent normal tissue as control, and samples were collected by laser-capture microdissection. After cDNA synthesis, reverse transcription-quantitative polymerase chain reaction was performed. Statistical analyses for patient clinicopathological characteristics, recurrence/metastasis, and survival rates were performed to discern their relationships with miR expression levels, and the 2−ΔΔCq method was used. miR-196a-5p levels were significantly upregulated in early-stage TSCC, particularly in the lymph node metastasis (LNM) group. The LNM-free survival rate in the low miR-196a-5p ΔΔCq value regulation group was found to be lower than that in the high ΔΔCq value regulation group (P=0.0079). Receiver operating characteristic analysis of ΔΔCq values revealed that miR-196a-5p had a P-value=0.0025, area under the curve=0.740, and a cut-off value=−0.875 for distinguishing LNM. To our knowledge, this is the first study to examine LNM-related miRs in early-stage TSCC as well as miRs and ‘delayed LNM’ in head and neck cancer. miR-196a-5p upregulation may predict delayed LNM. Our data serve as a foundation for future studies to evaluate miR levels and facilitate the prediction of delayed LNM during early-stage TSCC, which prevent metastasis when combined with close follow-up and aggressive adjuvant therapy or elective neck dissection. Moreover, our data will serve as a foundation for future studies to evaluate whether miR-196a-5p can serve as a therapeutic marker for preventing metastasis. PMID:29434944

Isolating LacZ-expressing cells from mouse inner ear tissues using flow cytometry.

PubMed

Jan, Taha A; Chai, Renjie; Sayyid, Zahra N; Cheng, Alan G

2011-12-23

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae. The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown. To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Tissue factor pathway inhibitor prevents airway obstruction, respiratory failure and death due to sulfur mustard analog inhalation

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

Rancourt, Raymond C., E-mail: raymond.rancourt@ucdenver.edu; Veress, Livia A., E-mail: livia.veress@ucdenver.edu; Ahmad, Aftab, E-mail: aftab.ahmad@ucdenver.edu

Sulfur mustard (SM) inhalation causes airway injury, with enhanced vascular permeability, coagulation, and airway obstruction. The objective of this study was to determine whether recombinant tissue factor pathway inhibitor (TFPI) could inhibit this pathogenic sequence. Methods: Rats were exposed to the SM analog 2-chloroethyl ethyl sulfide (CEES) via nose-only aerosol inhalation. One hour later, TFPI (1.5 mg/kg) in vehicle, or vehicle alone, was instilled into the trachea. Arterial O{sub 2} saturation was monitored using pulse oximetry. Twelve hours after exposure, animals were euthanized and bronchoalveolar lavage fluid (BALF) and plasma were analyzed for prothrombin, thrombin–antithrombin complex (TAT), active plasminogen activatormore » inhibitor-1 (PAI-1) levels, and fluid fibrinolytic capacity. Lung steady-state PAI-1 mRNA was measured by RT-PCR analysis. Airway-capillary leak was estimated by BALF protein and IgM, and by pleural fluid measurement. In additional animals, airway cast formation was assessed by microdissection and immunohistochemical detection of airway fibrin. Results: Airway obstruction in the form of fibrin-containing casts was evident in central conducting airways of rats receiving CEES. TFPI decreased cast formation, and limited severe hypoxemia. Findings of reduced prothrombin consumption, and lower TAT complexes in BALF, demonstrated that TFPI acted to limit thrombin activation in airways. TFPI, however, did not appreciably affect CEES-induced airway protein leak, PAI-1 mRNA induction, or inhibition of the fibrinolytic activity present in airway surface liquid. Conclusions: Intratracheal administration of TFPI limits airway obstruction, improves gas exchange, and prevents mortality in rats with sulfur mustard-analog-induced acute lung injury. - Highlights: • TFPI administration to rats after mustard inhalation reduces airway cast formation. • Inhibition of thrombin activation is the likely mechanism for limiting casts. • Rats given TFPI had improved tissue oxygenation, and mortality was prevented.« less

Extracellular matrix disruption is an early event in the pathogenesis of skeletal disease in mucopolysaccharidosis I.

PubMed

Heppner, Jonathan M; Zaucke, Frank; Clarke, Lorne A

2015-02-01

Progressive skeletal and connective tissue disease represents a significant clinical burden in all of the mucopolysaccharidoses. Despite the introduction of enzyme replacement strategies for many of the mucopolysaccharidoses, symptomatology related to bone and joint disease appears to be recalcitrant to current therapies. In order to address these unmet medical needs a clearer understanding of skeletal and connective tissue disease pathogenesis is required. Historically the pathogenesis of the mucopolysaccharidoses has been assumed to directly relate to progressive storage of glycosaminoglycans. It is now apparent for many lysosomal storage disorders that more complex pathogenic mechanisms underlie patients' clinical symptoms. We have used proteomic and genome wide expression studies in the murine mucopolysaccharidosis I model to identify early pathogenic events occurring in micro-dissected growth plate tissue. Studies were conducted using 3 and 5-week-old mice thus representing a time at which no obvious morphological changes of bone or joints have taken place. An unbiased iTRAQ differential proteomic approach was used to identify candidates followed by validation with multiple reaction monitoring mass spectrometry and immunohistochemistry. These studies reveal significant decreases in six key structural and signaling extracellular matrix proteins; biglycan, fibromodulin, PRELP, type I collagen, lactotransferrin, and SERPINF1. Genome-wide expression studies in embryonic day 13.5 limb cartilage and 5 week growth plate cartilage followed by specific gene candidate qPCR studies in the 5week growth plate identified fourteen significantly deregulated mRNAs (Adamts12, Aspn, Chad, Col2a1, Col9a1, Hapln4, Lum, Matn1, Mmp3, Ogn, Omd, P4ha2, Prelp, and Rab32). The involvement of biglycan, PRELP and fibromodulin; all members of the small leucine repeat proteoglycan family is intriguing, as this protein family is implicated in the pathogenesis of late onset osteoarthritis. Taken as a whole, our data indicates that alteration of the extracellular matrix represents a very early event in the pathogenesis of the mucopolysaccharidoses and implies that biomechanical failure of chondro-osseous tissue may underlie progressive bone and joint disease symptoms. These findings have important therapeutic implications. Copyright © 2014 Elsevier Inc. All rights reserved.

Enzymes of acetylcholine metabolism in the rat cochlea.

PubMed

Godfrey, D A; Ross, C D

1985-01-01

The distributions within the rat cochlea of choline acetyltransferase and acetylcholinesterase activities were measured to evaluate the prominence of cholinergic mechanisms in cochlear function. Samples obtained by microdissection of freeze-dried bony labyrinths were assayed radiometrically. Activities of both enzymes were highest in regions containing olivocochlear fibers and terminals, especially the organ of Corti and spiral ganglion. Within the organ of Corti, activities of both enzymes were consistently higher in the vicinity of the inner hair cells than in that of the outer hair cells and were much lower in the apical turn than in middle or basal turns. Surgical cuts in the brain stem transecting the olivocochlear pathway on one side led within seven days to total loss of choline acetyltransferase activity in the ipsilateral organ of Corti. It is concluded that all cholinergic structures in the rat organ of Corti derive from the brain stem and that synapses on or near both inner and outer hair cells are cholinergic.

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.

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.

Differential expression and regulation of vitamin D hydroxylases and inflammatory genes in prostate stroma and epithelium by 1,25-dihydroxyvitamin D in men with prostate cancer and an in vitro model.

PubMed

Giangreco, Angeline A; Dambal, Shweta; Wagner, Dennis; Van der Kwast, Theodorus; Vieth, Reinhold; Prins, Gail S; Nonn, Larisa

2015-04-01

Previous work on vitamin D in the prostate has focused on the prostatic epithelium, from which prostate cancer arises. Prostatic epithelial cells are surrounded by stroma, which has well-established regulatory control over epithelial proliferation, differentiation, and the inflammatory response. Here we examined the regulation of vitamin D-related genes and inflammatory genes by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D) in laser-capture microdissected prostate tissue from a vitamin D3 clinical trial and in an in vitro model that facilitates stromal-epithelial crosstalk. Analysis of the trial tissues showed that VDR was present in both cell types, whereas expression of the hydroxylases was the highest in the epithelium. Examination of gene expression by prostatic (1,25(OH)2D) concentrations showed that VDR was significantly lower in prostate tissues with the highest concentration of 1,25(OH)2D, and down-regulation of VDR by 1,25(OH) 2D was confirmed in the primary cell cultures. Analysis of inflammatory genes in the patient tissues revealed that IL-6 expression was the highest in the prostate stroma while PTGS2 (COX2) levels were lowest in the prostate cancer tissues from men in the highest tertile of prostatic 1,25(OH)2D. In vitro, TNF-α, IL-6 and IL-8 were suppressed by 1,25 (OH)2D in the primary epithelial cells, whereas TNF-α and PTGS2 were suppressed by 1,25(OH) 2D in the stromal cells. Importantly, the ability of 1,25(OH)2D to alter pro-inflammatory-induced changes in epithelial cell growth were dependent on the presence of the stromal cells. In summary, whereas both stromal and epithelial cells of the prostate express VDR and can presumably respond to 1,25(OH)2D, the prostatic epithelium appears to be the main producer of 1,25(OH)2D. Further, while the prostate epithelium was more responsive to the anti-inflammatory activity of 1,25 (OH)2D than stromal cells, stroma-epithelial crosstalk enhanced the phenotypic effects of 1,25(OH)2D and the inflammatory process in the prostate gland. Copyright © 2014 Elsevier Ltd. All rights reserved.

p53 expression and mutation analysis of odontogenic cysts with and without dysplasia.

PubMed

Cox, Darren P

2012-01-01

Overexpression of p53 protein is well described in odontogenic cystic lesions (OCLs), including those with epithelial dysplasia; however, most p53 antibodies stain both wild-type and mutated p53 protein and may not reflect genotype. Direct sequencing of the p53 gene has not identified mutations in OCLs with dysplasia. The purpose of this study was to determine the molecular basis of p53 expression in several types of OCLs with and without dysplasia. The study material comprised 13 OCLs: odontogenic keratocyst (n = 5), orthokeratinized odontogenic cyst (n = 5), dentigerous cyst (n = 2), lateral periodontal cyst (n = 1), and unspecified developmental odontogenic cyst (UDOC) (n = 1). Five of these had features of mild or moderate epithelial dysplasia. One intraosseous squamous cell carcinoma (SCC) that was believed to have arisen from an antecedent dysplastic orthokeratinized OC was also included. Immunohistochemistry was performed using the DO7 monoclonal antibody that recognizes wild-type and mutated p53. DNA was extracted from microdissected tissue for all samples and exons 4 to 8 of the p53 gene direct sequenced. In 4 of 5 OCLs with dysplasia there was strong nuclear staining of basal and suprabasal cells. In all cases without dysplasia, nuclear expression in basal cells was either negative or weak and was absent in suprabasal cell nuclei. A mutation in exon 6 of the p53 gene (E224D) was identified in both the dysplastic orthokeratinized OC and the subsequent intraosseous SCC. OCLs with features of dysplasia show increased expression of p53 protein that does not reflect p53 mutational status. One dysplastic OC shared the same p53 mutation with a subsequent intraosseous SCC, indicating that p53 mutation may be associated with malignant transformation in this case. Copyright © 2012 Elsevier Inc. All rights reserved.

Asporin stably expressed in the surface layer of mandibular condylar cartilage and augmented in the deeper layer with age.

PubMed

Miyamoto, Yutaka; Kanzaki, Hiroyuki; Wada, Satoshi; Tsuruoka, Sari; Itohiya, Kanako; Kumagai, Kenichi; Hamada, Yoshiki; Nakamura, Yoshiki

2017-12-01

Mandibular condylar cartilage (MCC) exhibits dual roles both articular cartilage and growth center. Of many growth factors, TGF-β has been implicated in the growth of articular cartilage including MCC. Recently, Asporin, decoy to TGF-β, was discovered and it blocks TGF-β signaling. Asporin is expressed in a variety of tissues including osteoarthritic articular cartilage, though there was no report of Asporin expression in MCC. In the present study, we investigated the temporal and spatial expression of Asporin in MCC. Gene expression profile of MCC and epiphyseal cartilage in tibia of 5 weeks old ICR mice were firstly compared with microarray analysis using the laser capture microdissected samples. Variance of gene expression was further confirmed by real-time RT-PCR and immunohistochemical staining at 1,3,10, and 20 weeks old. TGF-β and its signaling molecule, phosphorylated Smad-2/3 (p-Smad2/3), were also examined by immunohistochemical staining. Microarray analysis revealed that Asporin was highly expressed in MCC. Real-time RT-PCR analysis confirmed that the fibrous layer of MCC exhibited stable higher Asporin expression at any time points as compared to epiphyseal cartilage. This was also observed in immunohistochemical staining. Deeper layer in MCC augmented Asporin expression with age. Whereas, TGF-β was stably highly observed in the layer. The fibrous layer of MCC exhibited weak staining of p-Smad2/3, though the proliferating layer of MCC was strongly stained as compared to epiphyseal cartilage of tibia at early time point. Consistent with the increase of Asporin expression in the deeper layer of MCC, the intensity of p-Smad-2/3 staining was decreased with age. In conclusion, we discovered that Asporin was stably expressed at the fibrous layer of MCC, which makes it possible to manage both articular cartilage and growth center at the same time.

Physical structure and chromosomal localization of a gene encoding human p58[sup clk-1], a cell division control related protein kinase

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

Eipers, P.G.

1992-01-01

The gene for the human p58[sup clk[minus]1] protein kinase, a cell division control-related gene, has been mapped by somatic cell hybrid analyses, in situ localization with the chromosomal gene, and nested polymerase chain reaction amplification of microdissected chromosomes. These studies indicate that the expressed p58[sup clk[minus]1] chromosomal gene maps to 1p36, while a highly related p58[sup clk[minus]1] sequence of unknown nature maps to chromosome 15. Assignment of a p34[sup cdc2]-related gene to 1p36 region, including neuroblastoma, ductal carcinoma of the breast, malignant melanoma, Merkel cell carcinoma and endocrine neoplasia among others. Aberrant expression of this protein kinase negatively regulates normalmore » cellular growth. The p58[sup clk[minus]1] protein contains a central domain of 299 amino acids that is 46% identical to human p34[sup cdc2], the master mitotic protein kinase. This dissertation details the complete structure of the p58[sup clk[minus]1] chromosomal gene, including its putative promoter region, transcriptional start sites, exonic sequences, and intron/exon boundary sequences. The gene is 10 kb in size and contains 12 exons and 11 introns. Interestingly, the rather large 2.0 kb 3[prime] untranslated region is interrupted by an intron that separates a region containing numerous AUUUA destabilization motifs from the coding region. Furthermore, the expression of this gene in normal human tissues, as well as several human tumor cell samples and lines, is examined. The origin of multiple human transcripts from the same chromosomal gene, and the possible differential stability of these various transcripts, is discussed with regard to the transcriptional and post-transcriptional regulation of this gene. This is the first report of the chromosomal gene structure of a member of the p34[sup cdc2] supergene family.« less

Mi-1.2, an R gene for aphid resistance in tomato, has direct negative effects on a zoophytophagous biocontrol agent, Orius insidiosus.

PubMed

Pallipparambil, Godshen R; Sayler, Ronald J; Shapiro, Jeffrey P; Thomas, Jean M G; Kring, Timothy J; Goggin, Fiona L

2015-02-01

Mi-1.2 is a single dominant gene in tomato that confers race-specific resistance against certain phloem-feeding herbivores including aphids, whiteflies, psyllids, and root-knot nematodes. Few prior studies have considered the potential non-target effects of race-specific resistance genes (R genes), and this paper evaluates the compatibility of Mi-mediated resistance in tomato with a beneficial zoophytophagous predator, Orius insidiosus (Say). In addition to preying on aphids and other pests, this piercing-sucking insect also feeds from the xylem, epidermis, and/or mesophyll, and oviposits within plant tissues. Comparison of O. insidiosus confined to isogenic tomato plants with and without Mi-1.2 revealed that immatures of O. insidiosus had lower survival on resistant plants even when the immatures were provisioned with prey that did not feed on the host plant. Molecular gut content analysis confirmed that adults and immatures of O. insidiosus feed on both resistant (Mi-1.2+) and susceptible (Mi-1.2-) genotypes, and bioassays suggest that resistance does not affect oviposition rates, plant sampling, or prey acceptance by O. insidiosus adults. These results demonstrate a direct negative impact of R-gene-mediated host plant resistance on a non-target beneficial species, and reveal that Mi-mediated resistance can impact organisms that do not feed on phloem sap. Through laser capture microdissection and RT-PCR, Mi-1.2 transcripts were detected in the epidermis and mesophyll as well as the phloem of tomato plants, consistent with our observations that Mi-mediated resistance is active outside the phloem. These results suggest that the mode of action and potential ecological impacts of Mi-mediated resistance are broader than previously assumed. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Combined serial analysis of gene expression and transcription factor binding site prediction identifies novel-candidate-target genes of Nr2e1 in neocortex development.

PubMed

Schmouth, Jean-François; Arenillas, David; Corso-Díaz, Ximena; Xie, Yuan-Yun; Bohacec, Slavita; Banks, Kathleen G; Bonaguro, Russell J; Wong, Siaw H; Jones, Steven J M; Marra, Marco A; Simpson, Elizabeth M; Wasserman, Wyeth W

2015-07-24

Nr2e1 (nuclear receptor subfamily 2, group e, member 1) encodes a transcription factor important in neocortex development. Previous work has shown that nuclear receptors can have hundreds of target genes, and bind more than 300 co-interacting proteins. However, recognition of the critical role of Nr2e1 in neural stem cells and neocortex development is relatively recent, thus the molecular mechanisms involved for this nuclear receptor are only beginning to be understood. Serial analysis of gene expression (SAGE), has given researchers both qualitative and quantitative information pertaining to biological processes. Thus, in this work, six LongSAGE mouse libraries were generated from laser microdissected tissue samples of dorsal VZ/SVZ (ventricular zone and subventricular zone) from the telencephalon of wild-type (Wt) and Nr2e1-null embryos at the critical development ages E13.5, E15.5, and E17.5. We then used a novel approach, implementing multiple computational methods followed by biological validation to further our understanding of Nr2e1 in neocortex development. In this work, we have generated a list of 1279 genes that are differentially expressed in response to altered Nr2e1 expression during in vivo neocortex development. We have refined this list to 64 candidate direct-targets of NR2E1. Our data suggested distinct roles for Nr2e1 during different neocortex developmental stages. Most importantly, our results suggest a possible novel pathway by which Nr2e1 regulates neurogenesis, which includes Lhx2 as one of the candidate direct-target genes, and SOX9 as a co-interactor. In conclusion, we have provided new candidate interacting partners and numerous well-developed testable hypotheses for understanding the pathways by which Nr2e1 functions to regulate neocortex development.

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

Molecular profiling identifies prognostic markers of stage IA lung adenocarcinoma.

PubMed

Zhang, Jie; Shao, Jinchen; Zhu, Lei; Zhao, Ruiying; Xing, Jie; Wang, Jun; Guo, Xiaohui; Tu, Shichun; Han, Baohui; Yu, Keke

2017-09-26

We previously showed that different pathologic subtypes were associated with different prognostic values in patients with stage IA lung adenocarcinoma (AC). We hypothesize that differential gene expression profiles of different subtypes may be valuable factors for prognosis in stage IA lung adenocarcinoma. We performed microarray gene expression profiling on tumor tissues micro-dissected from patients with acinar and solid predominant subtypes of stage IA lung adenocarcinoma. These patients had undergone a lobectomy and mediastinal lymph node dissection at the Shanghai Chest Hospital, Shanghai, China in 2012. No patient had preoperative treatment. We performed the Gene Set Enrichment Analysis (GSEA) analysis to look for gene expression signatures associated with tumor subtypes. The histologic subtypes of all patients were classified according to the 2015 WHO lung Adenocarcinoma classification. We found that patients with the solid predominant subtype are enriched for genes involved in RNA polymerase activity as well as inactivation of the p53 pathway. Further, we identified a list of genes that may serve as prognostic markers for stage IA lung adenocarcinoma. Validation in the TCGA database shows that these genes are correlated with survival, suggesting that they are novel prognostic factors for stage IA lung adenocarcinoma. In conclusion, we have uncovered novel prognostic factors for stage IA lung adenocarcinoma using gene expression profiling in combination with histopathology subtyping.

Biochemical and molecular characterization of rice (Oryza sativa L.) roots forming a barrier to radial oxygen loss.

PubMed

Kulichikhin, Konstantin; Yamauchi, Takaki; Watanabe, Kohtaro; Nakazono, Mikio

2014-10-01

The formation of a barrier to radial oxygen (O2 ) loss (ROL) in the root is an important adaptation of plants to root flooding, but the biochemical changes in plant roots where the barrier is formed are unclear. In this study, we analysed metabolic profiles and gene expression profiles in roots of rice (Oryza sativa L.) plants grown under stagnant deoxygenated conditions, which induce suberization in the outer cell layers of the roots and formation of barrier to ROL. Under these conditions, two distinctive biochemical features of the roots were the accumulations of malic acid and very long chain fatty acids (VLCFAs). We also showed that the expressions of some genes encoding plastid-localized enzymes, which convert malic acid to acetyl coenzyme A (AcCoA), were simultaneously up-regulated under stagnant conditions. The expression levels of these genes in specific root tissues isolated by laser microdissection suggested that malic acid is converted to AcCoA predominantly in the plastids in the outer cell layers of rice roots. We propose that the physiological role of malic acid accumulation in rice roots grown under stagnant conditions is to provide a substrate for the biosynthesis of fatty acids, which, in turn, are used in the biosynthesis of suberin. © 2014 John Wiley & Sons Ltd.

Adult DRG Stem/Progenitor Cells Generate Pericytes in the Presence of Central Nervous System (CNS) Developmental Cues, and Schwann Cells in Response to CNS Demyelination.

PubMed

Vidal, Marie; Maniglier, Madlyne; Deboux, Cyrille; Bachelin, Corinne; Zujovic, Violetta; Baron-Van Evercooren, Anne

2015-06-01

It has been proposed that the adult dorsal root ganglia (DRG) harbor neural stem/progenitor cells (NPCs) derived from the neural crest. However, the thorough characterization of their stemness and differentiation plasticity was not addressed. In this study, we investigated adult DRG-NPC stem cell properties overtime, and their fate when ectopically grafted in the central nervous system. We compared them in vitro and in vivo to the well-characterized adult spinal cord-NPCs derived from the same donors. Using micro-dissection and neurosphere cultures, we demonstrate that adult DRG-NPCs have quasi unlimited self-expansion capacities without compromising their tissue specific molecular signature. Moreover, they differentiate into multiple peripheral lineages in vitro. After transplantation, adult DRG-NPCs generate pericytes in the developing forebrain but remyelinating Schwann cells in response to spinal cord demyelination. In addition, we show that axonal and endothelial/astrocytic factors as well astrocytes regulate the fate of adult DRG-NPCs in culture. Although the adult DRG-NPC multipotency is restricted to the neural crest lineage, their dual responsiveness to developmental and lesion cues highlights their impressive adaptive and repair potentials making them valuable targets for regenerative medicine. © 2015 AlphaMed Press.

Glioblastomas with oligodendroglial component - common origin of the different histological parts and genetic subclassification.

PubMed

Klink, Barbara; Schlingelhof, Ben; Klink, Martin; Stout-Weider, Karen; Patt, Stephan; Schrock, Evelin

2010-01-01

Glioblastomas are the most common and most malignant brain tumors in adults. A small subgroup of glioblastomas contains areas with histological features of oligodendroglial differentiation (GBMO). Our objective was to genetically characterize the oligodendroglial and the astrocytic parts of GBMOs and correlate morphologic and genetic features with clinical data. The oligodendroglial and the "classic" glioblastoma parts of 13 GBMO were analyzed separately by interphase fluorescence in situ hybridization (FISH) on paraffin sections using a custom probe set (regions 1p, 1q, 7q, 10q, 17p, 19q, cen18, 21q) and by comparative genomic hybridization (CGH) of microdissected paraffin embedded tumor tissue. We identified four distinct genetic subtypes in 13 GBMOs: an "astrocytic" subtype (9/13) characterized by +7/-10; an "oligodendroglial" subtype with -1p/-19q (1/13); an "intermediate" subtype showing +7/-1p (1/13), and an "other" subtype having none of the former aberrations typical for gliomas (2/13). The different histological tumor parts of GBMO revealed common genetic changes in all tumors and showed additional aberrations specific for each part. Our findings demonstrate the monoclonal origin of GBMO followed by the development of the astrocytic and oligodendroglial components. The diagnostic determination of the genetic signatures may allow for a better prognostication of the patients.

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.

Intraglomerular pressure and mesangial stretching stimulate extracellular matrix formation in the rat.

PubMed Central

Riser, B L; Cortes, P; Zhao, X; Bernstein, J; Dumler, F; Narins, R G

1992-01-01

To define the interplay of glomerular hypertension and hypertrophy with mesangial extracellular matrix (ECM) deposition, we examined the effects of glomerular capillary distention and mesangial cell stretching on ECM synthesis. The volume of microdissected rat glomeruli (Vg), perfused ex vivo at increasing flows, was quantified and related to the proximal intraglomerular pressure (PIP). Glomerular compliance, expressed as the slope of the positive linear relationship between PIP and Vg was 7.68 x 10(3) microns 3/mmHg. Total Vg increment (PIP 0-150 mmHg) was 1.162 x 10(6) microns 3 or 61% (n = 13). A 16% increase in Vg was obtained over the PIP range equivalent to the pathophysiological limits of mean transcapillary pressure difference. A similar effect of renal perfusion on Vg was also noted histologically in tissue from kidneys perfused/fixed in vivo. Cultured mesangial cells undergoing cyclic stretching increased their synthesis of protein, total collagen, and key components of ECM (collagen IV, collagen I, laminin, fibronectin). Synthetic rates were stimulated by cell growth and the degree of stretching. These results suggest that capillary expansion and stretching of mesangial cells by glomerular hypertension provokes increased ECM production which is accentuated by cell growth and glomerular hypertrophy. Mesangial expansion and glomerulosclerosis might result from this interplay of mechanical and metabolic forces. Images PMID:1430216

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.

Transient Gene Expression in Maize, Rice, and Wheat Cells Using an Airgun Apparatus 1

PubMed Central

Oard, James H.; Paige, David F.; Simmonds, John A.; Gradziel, Thomas M.

1990-01-01

An airgun apparatus has been constructed for transient gene expression studies of monocots. This device utilizes compressed air from a commercial airgun to propel macroprojectile and DNA-coated tungsten particles. The β-glucuronidase (GUS) reporter gene was used to monitor transient expression in three distinct cell types of maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum). The highest level of GUS activity in cultured maize cells was observed when distance between stopping plate and target cells was adjusted to 4.3 centimeters. Efficiency of transformation was estimated to be 4.4 × 10−3. In a partial vacuum of 700 millimeters Hg, velocity of macroprojectile was measured at 520 meters per second with a 6% reduction in velocity at atmospheric pressure. A polyethylene film placed in the breech before firing contributed to a 12% increase in muzzle velocity. A 700 millimeters Hg level of vacuum was necessary for maximum number of transfornants. GUS expression was also detected in wheat leaf base tissue of microdissected shoot apices. High levels of transient gene expression were also observed in hard, compact embryogenic callus of rice. These results show that the airgun apparatus is a convenient, safe, and low-cost device for rapid transient gene expression studies in cereals. Images Figure 7 Figure 8 Figure 9 PMID:16667278

Cell Wall Remodeling in Abscission Zone Cells during Ethylene-Promoted Fruit Abscission in Citrus

PubMed Central

Merelo, Paz; Agustí, Javier; Arbona, Vicent; Costa, Mário L.; Estornell, Leandro H.; Gómez-Cadenas, Aurelio; Coimbra, Silvia; Gómez, María D.; Pérez-Amador, Miguel A.; Domingo, Concha; Talón, Manuel; Tadeo, Francisco R.

2017-01-01

Abscission is a cell separation process by which plants can shed organs such as fruits, leaves, or flowers. The process takes place in specific locations termed abscission zones. In fruit crops like citrus, fruit abscission represents a high percentage of annual yield losses. Thus, understanding the molecular regulation of abscission is of capital relevance to control production. To identify genes preferentially expressed within the citrus fruit abscission zone (AZ-C), we performed a comparative transcriptomics assay at the cell type resolution level between the AZ-C and adjacent fruit rind cells (non-abscising tissue) during ethylene-promoted abscission. Our strategy combined laser microdissection with microarray analysis. Cell wall modification-related gene families displayed prominent representation in the AZ-C. Phylogenetic analyses of such gene families revealed a link between phylogenetic proximity and expression pattern during abscission suggesting highly conserved roles for specific members of these families in abscission. Our transcriptomic data was validated with (and strongly supported by) a parallel approach consisting on anatomical, histochemical and biochemical analyses on the AZ-C during fruit abscission. Our work identifies genes potentially involved in organ abscission and provides relevant data for future biotechnology approaches aimed at controlling such crucial process for citrus yield. PMID:28228766

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.

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.

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

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

DOEpatents

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

2003-12-16

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

Genome-wide allelotyping of a new in vitro model system reveals early events in breast cancer progression.

PubMed

Li, Zheng; Meng, Zhen Hang; Sayeed, Aejaz; Shalaby, Refaat; Ljung, Britt-Marie; Dairkee, Shanaz H

2002-10-15

Toward the goal of identifying early genetic losses, which mediate the release of human breast epithelium from replicative suppression leading to cellular immortalization, we have used a newly developed in vitro model system. This system consists of epithelial cultures derived from noncancerous breast tissue, treated with the chemical carcinogen N-ethyl-N-nitrosourea, and continuously passaged to yield cell populations culminating in the immortal phenotype. Genome-wide allelotyping of early passage N-ethyl-N-nitrosourea-exposed cell populations revealed aberrations at >10% (18 of 169) loci examined. Allelic losses encompassing chromosomes 6q24-6q27, implicating immortalization-associated candidate genes, hZAC and SEN6, occurred in two independently derived cell lines before the Hayflick limit. Additional LOH sites were present in one cell line at 3p11-3p26, 11p15, and 20p12-13. Allelic losses reported in this cell line preceded detectable levels of telomerase activity and the occurrence of p53-related aberrations. Information gained from the search for early immortalization-associated genetic deletions in cultured cells was applied in a novel approach toward the analysis of morphologically normal terminal ductal lobular units microdissected from 20 cases of ductal carcinoma in situ. Notably, clonal allelic losses at chromosome 3p24 and 6q24 were an early occurrence in adjoining terminal ductal lobular units of a proportion of primary tumors, which displayed loss of heterozygosity (3 of 11 and 3 of 6, respectively). The biological insights provided by the new model system reported here strongly suggest that early allelic losses delineated in immortalized cultures and validated in vivo could serve as surrogate endpoints to assist in the identification and intervention of high-risk benign breast tissue, which sustains the potential for continuous proliferation.

Glucagon-like peptide-1 regulates brown adipose tissue thermogenesis via the gut-brain axis in rats.

PubMed

Krieger, Jean-Philippe; Santos da Conceição, Ellen Paula; Sanchez-Watts, Graciela; Arnold, Myrtha; Pettersen, Klaus G; Mohammed, Mazher; Modica, Salvatore; Lossel, Pius; Morrison, Shaun F; Madden, Christopher J; Watts, Alan G; Langhans, Wolfgang; Lee, Shin J

2018-05-30

Endogenous intestinal glucagon-like peptide-1 (GLP-1) controls satiation and glucose metabolism via vagal afferent neurons (VAN). Recently, VAN have received increasing attention for their role in brown adipose tissue (BAT) thermogenesis. It is however unclear whether VAN GLP-1 receptor (GLP-1R) signaling affects BAT thermogenesis and energy expenditure (EE), and whether this VAN mechanism contributes to energy balance. First, we tested the effect of the GLP-1R agonist Exendin-4 (Ex4, 0.3 μg/kg IP) on EE and BAT thermogenesis, and whether these effects require VAN GLP-1R signaling, using a rat model with a selective Glp1r knockdown (kd) in VAN. Second, we examined the role of VAN GLP-1R in energy balance during chronic high-fat diet (HFD) feeding in VAN Glp1r kd rats. Lastly, we used viral transsynaptic tracers to identify the possible neuronal substrates of such a gut-BAT interaction. VAN Glp1r kd attenuated the acute suppressive effects of Ex4 on EE and BAT thermogenesis. Consistent with this finding, the VAN Glp1r kd increased EE and BAT activity, diminished body weight gain, and improved insulin sensitivity compared to HFD-fed controls. Anterograde transsynaptic viral tracing of VAN infected major hypothalamic and hindbrain areas involved in BAT sympathetic regulation. Moreover, retrograde tracing from BAT combined with laser capture microdissection revealed that a population of VAN expressing Glp1r is synaptically connected to the BAT. Our findings reveal a novel role of VAN GLP-1R signaling in the regulation of EE and BAT thermogenesis, and imply that through this gut-brain-BAT connection intestinal GLP-1 plays a role in HFD-induced metabolic syndrome.

Proteomic Profiling in the Brain of CLN1 Disease Model Reveals Affected Functional Modules.

PubMed

Tikka, Saara; Monogioudi, Evanthia; Gotsopoulos, Athanasios; Soliymani, Rabah; Pezzini, Francesco; Scifo, Enzo; Uusi-Rauva, Kristiina; Tyynelä, Jaana; Baumann, Marc; Jalanko, Anu; Simonati, Alessandro; Lalowski, Maciej

2016-03-01

Neuronal ceroid lipofuscinoses (NCL) are the most commonly inherited progressive encephalopathies of childhood. Pathologically, they are characterized by endolysosomal storage with different ultrastructural features and biochemical compositions. The molecular mechanisms causing progressive neurodegeneration and common molecular pathways linking expression of different NCL genes are largely unknown. We analyzed proteome alterations in the brains of a mouse model of human infantile CLN1 disease-palmitoyl-protein thioesterase 1 (Ppt1) gene knockout and its wild-type age-matched counterpart at different stages: pre-symptomatic, symptomatic and advanced. For this purpose, we utilized a combination of laser capture microdissection-based quantitative liquid chromatography tandem mass spectrometry (MS) and matrix-assisted laser desorption/ionization time-of-flight MS imaging to quantify/visualize the changes in protein expression in disease-affected brain thalamus and cerebral cortex tissue slices, respectively. Proteomic profiling of the pre-symptomatic stage thalamus revealed alterations mostly in metabolic processes and inhibition of various neuronal functions, i.e., neuritogenesis. Down-regulation in dynamics associated with growth of plasma projections and cellular protrusions was further corroborated by findings from RNA sequencing of CLN1 patients' fibroblasts. Changes detected at the symptomatic stage included: mitochondrial functions, synaptic vesicle transport, myelin proteome and signaling cascades, such as RhoA signaling. Considerable dysregulation of processes related to mitochondrial cell death, RhoA/Huntington's disease signaling and myelin sheath breakdown were observed at the advanced stage of the disease. The identified changes in protein levels were further substantiated by bioinformatics and network approaches, immunohistochemistry on brain tissues and literature knowledge, thus identifying various functional modules affected in the CLN1 childhood encephalopathy.

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

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

Truncated neurokinin-1 receptor is increased in colonic epithelial cells from patients with colitis-associated cancer

PubMed Central

Gillespie, Earl; Leeman, Susan E.; Watts, Luisa A.; Coukos, Jennifer A.; O'Brien, Michael J.; Cerda, Sandra R.; Farraye, Francis A.; Stucchi, Arthur F.; Becker, James M.

2011-01-01

Patients with chronic ulcerative colitis (UC) are at high risk for developing colorectal cancer. In this study, archival formalin-fixed paraffin-embedded colonic tissue from patients with UC who developed carcinoma (CA) or high-grade dysplasia (HGD) was examined for changes in expression of the proinflammatory and mitogenic neurokinin-1 receptor (NK-1R). Laser capture microscopy was used to microdissect epithelia from areas of colons that showed histologic evidence of CA, HGD, and epithelia that were not dysplastic or cancerous but did contain evidence of prior inflammation (quiescent colitis). mRNA was extracted from the dissected tissue, and PCR array analysis was performed on extracted mRNA. Two antibodies were necessary to separately estimate the protein levels of the truncated (tr-NK-1R) and full-length (fl-NK-1R) receptors by immunohistochemistry. mRNA expression of tr-NK-1R increased 14-fold (P = 0.02) when comparing the HGD and CA groups. In contrast, the fl-NK-1R transcript showed no significant differences among groups. The protein levels of the total NK-1R increased by 40% (P = 0.02) in HGD and 80% (P = 0.0007) in CA compared with quiescent colitis. There were no significant changes in protein levels of the fl-NK-1R. We conclude that the increase in total NK-1R protein in HGD and CA is attributable to an increase in tr-NK-1R, suggesting there may be a functional role for tr-NK-1R in malignant transformation in colitis-associated cancer. The tr-NK-1R could prove useful as a diagnostic marker to identify patients at risk for neoplasia and may serve as a useful therapeutic target in the treatment of colitis-associated cancer. PMID:21969570

Connective tissue growth factor and its regulation: a new element in diabetic glomerulosclerosis.

PubMed

Riser, B L; Cortes, P

2001-01-01

Connective tissue growth factor (CTGF), a member of the closely related CCN family of cytokines appears to be fibrotic in skin. To determine whether CTGF is implicated in diabetic glomerulosclerosis we studied cultured rat mesangial cells (MC) as well as kidney cortex and microdissected glomeruli from obese, diabetic db/db mice and their normal counterparts. Exposure of MC to rhCTGF significantly increased fibronectin and collagen type I secretion. Further, unstimulated MC expressed low levels of CTGF message and secreted minimal amounts of CTGF protein (36-38 kDa). However, exposure to TGF-beta, increased glucose concentrations, or cyclic mechanical strain, all causal factors in glomerulosclerosis, markedly induced the expression of CTGF transcripts. With all but mechanical strain there was a concomitant stimulation of CTGF protein secretion. TGF-beta also induced abundant quantities of a small molecular weight form of CTGF (18 kDa). The induction of CTGF protein by a high glucose concentration was mediated by TGF-beta, since a TGF-beta neutralizing antibody blocked this stimulation. In vivo studies using quantitative RT-PCR demonstrated that while CTGF transcripts were low in the glomeruli of control mice, expression was increased 27-fold after approximately 3.5 months of diabetes. These changes occurred early in diabetic nephropathy when mesangial expansion was mild, and interstitial disease and proteinuria were absent. A substantially reduced elevation of CTGF mRNA (2-fold) observed in whole kidney cortices indicted that the primary alteration of CTGF expression was in the glomerulus. These results suggest that CTGF upregulation is an important factor in the pathogenesis of mesangial matrix accumulation in both diabetic and non-diabetic glomerulosclerosis, acting downstream of TGF-beta.

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.

Tissue factor pathway inhibitor prevents airway obstruction, respiratory failure and death due to sulfur mustard analog inhalation.

PubMed

Rancourt, Raymond C; Veress, Livia A; Ahmad, Aftab; Hendry-Hofer, Tara B; Rioux, Jacqueline S; Garlick, Rhonda B; White, Carl W

2013-10-01

Sulfur mustard (SM) inhalation causes airway injury, with enhanced vascular permeability, coagulation, and airway obstruction. The objective of this study was to determine whether recombinant tissue factor pathway inhibitor (TFPI) could inhibit this pathogenic sequence. Rats were exposed to the SM analog 2-chloroethyl ethyl sulfide (CEES) via nose-only aerosol inhalation. One hour later, TFPI (1.5mg/kg) in vehicle, or vehicle alone, was instilled into the trachea. Arterial O2 saturation was monitored using pulse oximetry. Twelve hours after exposure, animals were euthanized and bronchoalveolar lavage fluid (BALF) and plasma were analyzed for prothrombin, thrombin-antithrombin complex (TAT), active plasminogen activator inhibitor-1 (PAI-1) levels, and fluid fibrinolytic capacity. Lung steady-state PAI-1 mRNA was measured by RT-PCR analysis. Airway-capillary leak was estimated by BALF protein and IgM, and by pleural fluid measurement. In additional animals, airway cast formation was assessed by microdissection and immunohistochemical detection of airway fibrin. Airway obstruction in the form of fibrin-containing casts was evident in central conducting airways of rats receiving CEES. TFPI decreased cast formation, and limited severe hypoxemia. Findings of reduced prothrombin consumption, and lower TAT complexes in BALF, demonstrated that TFPI acted to limit thrombin activation in airways. TFPI, however, did not appreciably affect CEES-induced airway protein leak, PAI-1 mRNA induction, or inhibition of the fibrinolytic activity present in airway surface liquid. Intratracheal administration of TFPI limits airway obstruction, improves gas exchange, and prevents mortality in rats with sulfur mustard-analog-induced acute lung injury. Copyright © 2013 Elsevier Inc. All rights reserved.

Circuit- and Diagnosis-Specific DNA Methylation Changes at γ-Aminobutyric Acid-Related Genes in Postmortem Human Hippocampus in Schizophrenia and Bipolar Disorder.

PubMed

Ruzicka, W Brad; Subburaju, Sivan; Benes, Francine M

2015-06-01

Dysfunction related to γ-aminobutyric acid (GABA)-ergic neurotransmission in the pathophysiology of major psychosis has been well established by the work of multiple groups across several decades, including the widely replicated downregulation of GAD1. Prior gene expression and network analyses within the human hippocampus implicate a broader network of genes, termed the GAD1 regulatory network, in regulation of GAD1 expression. Several genes within this GAD1 regulatory network show diagnosis- and sector-specific expression changes within the circuitry of the hippocampus, influencing abnormal GAD1 expression in schizophrenia and bipolar disorder. To investigate the hypothesis that aberrant DNA methylation contributes to circuit- and diagnosis-specific abnormal expression of GAD1 regulatory network genes in psychotic illness. This epigenetic association study targeting GAD1 regulatory network genes was conducted between July 1, 2012, and June 30, 2014. Postmortem human hippocampus tissue samples were obtained from 8 patients with schizophrenia, 8 patients with bipolar disorder, and 8 healthy control participants matched for age, sex, postmortem interval, and other potential confounds from the Harvard Brain Tissue Resource Center, McLean Hospital, Belmont, Massachusetts. We extracted DNA from laser-microdissected stratum oriens tissue of cornu ammonis 2/3 (CA2/3) and CA1 postmortem human hippocampus, bisulfite modified it, and assessed it with the Infinium HumanMethylation450 BeadChip (Illumina, Inc). The subset of CpG loci associated with GAD1 regulatory network genes was analyzed in R version 3.1.0 software (R Foundation) using the minfi package. Findings were validated using bisulfite pyrosequencing. Methylation levels at 1308 GAD1 regulatory network-associated CpG loci were assessed both as individual sites to identify differentially methylated positions and by sharing information among colocalized probes to identify differentially methylated regions. A total of 146 differentially methylated positions with a false detection rate lower than 0.05 were identified across all 6 groups (2 circuit locations in each of 3 diagnostic categories), and 54 differentially methylated regions with P < .01 were identified in single-group comparisons. Methylation changes were enriched in MSX1, CCND2, and DAXX at specific loci within the hippocampus of patients with schizophrenia and bipolar disorder. This work demonstrates diagnosis- and circuit-specific DNA methylation changes at a subset of GAD1 regulatory network genes in the human hippocampus in schizophrenia and bipolar disorder. These genes participate in chromatin regulation and cell cycle control, supporting the concept that the established GABAergic dysfunction in these disorders is related to disruption of GABAergic interneuron physiology at specific circuit locations within the human hippocampus.

Circuit- and Diagnosis-Specific DNA Methylation Changes at γ-Aminobutyric Acid–Related Genes in Postmortem Human Hippocampus in Schizophrenia and Bipolar Disorder

PubMed Central

Ruzicka, W. Brad; Subburaju, Sivan; Benes, Francine M.

2017-01-01

IMPORTANCE Dysfunction related to γ-aminobutyric acid (GABA)–ergic neurotransmission in the pathophysiology of major psychosis has been well established by the work of multiple groups across several decades, including the widely replicated downregulation of GAD1. Prior gene expression and network analyses within the human hippocampus implicate a broader network of genes, termed the GAD1 regulatory network, in regulation of GAD1 expression. Several genes within this GAD1 regulatory network show diagnosis- and sector-specific expression changes within the circuitry of the hippocampus, influencing abnormal GAD1 expression in schizophrenia and bipolar disorder. OBJECTIVE To investigate the hypothesis that aberrant DNA methylation contributes to circuit- and diagnosis-specific abnormal expression of GAD1 regulatory network genes in psychotic illness. DESIGN, SETTING, AND PARTICIPANTS This epigenetic association study targeting GAD1 regulatory network genes was conducted between July 1, 2012, and June 30, 2014. Postmortem human hippocampus tissue samples were obtained from 8patients with schizophrenia, 8 patients with bipolar disorder, and 8 healthy control participants matched for age, sex, postmortem interval, and other potential confounds from the Harvard Brain Tissue Resource Center, McLean Hospital, Belmont,Massachusetts. We extracted DNA from laser-microdissected stratum oriens tissue of cornu ammonis 2/3 (CA2/3) and CA1 postmortem human hippocampus, bisulfite modified it, and assessed it with the Infinium HumanMethylation450 BeadChip (Illumina, Inc). The subset of CpG loci associated with GAD1 regulatory network genes was analyzed in R version 3.1.0 software (R Foundation) using the minfi package. Findings were validated using bisulfite pyrosequencing. MAIN OUTCOMES AND MEASURES Methylation levels at 1308 GAD1 regulatory network–associated CpG loci were assessed both as individual sites to identify differentially methylated positions and by sharing information among colocalized probes to identify differentially methylated regions. RESULTS A total of 146 differentially methylated positions with a false detection rate lower than 0.05 were identified across all 6 groups (2 circuit locations in each of 3 diagnostic categories), and 54 differentially methylated regions with P < .01 were identified in single-group comparisons. Methylation changes were enriched in MSX1, CCND2, and DAXX at specific loci within the hippocampus of patients with schizophrenia and bipolar disorder. CONCLUSIONS AND RELEVANCE This work demonstrates diagnosis- and circuit-specific DNA methylation changes at a subset of GAD1 regulatory network genes in the human hippocampus in schizophrenia and bipolar disorder. These genes participate in chromatin regulation and cell cycle control, supporting the concept that the established GABAergic dysfunction in these disorders is related to disruption of GABAergic interneuron physiology at specific circuit locations within the human hippocampus. PMID:25738424

Anatomy of the clitoris.

PubMed

O'Connell, Helen E; Sanjeevan, Kalavampara V; Hutson, John M

2005-10-01

We present a comprehensive account of clitoral anatomy, including its component structures, neurovascular supply, relationship to adjacent structures (the urethra, vagina and vestibular glands, and connective tissue supports), histology and immunohistochemistry. We related recent anatomical findings to the historical literature to determine when data on accurate anatomy became available. An extensive review of the current and historical literature was done. The studies reviewed included dissection and microdissection, magnetic resonance imaging (MRI), 3-dimensional sectional anatomy reconstruction, histology and immunohistochemical studies. The clitoris is a multiplanar structure with a broad attachment to the pubic arch and via extensive supporting tissue to the mons pubis and labia. Centrally it is attached to the urethra and vagina. Its components include the erectile bodies (paired bulbs and paired corpora, which are continuous with the crura) and the glans clitoris. The glans is a midline, densely neural, non-erectile structure that is the only external manifestation of the clitoris. All other components are composed of erectile tissue with the composition of the bulbar erectile tissue differing from that of the corpora. The clitoral and perineal neurovascular bundles are large, paired terminations of the pudendal neurovascular bundles. The clitoral neurovascular bundles ascend along the ischiopubic rami to meet each other and pass along the superior surface of the clitoral body supplying the clitoris. The neural trunks pass largely intact into the glans. These nerves are at least 2 mm in diameter even in infancy. The cavernous or autonomic neural anatomy is microscopic and difficult to define consistently. MRI complements dissection studies and clarifies the anatomy. Clitoral pharmacology and histology appears to parallel those of penile tissue, although the clinical impact is vastly different. Typical textbook descriptions of the clitoris lack detail and include inaccuracies. It is impossible to convey clitoral anatomy in a single diagram showing only 1 plane, as is typically provided in textbooks, which reveal it as a flat structure. MRI provides a multiplanar representation of clitoral anatomy in the live state, which is a major advantage, and complements dissection materials. The work of Kobelt in the early 19th century provides a most comprehensive and accurate description of clitoral anatomy, and modern study provides objective images and few novel findings. The bulbs appear to be part of the clitoris. They are spongy in character and in continuity with the other parts of the clitoris. The distal urethra and vagina are intimately related structures, although they are not erectile in character. They form a tissue cluster with the clitoris. This cluster appears to be the locus of female sexual function and orgasm.

Microarray-based comparison of three amplification methods for nanogram amounts of total RNA

PubMed Central

Singh, Ruchira; Maganti, Rajanikanth J.; Jabba, Sairam V.; Wang, Martin; Deng, Glenn; Heath, Joe Don; Kurn, Nurith; Wangemann, Philine

2007-01-01

Gene expression profiling using microarrays requires microgram amounts of RNA, which limits its direct application for the study of nanogram RNA samples obtained using microdissection, laser capture microscopy, or needle biopsy. A novel system based on Ribo-SPIA technology (RS, Ovation-Biotin amplification and labeling system) was recently introduced. The utility of the RS system, an optimized prototype system for picogram RNA samples (pRS), and two T7-based systems involving one or two rounds of amplification (OneRA, Standard Protocol, or TwoRA, Small Sample Prototcol, version II) were evaluated in the present study. Mouse kidney (MK) and mouse universal reference (MUR) RNA samples, 0.3 ng to 10 μg, were analyzed using high-density Affymetrix Mouse Genome 430 2.0 GeneChip arrays. Call concordance between replicates, correlations of signal intensity, signal intensity ratios, and minimal fold increase necessary for significance were determined. All systems amplified partially overlapping sets of genes with similar signal intensity correlations. pRS amplified the highest number of genes from 10-ng RNA samples. We detected 24 of 26 genes verified by RT-PCR in samples prepared using pRS. TwoRA yielded somewhat higher call concordances than did RS and pRS (91.8% vs. 89.3% and 88.1%, respectively). Although all target preparation methods were suitable, pRS amplified the highest number of targets and was found to be suitable for amplification of as little as 0.3 ng of total RNA. In addition, RS and pRS were faster and simpler to use than the T7-based methods and resulted in the generation of cDNA, which is more stable than cRNA. PMID:15613496

Poplar Wood Rays Are Involved in Seasonal Remodeling of Tree Physiology1[C][W

PubMed Central

Larisch, Christina; Dittrich, Marcus; Wildhagen, Henning; Lautner, Silke; Fromm, Jörg; Polle, Andrea; Hedrich, Rainer; Rennenberg, Heinz; Müller, Tobias; Ache, Peter

2012-01-01

Understanding seasonality and longevity is a major challenge in tree biology. In woody species, growth phases and dormancy follow one another consecutively. In the oldest living individuals, the annual cycle may run for more than 1,000 years. So far, however, not much is known about the processes triggering reactivation from dormancy. In this study, we focused on wood rays, which are known to play an important role in tree development. The transition phase from dormancy to flowering in early spring was compared with the phase of active growth in summer. Rays from wood samples of poplar (Populus × canescens) were enriched by laser microdissection, and transcripts were monitored by poplar whole-genome microarrays. The resulting seasonally varying complex expression and metabolite patterns were subjected to pathway analyses. In February, the metabolic pathways related to flower induction were high, indicating that reactivation from dormancy was already taking place at this time of the year. In July, the pathways related to active growth, like lignin biosynthesis, nitrogen assimilation, and defense, were enriched. Based on “marker” genes identified in our pathway analyses, we were able to validate periodical changes in wood samples by quantitative polymerase chain reaction. These studies, and the resulting ray database, provide new insights into the steps underlying the seasonality of poplar trees. PMID:22992511

Investigation of the "true" extraction recovery of analytes from multiple types of tissues and its impact on tissue bioanalysis using two model compounds.

PubMed

Yuan, Long; Ma, Li; Dillon, Lisa; Fancher, R Marcus; Sun, Huadong; Zhu, Mingshe; Lehman-McKeeman, Lois; Aubry, Anne-Françoise; Ji, Qin C

2016-11-16

LC-MS/MS has been widely applied to the quantitative analysis of tissue samples. However, one key remaining issue is that the extraction recovery of analyte from spiked tissue calibration standard and quality control samples (QCs) may not accurately represent the "true" recovery of analyte from incurred tissue samples. This may affect the accuracy of LC-MS/MS tissue bioanalysis. Here, we investigated whether the recovery determined using tissue QCs by LC-MS/MS can accurately represent the "true" recovery from incurred tissue samples using two model compounds: BMS-986104, a S1P 1 receptor modulator drug candidate, and its phosphate metabolite, BMS-986104-P. We first developed a novel acid and surfactant assisted protein precipitation method for the extraction of BMS-986104 and BMS-986104-P from rat tissues, and determined their recoveries using tissue QCs by LC-MS/MS. We then used radioactive incurred samples from rats dosed with 3 H-labeled BMS-986104 to determine the absolute total radioactivity recovery in six different tissues. The recoveries determined using tissue QCs and incurred samples matched with each other very well. The results demonstrated that, in this assay, tissue QCs accurately represented the incurred tissue samples to determine the "true" recovery, and LC-MS/MS assay was accurate for tissue bioanalysis. Another aspect we investigated is how the tissue QCs should be prepared to better represent the incurred tissue samples. We compared two different QC preparation methods (analyte spiked in tissue homogenates or in intact tissues) and demonstrated that the two methods had no significant difference when a good sample preparation was in place. The developed assay showed excellent accuracy and precision, and was successfully applied to the quantitative determination of BMS-986104 and BMS-986104-P in tissues in a rat toxicology study. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of Noninvasive Biomarkers for Diagnosing and Monitoring Nonindolent Prostate Cancer

DTIC Science & Technology

2013-04-01

of higher-grade non-indolent tumors. By gene expression analysis (from microdissected Gleason-pattern (GP) 3 and GP4 PCa), in combination with...publically available Gleason-associated transcriptional profiles, we have created a 46- gene panel that differentiates high Gleason from low Gleason...We validated the GP4-associated upregulation of candidate genes by qPCR. Additionally, we have started to measure by qPCR the transcript levels for

Principles, Techniques, and Applications of Tissue Microfluidics

NASA Technical Reports Server (NTRS)

Wade, Lawrence A.; Kartalov, Emil P.; Shibata, Darryl; Taylor, Clive

2011-01-01

The principle of tissue microfluidics and its resultant techniques has been applied to cell analysis. Building microfluidics to suit a particular tissue sample would allow the rapid, reliable, inexpensive, highly parallelized, selective extraction of chosen regions of tissue for purposes of further biochemical analysis. Furthermore, the applicability of the techniques ranges beyond the described pathology application. For example, they would also allow the posing and successful answering of new sets of questions in many areas of fundamental research. The proposed integration of microfluidic techniques and tissue slice samples is called tissue microfluidics because it molds the microfluidic architectures in accordance with each particular structure of each specific tissue sample. Thus, microfluidics can be built around the tissues, following the tissue structure, or alternatively, the microfluidics can be adapted to the specific geometry of particular tissues. By contrast, the traditional approach is that microfluidic devices are structured in accordance with engineering considerations, while the biological components in applied devices are forced to comply with these engineering presets. The proposed principles represent a paradigm shift in microfluidic technology in three important ways: Microfluidic devices are to be directly integrated with, onto, or around tissue samples, in contrast to the conventional method of off-chip sample extraction followed by sample insertion in microfluidic devices. Architectural and operational principles of microfluidic devices are to be subordinated to suit specific tissue structure and needs, in contrast to the conventional method of building devices according to fluidic function alone and without regard to tissue structure. Sample acquisition from tissue is to be performed on-chip and is to be integrated with the diagnostic measurement within the same device, in contrast to the conventional method of off-chip sample prep and subsequent insertion into a diagnostic device. A more advanced form of tissue integration with microfluidics is tissue encapsulation, wherein the sample is completely encapsulated within a microfluidic device, to allow for full surface access. The immediate applications of these approaches lie with diagnostics of tissue slices and biopsy samples e.g. for cancer but the approaches would also be very useful in comparative genomics and other areas of fundamental research involving heterogeneous tissue samples.

Conjunct SEP and MEP monitoring in resection of infratentorial lesions: lessons learned in a cohort of 210 patients.

PubMed

Kodama, Kunihiko; Javadi, Mani; Seifert, Volker; Szelényi, Andrea

2014-12-01

During the surgical removal of infratentorial lesions, intraoperative neuromonitoring is mostly focused on cranial nerve assessment and brainstem auditory potentials. Despite the known risk of perforating vessel injury during microdissection within the vicinity of the brainstem, there are few reports about intraoperative neuromonitoring with somatosensory evoked potentials (SEPs) and motor evoked potentials (MEPs) assessing the medial lemniscus and corticospinal tract. This study analyses the occurrence of intraoperative changes in MEPs and SEPs with regard to lesion location and postoperative neurological outcome. The authors analyzed 210 cases in which patients (mean age 49 ± 13 years, 109 female) underwent surgeries involving the skull base (n = 104), cerebellum (n = 63), fourth ventricle (n = 28), brainstem (n = 12), and foramen magnum (n = 3). Of 210 surgeries, 171 (81.4%) were uneventful with respect to long-tract monitoring. Nine (23%) of the 39 SEP and/or MEP alterations were transient and were only followed by a slight permanent deficit in 1 case. Permanent deterioration only was seen in 19 (49%) of 39 cases; the deterioration was related to tumor dissection in 4 of these cases, and permanent deficit (moderate-severe) was seen in only 1 of these 4 cases. Eleven patients (28%) had losses of at least 1 modality, and in 9 of these 11 cases, the loss was related to surgical microdissection within the vicinity of the brainstem. Four of these 9 patients suffered a moderate-to-severe long-term deficit. For permanent changes, the positive predictive value for neuromonitoring of the long tracts was 0.467, the negative predictive value was 0.989, the sensitivity was 0.875, and the specificity 0.918. Twenty-eight (72%) of 39 SEP and MEP alterations occurred in 66 cases involving intrinsic brainstem tumors or tumors adjacent to the brainstem. Lesion location and alterations in intraoperative neuromonitoring significantly correlated with patients' outcome (p < 0.001, chi-square test). In summary, long-tract monitoring with SEPs and MEPs in infratentorial surgeries has a high sensitivity and negative predictive value with respect to postoperative neurological status. It is recommended especially in those surgeries in which microdissection within and in the vicinity of the brainstem might lead to injury of the brainstem parenchyma or perforating vessels and a subsequent perfusion deficit within the brainstem.

The role of miRNAs in human papilloma virus (HPV)-associated cancers: bridging between HPV-related head and neck cancer and cervical cancer

PubMed Central

Lajer, C B; Garnæs, E; Friis-Hansen, L; Norrild, B; Therkildsen, M H; Glud, M; Rossing, M; Lajer, H; Svane, D; Skotte, L; Specht, L; Buchwald, C; Nielsen, F C

2012-01-01

Background: Although the role of human papilloma virus (HPV) in cervical squamous cell carcinoma (CSCC) is well established, the role in head and neck SCC (HNSCC) is less clear. MicroRNAs (miRNAs) have a role in the cancer development, and HPV status may affect the miRNA expression pattern in HNSCC. To explore the influence of HPV in HNSCC, we made a comparative miRNA profile of HPV-positive (HPV+) and HPV-negative (HPV−) HNSCC against CSCC. Methods: Fresh frozen and laser microdissected-paraffin-embedded samples obtained from patients with HPV+/HPV− HNSCC, CSCC and controls were used for microarray analysis. Differentially expressed miRNAs in the HPV+ and HPV− HNSCC samples were compared with the differentially expressed miRNAs in the CSCC samples. Results: Human papilloma virus positive (+) HNSCC had a distinct miRNA profile compared with HPV− HNSCC. Significantly more similarity was seen between HPV+ HNSCC and CSCC than HPV− and CSCC. A set of HPV core miRNAs were identified. Of these especially the miR-15a/miR-16/miR195/miR-497 family, miR-143/miR-145 and the miR-106-363 cluster appear to be important within the known HPV pathogenesis. Conclusion: This study adds new knowledge to the known pathogenic pathways of HPV and substantiates the oncogenic role of HPV in subsets of HNSCCs. PMID:22472886

The anatomy of the intralingual neural interconnections.

PubMed

Păduraru, Dumitru; Rusu, Mugurel Constantin

2013-08-01

The intrinsic lingual neural interconnections are overlooked. It was hypothesized that intralingual anatomically well defined anastomoses interconnect the somatic and autonomic neural systems of the tongue. It was thus aimed to evaluate the intralingual neural scaffold in human tongues. Human tongue samples (ten adult and one pediatric) were microdissected (4.5 magnification). In the interstitium between the genioglossus and hyoglossus muscles, the branches of the lingual nerve (LN) and the medial trunk of the hypoglossal nerve (HN) had a layered disposition of the outer and inner side, respectively, of the lingual artery with its periarterial plexus. Anastomoses of these three distinctive neural suppliers of tongue were recorded, as also were those of the LN with the lateral trunk of the HN and the anastomoses between successive terminal branches of the LN. Successive ansae linguales were joining the LN branches and the medial trunk of the HN. The intrinsic neural system of the tongue supports integrative functions and allows a better retrospective understanding of various experimental studies. The topographical pattern is useful for an accurate diagnosis of intralingual nerves on microscopic slides.

Phospholipid composition of the plasma membrane of the green alga, Hydrodictyon africanum.

PubMed Central

Bailey, D S; Northcote, D H

1976-01-01

A plasma-membrane fraction was isolated from the alga Hydrodictyon africanum by micro-dissection and its phospholipid components were analysed. Phosphatidylcholine was the major phospholipid of the preparation. Both phosphatidylserine and diphosphatidylglycerol were enriched in the fraction compared with the whole cell, but the relative amount of phosphatidylglycerol present was less than that in the whole cell. Phosphatidylinositol was absent from the plasma-membrane preparation. Images PLATE 1 PLATE 2 PMID:182144

Subcellular analysis by laser ablation electrospray ionization mass spectrometry

DOEpatents

Vertes, Akos; Stolee, Jessica A; Shrestha, Bindesh

2014-12-02

In various embodiments, a method of laser ablation electrospray ionization mass spectrometry (LAESI-MS) may generally comprise micro-dissecting a cell comprising at least one of a cell wall and a cell membrane to expose at least one subcellular component therein, ablating the at least one subcellular component by an infrared laser pulse to form an ablation plume, intercepting the ablation plume by an electrospray plume to form ions, and detecting the ions by mass spectrometry.

Disruption of copper-dependent signaling pathway in the nitrofen-induced congenital diaphragmatic hernia.

PubMed

Takahashi, Toshiaki; Friedmacher, Florian; Takahashi, Hiromizu; Hofmann, Alejandro Daniel; Puri, Prem

2015-01-01

Normal development of the fetal diaphragm requires muscularization of the diaphragm as well as the structural integrity of its underlying connective tissue components. Developmental mutations that inhibit the formation of extracellular matrix (ECM) have been shown to result in congenital diaphragmatic hernia (CDH). Copper (Cu) is an important element during diaphragm morphogenesis by participating in cross-linking of collagen and elastin fibers. Cu transport is strictly regulated by two membrane proteins: Cu-uptake transporter 1 (CTR1) and the Cu-efflux pump ATP7A. Animals lacking Cu-dependent enzymes exhibit abnormal connective tissue with diaphragmatic defects. However, the molecular basis of disruptions in Cu-mediated ECM formation in CDH remains unclear. We designed this study to investigate the hypothesis that diaphragmatic expression of CTR1 and ATP7A is decreased in the nitrofen-induced CDH model. Timed-pregnant rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on selected time-points D15 and D18. Microdissected fetal diaphragms (n = 48) were divided into control and nitrofen-induced CDH samples (n = 12 per experimental group and time-point). Diaphragmatic gene expression levels of CTR1 and ATP7A were analyzed by quantitative real-time polymerase chain reaction. Immunohistochemistry was performed to evaluate CTR1 and ATP7A protein expression in fetal diaphragms, which was combined with specific rhodanine staining to determine diaphragmatic Cu content. Relative mRNA levels of CTR1 and ATP7A were significantly reduced in diaphragms of nitrofen-exposed fetuses on D15 (0.06 ± 0.02 vs. 0.18 ± 0.08; p < 0.05 and 0.04 ± 0.02 vs. 0.08 ± 0.02; p < 0.05) and D18 (0.10 ± 0.03 vs. 0.17 ± 0.02; p < 0.05 and 0.09 ± 0.03 vs. 0.16 ± 0.04; p < 0.05) compared to controls. Immunoreactivity of CTR1 and ATP7A was markedly decreased in the malformed diaphragmatic ECM of nitrofen-exposed fetuses on D15 and D18, which was associated with a significantly decreased diaphragmatic Cu content on D15 (7.22 ± 2.91 vs. 17.50 ± 3.09; p < 0.05) and D18 (17.60 ± 3.54 vs. 28.20 ± 4.63; p < 0.05) compared to controls. Reduced diaphragmatic expression of CTR1 and ATP7A during morphogenesis may impair the activity of Cu-dependent enzymes and thus contribute to defective ECM during diaphragmatic development.

Fibrillin-1 Expression Is Decreased in the Diaphragmatic Muscle Connective Tissue of Nitrofen-Induced Congenital Diaphragmatic Hernia.

PubMed

Takahashi, Toshiaki; Friedmacher, Florian; Zimmer, Julia; Puri, Prem

2017-02-01

Introduction  Diaphragmatic morphogenesis depends on proper formation of muscle connective tissue (MCT) and underlying extracellular matrix (ECM). Fibrillin-1 is an essential ECM protein and crucial for the structural integrity of MCT in the developing diaphragm. Recently, mutations in the fibrillin-1 gene (FBN1) have been identified in cases of congenital diaphragmatic hernia (CDH), thus suggesting that alterations in FBN1 gene expression may lead to diaphragmatic defects. We designed this study to investigate the hypothesis that the diaphragmatic expression of fibrillin-1 is decreased in the MCT of nitrofen-induced CDH. Materials and Methods  Time-mated rats were exposed to nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms ( n  = 72) were harvested on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Laser-capture microdissection was used to obtain diaphragmatic tissue cells. Gene expression levels of FBN1 were analyzed by qRT-PCR. Immunofluorescence-double-staining for fibrillin-1 and the mesenchymal marker Gata4 was performed to evaluate protein expression and localization. Results  Relative mRNA expression of FBN1 was significantly decreased in pleuroperitoneal folds on D13 (3.39 ± 1.29 vs. 5.47 ± 1.92; p  < 0.05), developing diaphragms on D15 (2.48 ± 0.89 vs. 4.03 ± 1.62; p  < 0.05), and fully muscularized diaphragms on D18 (2.49 ± 0.69 vs. 3.93 ± 1.55; p  < 0.05) of nitrofen-exposed fetuses compared with controls. Confocal-laser-scanning microscopy revealed markedly diminished fibrillin-1 immunofluorescence mainly in MCT, associated with a reduction of proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15, and D18 compared with controls. Conclusions  Decreased expression of fibrillin-1 during morphogenesis of the fetal diaphragm may disrupt mesenchymal cell proliferation, causing malformed MCT and thus resulting in diaphragmatic defects in the nitrofen-induced CDH model. Georg Thieme Verlag KG Stuttgart · New York.

Wound healing, calcium signaling, and other novel pathways are associated with the formation of butterfly eyespots.

PubMed

Özsu, Nesibe; Monteiro, Antónia

2017-10-16

One hypothesis surrounding the origin of novel traits is that they originate from the co-option of pre-existing genes or larger gene regulatory networks into novel developmental contexts. Insights into a trait's evolutionary origins can, thus, be gained via identification of the genes underlying trait development, and exploring whether those genes also function in other developmental contexts. Here we investigate the set of genes associated with the development of eyespot color patterns, a trait that originated once within the Nymphalid family of butterflies. Although several genes associated with eyespot development have been identified, the eyespot gene regulatory network remains largely unknown. In this study, next-generation sequencing and transcriptome analyses were used to identify a large set of genes associated with eyespot development of Bicyclus anynana butterflies, at 3-6 h after pupation, prior to the differentiation of the color rings. Eyespot-associated genes were identified by comparing the transcriptomes of homologous micro-dissected wing tissues that either develop or do not develop eyespots in wild-type and a mutant line of butterflies, Spotty, with extra eyespots. Overall, 186 genes were significantly up and down-regulated in wing tissues that develop eyespots compared to wing tissues that do not. Many of the differentially expressed genes have yet to be annotated. New signaling pathways, including the Toll, Fibroblast Growth Factor (FGF), extracellular signal-regulated kinase (ERK) and/or Jun N-terminal kinase (JNK) signaling pathways are associated for the first time with eyespot development. In addition, several genes involved in wound healing and calcium signaling were also found to be associated with eyespots. Overall, this study provides the identity of many new genes and signaling pathways associated with eyespots, and suggests that the ancient wound healing gene regulatory network may have been co-opted to cells at the center of the pattern to aid in eyespot origins. New transcription factors that may be providing different identities to distinct wing sectors, and genes with sexually dimorphic expression in the eyespots were also identified.

Autoradiographic identification of acetylcholine in the rabbit retina

PubMed Central

1979-01-01

Rabbit retinas were studied in vitro under conditions known to maintain their physiological function. Retinas incubated in the presence of [3H]choline synthesized substantial amounts of both [3H]phosphorylcholine and [3H]acetylcholine. With time, [3H]phosphorylcholine proceeded into phospholipids, primarily phosphatidylcholine. Retinas pulse-labeled by a 15-min exposure to 0.3 microM [3H]choline were incubated for a subsequent hour under chase conditions designed either to retain newly synthesized acetylcholine within synapses or to promote its release. At the end of this time the two groups of retinas were found to contain equal amounts of radioactivity in the phospholipid pathway, but only the retinas incubated under the acetylcholine-protecting conditions contained [3H]acetylcholine. Freeze-dried, vacuum-embedded tissue from each retina was autoradiographed on dry emulsion. All retinas showed silver grains over the photoreceptor cells and faint labeling of all ganglion cells. In the retinas that contained [3H]acetylcholine, silver grains also accumulated densely over a few cells with the position of amacrine cells, over a subset of the cells of the ganglion cell layer, and in two bands over the inner plexiform layer. Fixation of the retina with aqueous osmium tetroxide retained only the radioactive compounds located in the photoreceptor and ganglion cells. Sections from freeze- dried tissue lost their water-soluble choline metabolites when exposed to water, and autoradiography of such sections again revealed radioactivity primarily in the photoreceptor and ganglion cells. Radioactive compounds extracted from the sections were found to faithfully reflect those present in the tissue before processing; analysis of the compounds eluted from sections microdissected along the outer plexiform layer showed [3H]acetylcholine to have been synthesized only by cells of the inner retina. Taken together, these results indicate that the photoreceptor and ganglion cells are distinguished by a rapid synthesis of choline-containing phospholipids, while acetylcholine synthesis is restricted to a few cells at both margins of the inner plexiform layer. They imply that the only neurons to release acetylcholine within the rabbit retina are a small group of probable amacrine cells. PMID:92476

Development of an Ammonium Sulfate DNA Extraction Method for Obtaining Amplifiable DNA in a Small Number of Cells and Its Application to Clinical Specimens

PubMed Central

Oh, Seo Young; Kim, Wook Youn; Hwang, Tae Sook; Han, Hye Seung; Lim, So Dug; Kim, Wan Seop

2013-01-01

DNA extraction from microdissected cells has become essential for handling clinical specimens with advances in molecular pathology. Conventional methods have limitations for extracting amplifiable DNA from specimens containing a small number of cells. We developed an ammonium sulfate DNA extraction method (A) and compared it with two other methods (B and C). DNA quality and quantity, β-globin amplification, and detectability of two cancer associated gene mutations were evaluated. Method A showed the best DNA yield, particularly when the cell number was very low. Amplification of the β-globin gene using DNA from the SNU 790 cell line and papillary thyroid carcinoma (PTC) cells extracted with Method A demonstrated the strongest band. BRAF V600E mutation analysis using ethanol-fixed PTC cells from a patient demonstrated both a “T” peak increase and an adjacent “A” peak decrease when 25 and 50 cells were extracted, whereas mutant peaks were too low to be analyzed using the other two methods. EGFR mutation analysis using formalin-fixed paraffin-embedded lung cancer tissues demonstrated a mutant peak with Method A, whereas the mutant peak was undetectable with Methods B or C. Method A yielded the best DNA quantity and quality with outstanding efficiency, particularly when paucicellular specimens were used. PMID:23691506

Anatomic documentation of the G-spot complex role in the genesis of anterior vaginal wall ballooning.

PubMed

Ostrzenski, Adam

2014-09-01

To expand previous G-spot anatomical and histological investigations; to examine the G-spot complex anatomic role in the anterior vaginal wall ballooning bio-mechanisms; and to determine, which division of autonomic nervous system (sympathetic or parasympathetic) dominates at the time of female sudden death. A prospective-descriptive case series anatomical study on eleven consecutive fresh humane female cadavers was conducted. Anterior vaginal wall stratum-by-stratum macro-dissections were executed in axial, coronal and sagittal plains. Upon G-spot extirpations, micro-dissections were performed. The G-spot tissues were stained with hematoxilin and eosin for histological examinations to authenticate the G-spot anatomical and histological characteristic features. The G-spot complex was identified and present in all subjects on either the distal vaginal left (more often) or on the right side from the lateral margin of the urethra; the G-spot anatomical and microscopic characteristic features have been authenticated; the G-spot complex expansion elevated anterior vaginal walls in each subject; the autonomic parasympathetic nervous system was the dominant division at the time of female subject sudden death. This study advances our anatomical and histological understanding of the G-spot complex and its role in the genesis of anterior vaginal ballooning bio-mechanisms. The G-spot complex is under parasympathetic nervous system domination at the time of female sudden death. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

Glioblastomas with Oligodendroglial Component – Common Origin of the Different Histological Parts and Genetic Subclassification

PubMed Central

Klink, Barbara; Schlingelhof, Ben; Klink, Martin; Stout-Weider, Karen; Patt, Stephan; Schrock, Evelin

2010-01-01

Background: Glioblastomas are the most common and most malignant brain tumors in adults. A small subgroup of glioblastomas contains areas with histological features of oligodendroglial differentiation (GBMO). Our objective was to genetically characterize the oligodendroglial and the astrocytic parts of GBMOs and correlate morphologic and genetic features with clinical data. Methods: The oligodendroglial and the “classic” glioblastoma parts of 13 GBMO were analyzed separately by interphase fluorescence in situ hybridization (FISH) on paraffin sections using a custom probe set (regions 1p, 1q, 7q, 10q, 17p, 19q, cen18, 21q) and by comparative genomic hybridization (CGH) of microdissected paraffin embedded tumor tissue. Results: We identified four distinct genetic subtypes in 13 GBMOs: an “astrocytic” subtype (9/13) characterized by +7/−10; an “oligodendroglial” subtype with −1p/−19q (1/13); an “intermediate” subtype showing +7/−1p (1/13), and an “other” subtype having none of the former aberrations typical for gliomas (2/13). The different histological tumor parts of GBMO revealed common genetic changes in all tumors and showed additional aberrations specific for each part. Conclusion: Our findings demonstrate the monoclonal origin of GBMO followed by the development of the astrocytic and oligodendroglial components. The diagnostic determination of the genetic signatures may allow for a better prognostication of the patients. PMID:20966543

IDENTIFICATION OF NOVEL FIBROBLAST GROWTH FACTOR RECEPTOR 3 GENE MUTATIONS IN ACTINIC CHEILITIS

PubMed Central

Chou, Annie; Dekker, Nusi; Jordan, Richard C.K.

2009-01-01

Objective Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are responsible for several craniosynostosis and chondrodysplasia syndromes as well as some human cancers including bladder and cervical carcinoma. Despite a high frequency in some benign skin disorders, FGFR3 mutations have not been reported in cutaneous malignancies. Actinic cheilitis (AC) is a sun-induced premalignancy affecting the lower lip that frequently progresses to squamous cell carcinoma (SCC). The objective of this study was to determine if FGFR3 gene mutations are present in AC and SCC of the lip. Study Design DNA was extracted and purified from micro-dissected, formalin-fixed, paraffin-embedded tissue sections of 20 cases of AC and SCC arising in AC. Exons 7, 15, and 17 were PCR amplified and direct sequenced. Results Four novel somatic mutations in the FGFR3 gene were identified: exon 7 mutation 742C→T (amino acid change R248C), exon 15 mutations 1850A→G (D617G) and 1888G→A (V630M), and exon 17 mutation 2056G→A (E686K). Grade of dysplasia did not correlate with presence of mutations. Conclusion The frequency of FGFR3 receptor mutations suggests a functional role for the FGFR3 receptor in the development of epithelial disorders and perhaps a change may contribute to the pathogenesis of some AC and SCC. PMID:19327639

Molecular Characteristics of Mantle Cell Lymphoma Presenting with Clonal Plasma Cell Component

PubMed Central

Visco, Carlo; Hoeller, Sylvia; Malik, Jeffrey T.; Xu-Monette, Zijun Y.; Wiggins, Michele L.; Liu, Jessica; Sanger, Warren G.; Liu, Zhongfeng; Chang, Julie; Ranheim, Erik A.; Gradowski, Joel F.; Serrrano, Sergio; Wang, Huan-You; Liu, Qingquan; Dave, Sandeep; Olsen, Brian; Gascoyne, Randy D.; Campo, Elias; Swerdlow, Steven H.; Chan, Wing C.; Tzankov, Alexander; Young, Ken H.

2011-01-01

The normal counterparts of mantle cell lymphoma (MCL) are naïve quiescent B-cells that have not been processed through the germinal center (GC). For this reason, while lymphomas arising from GC or post-GC B-cells often exhibit plasmacytic differentiation, MCL rarely presents with plasmacytic features. Seven cases of MCL with a monotypic plasma cell (PC) population were collected from six centers and studied by immunohistochemistry, FICTION (Fluorescence immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Neoplasms), capillary gel electrophoresis, and restriction fragment length polymorphism of immunoglobulin heavy chain analysis (RFLP/IgH) of microdissections of each of the MCL and PC populations to assess their clonal relationship. Clinical presentation was rather unusual compared to typical MCL, with two cases arising from extranodal soft-tissues of the head. All MCL cases were morphologically and immunohistochemically typical, bearing the t(11;14)(q13;q32). In all cases PC populations were clonal. In 5 of the 7 cases, the MCL and PC clones showed identical restriction fragments, indicating a common clonal origin of the neoplastic populations. The two cases with clonal diversity denoted the coexistence of two different tumors in a composite lymphoma/plasma cell neoplasm. Our findings suggest that MCL can present with a PC component that is often clonally related to the lymphoma, representing a rare but unique biological variant of this tumor. PMID:21263238

Trace-Element Concentrations in Tissues of Aquatic Organisms from Rivers and Streams of the United States, 1992-1999

USGS Publications Warehouse

DeWeese, Lawrence R.; Stephens, Verlin C.; Short, Terry M.; Dubrovsky, Neil M.

2007-01-01

The U.S. Geological Survey National Water-Quality Assessment Program collected tissue samples from a variety of aquatic organisms during 1992-1999 within 47 study units across the United States. These tissue samples were collected to determine the occurrence and distribution of 20 major and minor trace elements in aquatic organisms. This report presents the tissue trace-element concentration data, sample summaries, and concentration statistics for 1,457 tissue samples representing 76 species or groups of fish, aquatic invertebrates, and plants were collected at 824 sampling sites.

Anatomic renal artery branch microdissection to facilitate zero-ischemia partial nephrectomy.

PubMed

Ng, Casey K; Gill, Inderbir S; Patil, Mukul B; Hung, Andrew J; Berger, Andre K; de Castro Abreu, Andre Luis; Nakamoto, Masahiko; Eisenberg, Manuel S; Ukimura, Osamu; Thangathurai, Duraiyah; Aron, Monish; Desai, Mihir M

2012-01-01

Robot-assisted and laparoscopic partial nephrectomies (PNs) for medial tumors are technically challenging even with the hilum clamped and, until now, were impossible to perform with the hilum unclamped. Evaluate whether targeted vascular microdissection (VMD) of renal artery branches allows zero-ischemia PN to be performed even for challenging medial tumors. A prospective cohort evaluation of 44 patients with renal masses who underwent robot-assisted or laparoscopic zero-ischemia PN either with anatomic VMD (group 1; n=22) or without anatomic VMD (group 2; n=22) performed by a single surgeon from April 2010 to January 2011. Zero-ischemia PN with VMD incorporates four maneuvers: (1) preoperative computed tomographic reconstruction of renal arterial branch anatomy, (2) anatomic dissection of targeted, tumor-specific tertiary or higher-order renal arterial branches, (3) neurosurgical aneurysm microsurgical bulldog clamp(s) for superselective tumor devascularization, and (4) transient, controlled reduction of blood pressure, if necessary. Baseline, perioperative, and postoperative data were collected prospectively. Group 1 tumors were larger (4.3 vs 2.6 cm; p=0.011), were more often hilar (41% vs 9%; p=0.09), were medial (59% and 23%; p=0.017), were closer to the hilum (1.46 vs 3.26 cm; p=0.0002), and had a lower C index score (2.1 vs 3.9; p=0.004) and higher RENAL nephrometry scores (7.7 vs 6.2; p=0.013). Despite greater complexity, no group 1 tumor required hilar clamping, and perioperative outcomes were similar to those of group 2: operating room time (4.7 and 4.1h), median blood loss (200 and 100ml), surgical margins for cancer (all negative), major complications (0% and 9%), and minor complications (18% and 14%). The median serum creatinine level was similar 2 mo postoperatively (1.2 and 1.3mg/dl). The study was limited by the relatively small sample size. Anatomic targeted dissection and superselective control of tumor-specific renal arterial branches facilitate zero-ischemia PN. Even challenging medial and hilar tumors can be excised without hilar clamping. Global surgical renal ischemia has been eliminated for most patients undergoing PN at our institution. Copyright © 2011 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Quantification of HCV RNA in Liver Tissue by bDNA Assay.

PubMed

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

1999-01-01

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

Combined Bisulfite Restriction Analysis for brain tissue identification.

PubMed

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

2018-05-01

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

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

NASA Astrophysics Data System (ADS)

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

2015-05-01

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

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

PubMed Central

Yu, Cheng-Chia; Chen, Chin-Chuan

2018-01-01

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

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

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

Demos, Stavros; Levenson, Richard

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

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

NASA Astrophysics Data System (ADS)

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

2017-07-01

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

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

PubMed

Varettas, Kerry

2013-12-01

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

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

PubMed

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

2018-01-31

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

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

PubMed

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

2015-06-01

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

Anatomical and echocardiographic correlates of normal cardiac morphology in the late first trimester fetus.

PubMed Central

Allan, L. D.; Santos, R.; Pexieder, T.

1997-01-01

OBJECTIVES: To describe the normal cardiac morphology as seen by transvaginal ultrasound imaging in the first trimester fetus and to compare it with the morphology of the heart as seen by microdissection at the same gestational age. DESIGN: In 53 mothers undergoing early sonography, the fetal heart was examined and the images recorded. The gestational age range was 5-12 weeks of gestation, which represents 21 to 70 days after conception. Images were analysed frame by frame and compared with the anatomy of embryos and fetuses at the same gestational ages. RESULTS: After the 9th week of gestation, four cardiac chambers, the aortic origin, and the pulmonary artery could be identified on cross sectional echocardiography in conjunction with colour flow Doppler. At 9 weeks, the apex pointed anteriorly and the right ventricle and pulmonary artery lay to the right of the midline. By the 11th week of gestation, the apex pointed to the left and the pulmonary artery lay to the left of the midline as in the older fetus. Between 9 and 12 weeks' gestation the aorta was larger than the pulmonary artery. These findings were confirmed in the microdissected hearts. CONCLUSIONS: The current quality of ultrasound images obtained using transvaginal transducers in the first trimester fetus allows the study of fetal cardiac anatomy. Some of the later developmental changes can be demonstrated. As technology improves further the details of earlier cardiac morphogenesis may also become visible. Images PMID:9038698

BCL2 expression in CD105 positive neoangiogenic cells and tumor progression in angioimmunoblastic T-cell lymphoma.

PubMed

Ratajczak, Philippe; Leboeuf, Christophe; Wang, Li; Brière, Josette; Loisel-Ferreira, Irmine; Thiéblemont, Catherine; Zhao, Wei-Li; Janin, Anne

2012-06-01

The angiogenic microenvironment has been known to be a component of angioimmunoblastic T-cell lymphoma since its initial characterization. We have shown that angioimmunoblastic T-cell lymphoma endothelial cells produce vascular endothelial growth factor-A (VEGFA), and participate in lymphoma progression. In squamous cell carcinoma, endothelial BCL2 expression induces a crosstalk with tumor cells through VEGFA, a major mediator of tumoral angiogenesis. In the present study, we analyzed BCL2 and VEGFA in 30 angioimmunoblastic T-cell lymphomas, using triple immunofluorescence to identify protein coexpression in well-characterized lymphoma cells and microenvironment neoangiogenic endothelial cells. Using quantitative real-time PCR, we assessed mRNA expression levels in laser-microdissected endothelial and lymphoma cells. In lymphoma cells, as in endothelial cells, BCL2 and VEGFA proteins were coexpressed. BCL2 was expressed only in neoangiogenic CD34(+)CD105(+) endothelial cells. In laser-microdissected cells, mRNA studies showed a significant relationship between BCL2 and VEGFA levels in CD34(+) endothelial cells, but not in CD3(+)CD10(+)lymphoma cells, or in CD34(+) endothelial cells from lymph node hyperplasia. Further study showed that, in AITL, BCL2 mRNA levels in CD34(+)CD105(+) neoangiogenic endothelial cells also correlated with microvessel density, International Prognostic Index, Ann Arbor stage, bone marrow involvement and elevated LDH. BCL2 expression by CD105(+) neoangiogenic endothelial cells is related to tumor progression in angioimmunoblastic T-cell lymphoma.

Enterochromaffin cells of the human gut: sensors for spices and odorants.

PubMed

Braun, Thomas; Voland, Petra; Kunz, Lars; Prinz, Christian; Gratzl, Manfred

2007-05-01

Release of serotonin from mucosal enterochromaffin cells triggered by luminal substances is the key event in the regulation of gut motility and secretion. We were interested to know whether nasal olfactory receptors are also expressed in the human gut mucosa by enterochromaffin cells and whether their ligands and odorants present in spices, fragrances, detergents, and cosmetics cause serotonin release. Receptor expression was studied by the reverse-transcription polymerase chain reaction method in human mucosal enterochromaffin cells isolated by laser microdissection and in a cell line derived from human enterochromaffin cells. Activation of the cells by odorants was investigated by digital fluorescence imaging using the fluorescent Ca(2+) indicator Fluo-4. Serotonin release was measured in culture supernatants by a serotonin enzyme immunoassay and amperometry using carbon fiber microelectrodes placed on single cells. We found expression of 4 olfactory receptors in microdissected human mucosal enterochromaffin cells and in a cell line derived from human enterochromaffin cells. Ca(2+) imaging studies revealed that odorant ligands of the identified olfactory receptors cause Ca(2+) influx, elevation of intracellular free Ca(2+) levels, and, consequently, serotonin release. Our results show that odorants present in the luminal environment of the gut may stimulate serotonin release via olfactory receptors present in human enterochromaffin cells. Serotonin controls both gut motility and secretion and is implicated in pathologic conditions such as vomiting, diarrhea, and irritable bowel syndrome. Thus, olfactory receptors are potential novel targets for the treatment of gastrointestinal diseases and motility disorders.

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

PubMed

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

2016-09-01

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

Blind Biobanking of the Prostatectomy Specimen: Critical Evaluation of the Existing Techniques and Development of the New 4-Level Tissue Extraction Model With High Sampling Efficacy.

PubMed

Tolkach, Yuri; Eminaga, Okyaz; Wötzel, Fabian; Huss, Sebastian; Bettendorf, Olaf; Eltze, Elke; Abbas, Mahmoud; Imkamp, Florian; Semjonow, Axel

2017-03-01

Fresh tissue is mandatory to perform high-quality translation studies. Several models for tissue extraction from prostatectomy specimens without guidance by frozen sections are already introduced. However, little is known about the sampling efficacy of these models, which should provide representative tissue in adequate volumes, account for multifocality and heterogeneity of tumor, not violate the routine final pathological examination, and perform quickly without frozen section-based histological control. The aim of the study was to evaluate the sampling efficacy of the existing tissue extraction models without guidance by frozen sections ("blind") and to develop an optimized model for tissue extraction. Five hundred thirty-three electronic maps of the tumor distribution in prostates from a single-center cohort of the patients subjected to radical prostatectomy were used for analysis. Six available models were evaluated in silico for their sampling efficacy. Additionally, a novel model achieving the best sampling efficacy was developed. The available models showed high efficacies for sampling "any part" from the tumor (up to 100%), but were uniformly low in efficacy to sample all tumor foci from the specimens (with the best technique sampling only 51.6% of the all tumor foci). The novel 4-level extraction model achieved a sampling efficacy of 93.1% for all tumor foci. The existing "blind" tissue extraction models from prostatectomy specimens without frozen sections control are suitable to target tumor tissues but these tissues do not represent the whole tumor. The novel 4-level model provides the highest sampling efficacy and a promising potential for integration into routine. Prostate 77: 396-405, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

PubMed

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

2015-11-01

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

Characteristic Changes in Decidual Gene Expression Signature in Spontaneous Term Parturition

PubMed Central

El-Azzamy, Haidy; Balogh, Andrea; Romero, Roberto; Xu, Yi; LaJeunesse, Christopher; Plazyo, Olesya; Xu, Zhonghui; Price, Theodore G.; Dong, Zhong; Tarca, Adi L.; Papp, Zoltan; Hassan, Sonia S.; Chaiworapongsa, Tinnakorn; Kim, Chong Jai; Gomez-Lopez, Nardhy; Than, Nandor Gabor

2017-01-01

Background The decidua has been implicated in the “terminal pathway” of human term parturition, which is characterized by the activation of pro-inflammatory pathways in gestational tissues. However, the transcriptomic changes in the decidua leading to terminal pathway activation have not been systematically explored. This study aimed to compare the decidual expression of developmental signaling and inflammation-related genes before and after spontaneous term labor in order to reveal their involvement in this process. Methods Chorioamniotic membranes were obtained from normal pregnant women who delivered at term with spontaneous labor (TIL, n = 14) or without labor (TNL, n = 15). Decidual cells were isolated from snap-frozen chorioamniotic membranes with laser microdissection. The expression of 46 genes involved in decidual development, sex steroid and prostaglandin signaling, as well as pro- and anti-inflammatory pathways, was analyzed using high-throughput quantitative real-time polymerase chain reaction (qRT-PCR). Chorioamniotic membrane sections were immunostained and then semi-quantified for five proteins, and immunoassays for three chemokines were performed on maternal plasma samples. Results The genes with the highest expression in the decidua at term gestation included insulin-like growth factor-binding protein 1 (IGFBP1), galectin-1 (LGALS1), and progestogen-associated endometrial protein (PAEP); the expression of estrogen receptor 1 (ESR1), homeobox A11 (HOXA11), interleukin 1β (IL1B), IL8, progesterone receptor membrane component 2 (PGRMC2), and prostaglandin E synthase (PTGES) was higher in TIL than in TNL cases; the expression of chemokine C-C motif ligand 2 (CCL2), CCL5, LGALS1, LGALS3, and PAEP was lower in TIL than in TNL cases; immunostaining confirmed qRT-PCR data for IL-8, CCL2, galectin-1, galectin-3, and PAEP; and no correlations between the decidual gene expression and the maternal plasma protein concentrations of CCL2, CCL5, and IL-8 were found. Conclusions Our data suggests that with the initiation of parturition, the decidual expression of anti-inflammatory mediators decreases, while the expression of pro-inflammatory mediators and steroid receptors increases. This shift may affect downstream signaling pathways that can lead to parturition. PMID:28226203

Residual antibiotics in decontaminated human cardiovascular tissues intended for transplantation and risk of falsely negative microbiological analyses.

PubMed

Buzzi, Marina; Guarino, Anna; Gatto, Claudio; Manara, Sabrina; Dainese, Luca; Polvani, Gianluca; Tóthová, Jana D'Amato

2014-01-01

We investigated the presence of antibiotics in cryopreserved cardiovascular tissues and cryopreservation media, after tissue decontamination with antibiotic cocktails, and the impact of antibiotic residues on standard tissue bank microbiological analyses. Sixteen cardiovascular tissues were decontaminated with bank-prepared cocktails and cryopreserved by two different tissue banks according to their standard operating procedures. Before and after decontamination, samples underwent microbiological analysis by standard tissue bank methods. Cryopreserved samples were tested again with and without the removal of antibiotic residues using a RESEP tube, after thawing. Presence of antibiotics in tissue homogenates and processing liquids was determined by a modified agar diffusion test. All cryopreserved tissue homogenates and cryopreservation media induced important inhibition zones on both Staphylococcus aureus- and Pseudomonas aeruginosa-seeded plates, immediately after thawing and at the end of the sterility test. The RESEP tube treatment markedly reduced or totally eliminated the antimicrobial activity of tested tissues and media. Based on standard tissue bank analysis, 50% of tissues were found positive for bacteria and/or fungi, before decontamination and 2 out of 16 tested samples (13%) still contained microorganisms after decontamination. After thawing, none of the 16 cryopreserved samples resulted positive with direct inoculum method. When the same samples were tested after removal of antibiotic residues, 8 out of 16 (50%) were contaminated. Antibiotic residues present in tissue allografts and processing liquids after decontamination may mask microbial contamination during microbiological analysis performed with standard tissue bank methods, thus resulting in false negatives.

Residual Antibiotics in Decontaminated Human Cardiovascular Tissues Intended for Transplantation and Risk of Falsely Negative Microbiological Analyses

PubMed Central

Gatto, Claudio; Manara, Sabrina; Dainese, Luca; Polvani, Gianluca; Tóthová, Jana D'Amato

2014-01-01

We investigated the presence of antibiotics in cryopreserved cardiovascular tissues and cryopreservation media, after tissue decontamination with antibiotic cocktails, and the impact of antibiotic residues on standard tissue bank microbiological analyses. Sixteen cardiovascular tissues were decontaminated with bank-prepared cocktails and cryopreserved by two different tissue banks according to their standard operating procedures. Before and after decontamination, samples underwent microbiological analysis by standard tissue bank methods. Cryopreserved samples were tested again with and without the removal of antibiotic residues using a RESEP tube, after thawing. Presence of antibiotics in tissue homogenates and processing liquids was determined by a modified agar diffusion test. All cryopreserved tissue homogenates and cryopreservation media induced important inhibition zones on both Staphylococcus aureus- and Pseudomonas aeruginosa-seeded plates, immediately after thawing and at the end of the sterility test. The RESEP tube treatment markedly reduced or totally eliminated the antimicrobial activity of tested tissues and media. Based on standard tissue bank analysis, 50% of tissues were found positive for bacteria and/or fungi, before decontamination and 2 out of 16 tested samples (13%) still contained microorganisms after decontamination. After thawing, none of the 16 cryopreserved samples resulted positive with direct inoculum method. When the same samples were tested after removal of antibiotic residues, 8 out of 16 (50%) were contaminated. Antibiotic residues present in tissue allografts and processing liquids after decontamination may mask microbial contamination during microbiological analysis performed with standard tissue bank methods, thus resulting in false negatives. PMID:25397402

Fit for purpose frozen tissue collections by RNA integrity number-based quality control assurance at the Erasmus MC tissue bank.

PubMed

Kap, Marcel; Oomen, Monique; Arshad, Shazia; de Jong, Bas; Riegman, Peter

2014-04-01

About 5000 frozen tissue samples are collected each year by the Erasmus Medical Center tissue bank. Two percent of these samples are randomly selected annually for RNA isolation and RNA Integrity Number (RIN) measurement. A similar quality assessment was conducted during centralization of a 20-year-old tissue collection from the cancer institute, a 15-year-old liver sample archive (-80°C), and a 13-year-old clinical pathology frozen biopsy archive (Liquid Nitrogen). Samples were divided into either high-quality (RIN ≥6.5) or low-quality overall categories, or into four "fit-for-purpose" quality groups: RIN <5: not reliable for demanding downstream analysis; 5 ≤RIN <6: suitable for RT-qPCR; 6 ≤RIN <8: suitable for gene array analysis; and RIN ≥8: suitable for all downstream techniques. In general, low RIN values were correlated with fatty, fibrous, pancreatic, or necrotic tissue. When the percentage of samples with RIN ≥6.5 is higher than 90%, the tissue bank performance is adequate. The annual 2011 quality control assessment showed that 90.3% (n=93) of all samples had acceptable RIN values; 97.4% (n=39) of the cancer institute collection had RIN values above 6.5; and 88.6% (n=123) of samples from the liver sample archive collection had RIN values higher than 6.5. As the clinical pathology biopsy collection contained only 58.8% (n=24) acceptable samples, the procurement protocols used for these samples needed immediate evaluation. When the distribution of RIN values of the different collections were compared, no significant differences were found, despite differences in average storage time and temperature. According to the principle of "fit-for-purpose" distribution, the vast majority of samples are considered good enough for most downstream techniques. In conclusion, an annual tissue bank quality control procedure provides useful information on tissue sample quality and sheds light on where and if improvements need to be made.

Swab or biopsy samples for bioburden testing of allograft musculoskeletal tissue?

PubMed

Varettas, Kerry

2014-12-01

Swab and biopsy samples of allograft musculoskeletal tissue are most commonly collected by tissue banks for bacterial and fungal bioburden testing. An in vitro study was performed using the National Committee for Clinical Laboratory Standards standard 'Quality control of microbiological transport systems' (2003) to validate and evaluate the recovery of six challenge organisms from swab and biopsy samples of allograft musculoskeletal tissue. On average, 8.4 to >100 and 7.2 to >100 % of the inoculum was recovered from swab and biopsy samples respectively. A retrospective review of donor episodes was also performed, consisting of paired swab and biopsy samples received in this laboratory during the period 2001-2012. Samples of allograft femoral heads were collected from living donors during hip operations. From the 3,859 donor episodes received, 21 paired swab and biopsy samples each recovered an isolate, 247 swab samples only and 79 biopsy samples only were culture positive. Low numbers of challenge organisms were recovered from inoculated swab and biopsy samples in the in vitro study and validated their use for bioburden testing of allograft musculoskeletal tissue. Skin commensals were the most common group of organisms isolated during a 12-year retrospective review of paired swab and biopsy samples from living donor allograft femoral heads. Paired swab and biopsy samples are a suitable representative sample of allograft musculoskeletal tissue for bioburden testing.

Tritiated-nicotine- and /sup 125/I-alpha-bungarotoxin-labeled nicotinic receptors in the interpeduncular nucleus of rats. II. Effects of habenular destruction

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

Clarke, P.B.; Hamill, G.S.; Nadi, N.S.

1986-09-15

The cholinergic innervation of the interpeduncular nucleus (IPN) is wholly extrinsic and is greatly attenuated by bilateral habenular destruction. We describe changes in the labeling of putative nicotinic receptors within this nucleus at 3, 5, or 11 days after bilateral habenular lesions. Adjacent tissue sections of the rat IPN were utilized for /sup 3/H-nicotine and /sup 125/I-alpha-bungarotoxin (/sup 125/I-BTX) receptor autoradiography. Compared to sham-operated controls, habenular destruction significantly reduced autoradiographic /sup 3/H-nicotine labeling in rostral (-25%), intermediate (-13%), and lateral subnuclei (-36%). Labeling in the central subnucleus was unchanged. Loss of labeling was maximal at the shortest survival time (3more » days) and did not change thereafter. In order to establish whether this loss was due to a reduction in the number or the affinity of /sup 3/H-nicotine-binding sites, a membrane assay was performed on microdissected IPN tissue from rats that had received surgery 3 days previously. Bilateral habenular lesions produced a 35% reduction of high-affinity /sup 3/H-nicotine-binding sites, with no change in binding affinity. Bilateral habenular lesions reduced /sup 125/I-BTX labeling in the intermediate subnuclei, and a slight increase occurred in the rostral subnucleus. In the lateral subnuclei, /sup 125/I-BTX labeling was significantly reduced (27%) at 3 days but not at later survival times. In view of the known synaptic morphology of the habenulointerpeduncular tract, it is concluded that a subpopulation of /sup 3/H-nicotine binding sites within the IPN is located on afferent axons and/or terminals. This subpopulation, located within rostral, intermediate, and lateral subnuclei, may correspond to presynaptic nicotinic cholinergic receptors. Sites that bind /sup 125/I-BTX may include a presynaptic subpopulation located in the lateral and possibly the intermediate subnuclei.« less

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.

Keratin 17 in premalignant and malignant squamous lesions of the cervix: proteomic discovery and immunohistochemical validation as a diagnostic and prognostic biomarker

PubMed Central

Escobar-Hoyos, Luisa F; Yang, Jie; Zhu, Jiawen; Cavallo, Julie-Ann; Zhai, Haiyan; Burke, Stephanie; Koller, Antonius; Chen, Emily I; Shroyer, Kenneth R

2014-01-01

Most previously described immunohistochemical markers of cervical high-grade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma may help to improve diagnostic accuracy but have a minimal prognostic value. The goals of the current study were to identify and validate novel candidate biomarkers that could potentially improve diagnostic and prognostic accuracy for cervical HSIL and squamous cell carcinoma. Microdissected tissue sections from formalin-fixed paraffin-embedded normal ectocervical squamous mucosa, low-grade squamous intraepithelial lesion (LSIL), HSIL and squamous cell carcinoma sections were analyzed by mass spectrometry-based shotgun proteomics for biomarker discovery. The diagnostic specificity of candidate biomarkers was subsequently evaluated by immunohistochemical analysis of tissue microarrays. Among 1750 proteins identified by proteomic analyses, keratin 4 (KRT4) and keratin 17 (KRT17) showed reciprocal patterns of expression in the spectrum of cases ranging from normal ectocervical squamous mucosa to squamous cell carcinoma. Immunohistochemical studies confirmed that KRT4 expression was significantly decreased in squamous cell carcinoma compared with the other diagnostic categories. By contrast, KRT17 expression was significantly increased in HSIL and squamous cell carcinoma compared with normal ectocervical squamous mucosa and LSIL. KRT17 was also highly expressed in immature squamous metaplasia and in endocervical reserve cells but was generally not detected in mature squamous metaplasia. Furthermore, high levels of KRT17 expression were significantly associated with poor survival of squamous cell carcinoma patients (Hazard ratio = 14.76, P = 0.01). In summary, both KRT4 and KRT17 expressions are related to the histopathology of the cervical squamous mucosa; KRT17 is highly overexpressed in immature squamous metaplasia, in HSIL, and in squamous cell carcinoma and the level of KRT17 in squamous cell carcinoma may help to identify patients who are at greatest risk for cervical cancer mortality. PMID:24051697

454 Transcriptome sequencing suggests a role for two-component signalling in cellularization and differentiation of barley endosperm transfer cells.

PubMed

Thiel, Johannes; Hollmann, Julien; Rutten, Twan; Weber, Hans; Scholz, Uwe; Weschke, Winfriede

2012-01-01

Cell specification and differentiation in the endosperm of cereals starts at the maternal-filial boundary and generates the endosperm transfer cells (ETCs). Besides the importance in assimilate transfer, ETCs are proposed to play an essential role in the regulation of endosperm differentiation by affecting development of proximate endosperm tissues. We attempted to identify signalling elements involved in early endosperm differentiation by using a combination of laser-assisted microdissection and 454 transcriptome sequencing. 454 sequencing of the differentiating ETC region from the syncytial state until functionality in transfer processes captured a high proportion of novel transcripts which are not available in existing barley EST databases. Intriguingly, the ETC-transcriptome showed a high abundance of elements of the two-component signalling (TCS) system suggesting an outstanding role in ETC differentiation. All components and subfamilies of the TCS, including distinct kinds of membrane-bound receptors, have been identified to be expressed in ETCs. The TCS system represents an ancient signal transduction system firstly discovered in bacteria and has previously been shown to be co-opted by eukaryotes, like fungi and plants, whereas in animals and humans this signalling route does not exist. Transcript profiling of TCS elements by qRT-PCR suggested pivotal roles for specific phosphorelays activated in a coordinated time flow during ETC cellularization and differentiation. ETC-specificity of transcriptionally activated TCS phosphorelays was assessed for early differentiation and cellularization contrasting to an extension of expression to other grain tissues at the beginning of ETC maturation. Features of candidate genes of distinct phosphorelays and transcriptional activation of genes putatively implicated in hormone signalling pathways hint at a crosstalk of hormonal influences, putatively ABA and ethylene, and TCS signalling. Our findings suggest an integral function for the TCS in ETC differentiation possibly coupled to sequent hormonal regulation by ABA and ethylene.

454 Transcriptome Sequencing Suggests a Role for Two-Component Signalling in Cellularization and Differentiation of Barley Endosperm Transfer Cells

PubMed Central

Thiel, Johannes; Hollmann, Julien; Rutten, Twan; Weber, Hans; Scholz, Uwe; Weschke, Winfriede

2012-01-01

Background Cell specification and differentiation in the endosperm of cereals starts at the maternal-filial boundary and generates the endosperm transfer cells (ETCs). Besides the importance in assimilate transfer, ETCs are proposed to play an essential role in the regulation of endosperm differentiation by affecting development of proximate endosperm tissues. We attempted to identify signalling elements involved in early endosperm differentiation by using a combination of laser-assisted microdissection and 454 transcriptome sequencing. Principal Findings 454 sequencing of the differentiating ETC region from the syncytial state until functionality in transfer processes captured a high proportion of novel transcripts which are not available in existing barley EST databases. Intriguingly, the ETC-transcriptome showed a high abundance of elements of the two-component signalling (TCS) system suggesting an outstanding role in ETC differentiation. All components and subfamilies of the TCS, including distinct kinds of membrane-bound receptors, have been identified to be expressed in ETCs. The TCS system represents an ancient signal transduction system firstly discovered in bacteria and has previously been shown to be co-opted by eukaryotes, like fungi and plants, whereas in animals and humans this signalling route does not exist. Transcript profiling of TCS elements by qRT-PCR suggested pivotal roles for specific phosphorelays activated in a coordinated time flow during ETC cellularization and differentiation. ETC-specificity of transcriptionally activated TCS phosphorelays was assessed for early differentiation and cellularization contrasting to an extension of expression to other grain tissues at the beginning of ETC maturation. Features of candidate genes of distinct phosphorelays and transcriptional activation of genes putatively implicated in hormone signalling pathways hint at a crosstalk of hormonal influences, putatively ABA and ethylene, and TCS signalling. Significance Our findings suggest an integral function for the TCS in ETC differentiation possibly coupled to sequent hormonal regulation by ABA and ethylene. PMID:22848641

Frequent epigenetic inactivation of chromosome 3p candidate tumor suppressor genes in gallbladder carcinoma.

PubMed

Riquelme, Erick; Tang, Moying; Baez, Sergio; Diaz, Alfonso; Pruyas, Martha; Wistuba, Ignacio I; Corvalan, Alejandro

2007-05-18

Gallbladder carcinoma (GBC) is a highly malignant neoplasm that represents the leading cause of death for cancer in Chilean females. There is limited information about the molecular abnormalities involved in its pathogenesis. We have identified a number of molecular changes in GBC, including frequent allelic losses at chromosome 3p regions. Four distinct 3p sites (3p12, 3p14.2, 3p21.3 and 3p22-24) with frequent and early allelic losses in the sequential pathogenesis of this neoplasm have been detected. We investigated epigenetic and genetic abnormalities in GBC affecting 6 candidate tumor suppressor genes (TSG) located in chromosome 3p, including DUTT1 (3p12), FHIT (3p14.2), BLU, RASSF1A, SEMA3B and hMLH1 (3p21.3). DNA extracted from frozen tissue obtained from 50 surgical resected GBCs was examined for gene promoter methylation using MSP (methylation-specific PCR) technique after bisulfite treatment in all 6 genes. In addition, we performed PCR-based mutation examination using SSCP in FHIT and RASSF1A genes and loss of heterozygosity (LOH) analysis using microdissected tissue in a subset of tumors for the 3p21.3 region with 8 microsatellite markers. A very high frequency of GBC methylation was detected in SEMA3B (46/50, 92%) and FHIT (33/50, 66%), intermediate incidences in BLU (13/50, 26%) and DUTT1 (11/50, 22%) and very low frequencies in RASSF1A (4/50, 8%) and hMLH1 (2/50, 4%). Allelic loss at 3p21.3 was found in nearly half of the GBCs examined. We conclude that epigenetic inactivation by abnormal promoter methylation is a frequent event in chromosome 3p candidate TSGs in GBC pathogenesis, especially affecting genes SEMA3B (3p21.3) and FHIT (3p14.2).

The role of human papillomavirus in p16-positive oral cancers.

PubMed

Belobrov, Simone; Cornall, Alyssa M; Young, Richard J; Koo, Kendrick; Angel, Christopher; Wiesenfeld, David; Rischin, Danny; Garland, Suzanne M; McCullough, Michael

2018-01-01

The aim of this study was to identify the presence and frequency of human papillomavirus (HPV) nucleic acid in p16-positive oral squamous cell carcinomas (OSCCs), to assess whether the virus was transcriptionally active and to assess the utility of p16 overexpression as a surrogate marker for HPV in OSCC. Forty-six OSCC patients treated between 2007 and 2011 with available formalin-fixed paraffin-embedded (FFPE) specimens were included. Twenty-three patients were positive for p16 by immunohistochemistry (IHC) and these were matched with 23 patients with p16-negative tumours. Laser capture microdissection of the FFPE OSCC tissues was undertaken to isolate invasive tumour tissue. DNA was extracted and tested for high-risk HPV types using a PCR-ELISA method based on the L1 SPF10 consensus primers, and a real-time PCR method targeting HPV-16 and HPV-18 E6 region. Genotyping of HPV-positive cases was performed using a reverse line blot hybridization assay (Inno-LiPA). RNAScope ® (a chromogenic RNA in situ hybridization assay) was utilized to detect E6/E7 mRNA of known high-risk HPV types for detection of transcriptionally active virus. HPV DNA was found in 3 OSCC cases, all of which were p16 IHC-positive. Two cases were genotyped as HPV-16 and one as HPV-33. Only one of the HPV-16 cases was confirmed to harbour transcriptionally active virus via HPV RNA ISH. We have shown that the presence of transcriptionally active HPV rarely occurs in OSCC and that p16 is not an appropriate surrogate marker for HPV in OSCC cases. We propose that non-viral mechanisms are responsible for the majority of IHC p16 overexpression in OSCC. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Postnatal establishment of allelic Gαs silencing as a plausible explanation for delayed onset of parathyroid hormone-resistance due to heterozygous Gαs disruption

PubMed Central

Turan, Serap; Fernandez-Rebollo, Eduardo; Aydin, Cumhur; Zoto, Teuta; Reyes, Monica; Bounoutas, George; Chen, Min; Weinstein, Lee S.; Erben, Reinhold G.; Marshansky, Vladimir; Bastepe, Murat

2013-01-01

Pseudohypoparathyroidism type-Ia (PHP-Ia), characterized by renal proximal tubular resistance to parathyroid hormone (PTH), results from maternal mutations of GNAS that lead to loss of Gαs activity. Gαs expression is paternally silenced in the renal proximal tubule, and this genomic event is critical for the development of PTH-resistance, as patients display impaired hormone action only if the mutation is inherited maternally. The primary clinical finding of PHP-Ia is hypocalcemia, which can lead to various neuromuscular defects including seizures. PHP-Ia patients frequently do not present with hypocalcemia until after infancy, but it has remained uncertain whether PTH-resistance occurs in a delayed fashion. Analyzing reported cases of PHP-Ia with documented GNAS mutations and mice heterozygous for disruption of Gnas, we herein determined that the manifestation of PTH-resistance caused by the maternal loss of Gαs, i.e. hypocalcemia and elevated serum PTH, occurs after early postnatal life. To investigate whether this delay could reflect gradual development of paternal Gαs silencing, we then analyzed renal proximal tubules isolated by laser capture microdissection from mice with either maternal or paternal disruption of Gnas. Our results revealed that, whereas expression of Gαs mRNA in this tissue is predominantly from the maternal Gnas allele at weaning (three-weeks postnatal) and in adulthood, the contributions of the maternal and paternal Gnas alleles to Gαs mRNA expression are equal at postnatal day 3. In contrast, we found that paternal Gαs expression is already markedly repressed in brown adipose tissue at birth. Thus, the mechanisms silencing the paternal Gαs allele in renal proximal tubules are not operational during early postnatal development, and this finding correlates well with the latency of PTH-resistance in patients with PHP-Ia. PMID:23956044

Macrophage-derived LIF and IL1B regulate alpha(1,2)fucosyltransferase 2 (Fut2) expression in mouse uterine epithelial cells during early pregnancy.

PubMed

Jasper, Melinda J; Care, Alison S; Sullivan, Brad; Ingman, Wendy V; Aplin, John D; Robertson, Sarah A

2011-01-01

Macrophages accumulate within stromal tissue subjacent to the luminal epithelium in the mouse uterus during early pregnancy after seminal fluid exposure at coitus. To investigate their role in regulating epithelial cell expression of fucosylated structures required for embryo attachment and implantation, fucosyltransferase enzymes Fut1, Fut2 (Enzyme Commission number [EC] 2.4.1.69), and Fut4 (EC 2.4.1.214) and Muc1 and Muc4 mRNAs were quantified by quantitative real-time PCR in uterine epithelial cells after laser capture microdissection in situ or after epithelial cell coculture with macrophages or macrophage-secreted factors. When uterine macrophage recruitment was impaired by mating with seminal plasma-deficient males, epithelial cell Fut2 expression on Day 3.5 postcoitus (pc) was reduced compared to intact-mated controls. Epithelial cell Fut2 was upregulated in vitro by coculture with macrophages or macrophage-conditioned medium (MCM). Macrophage-derived cytokines LIF, IL1B, and IL12 replicated the effect of MCM on Fut2 mRNA expression, and MCM-stimulated expression was inhibited by anti-LIF and anti-IL1B neutralizing antibodies. The effects of acute macrophage depletion on fucosylated structures detected with lectins Ulex europaeus 1 (UEA-1) and Lotus tetragonolobus purpureas (LTP), or LewisX immunoreactivity, were quantified in vivo in Cd11b-dtr transgenic mice. Depletion of macrophages caused a 30% reduction in luminal epithelial UEA-1 staining and a 67% reduction in LewisX staining in uterine tissues of mice hormonally treated to mimic early pregnancy. Together, these data demonstrate that uterine epithelial Fut2 mRNA expression and terminal fucosylation of embryo attachment ligands is regulated in preparation for implantation by factors including LIF and IL1B secreted from macrophages recruited during the inflammatory response to insemination.

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.

Sequence independent amplification of DNA

DOEpatents

Bohlander, S.K.

1998-03-24

The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example, the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei. 25 figs.

Sequence independent amplification of DNA

DOEpatents

Bohlander, Stefan K.

1998-01-01

The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei.

A workflow to preserve genome-quality tissue samples from plants in botanical gardens and arboreta1

PubMed Central

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

2016-01-01

Premise of the study: Internationally, gardens hold diverse living collections that can be preserved for genomic research. Workflows have been developed for genomic tissue sampling in other taxa (e.g., vertebrates), but are inadequate for plants. We outline a workflow for tissue sampling intended for two audiences: botanists interested in genomics research and garden staff who plan to voucher living collections. Methods and Results: Standard herbarium methods are used to collect vouchers, label information and images are entered into a publicly accessible database, and leaf tissue is preserved in silica and liquid nitrogen. A five-step approach for genomic tissue sampling is presented for sampling from living collections according to current best practices. Conclusions: Collecting genome-quality samples from gardens is an economical and rapid way to make available for scientific research tissue from the diversity of plants on Earth. The Global Genome Initiative will facilitate and lead this endeavor through international partnerships. PMID:27672517

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.

Human papilloma virus status and chromosomal imbalances in primary cervical carcinomas and tumour cell lines.

PubMed

Hidalgo, A; Schewe, C; Petersen, S; Salcedo, M; Gariglio, P; Schlüns, K; Dietel, M; Petersen, I

2000-03-01

Human papilloma virus (HPV) infection is the crucial step in the initiation of cervical carcinomas. In addition, HPV18 has been implicated in tumour progression and adverse clinical outcome. We determined the HPV types in 12 primary cervical carcinomas and 12 cell lines and compared the findings with the comparative genetic hybridisation (CGH) pattern of chromosomal alterations. The most frequent alteration was the deletion at 3p14 followed by the loss of 2q34-q36 along with 3q gain. High risk HPV types were detected in all samples except one primary tumour. In contrast to the normal distribution, HPV18 was present in 75% of cases including all cell lines. The cell lines carried a higher number of genetic alterations and a different CGH pattern for several chromosomes than the primary tumours, despite microdissection. Purely HPV18 positive cases indicated a high incidence of imbalances at specific loci with peaks of the histogram coinciding with known HPV integration sites. The study suggests that HPV infection is associated with a recurrent pattern of chromosomal changes in cervical carcinomas and that the development and progression of these alterations is triggered by integration into the host genome.

Liquid Microjunction Surface Sampling Probe Electrospray Mass Spectrometry for Detection of Drugs and Metabolites in Thin Tissue Sections

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

Van Berkel, Gary J; Kertesz, Vilmos; Koeplinger, Kenneth A.

2008-01-01

A self-aspirating, liquid micro-junction surface sampling probe/electrospray emitter mass spectrometry system was demonstrated for use in the direct analysis of spotted and dosed drugs and their metabolites in thin tissue sections. Proof-of-principle sampling and analysis directly from tissue without the need for sample preparation was demonstrated first by raster scanning a region on a section of rat liver onto which reserpine was spotted. The mass spectral signal from selected reaction monitoring was used to develop a chemical image of the spotted drug on the tissue. The probe was also used to selectively spot sample areas of sagittal whole mouse bodymore » tissue sections that had been dosed orally (90 mg/kg) with R,S-sulforaphane 3 hrs prior to sacrifice. Sulforaphane and its glutathione and N-acetyl cysteine conjugates were monitored with selected reaction monitoring and detected in the stomach and various other tissues from the dosed mouse. No signal for these species was observed in the tissue from a control mouse. The same dosed tissue section was used to illustrate the possibility of obtaining a line scan across the whole body section. In total these results illustrate the potential for rapid screening of the distribution of drugs and metabolites in tissue sections with the micro-liquid junction surface sampling probe/electrospray mass spectrometry approach.« less

The value of intraoperative ultrasonography during the resection of relapsed irradiated malignant gliomas in the brain.

PubMed

Mursch, Kay; Scholz, Martin; Brück, Wolfgang; Behnke-Mursch, Julianne

2017-01-01

The aim of this study was to investigate whether intraoperative ultrasonography (IOUS) helped the surgeon navigate towards the tumor as seen in preoperative magnetic resonance imaging and whether IOUS was able to distinguish between tumor margins and the surrounding tissue. Twenty-five patients suffering from high-grade gliomas who were previously treated by surgery and radiotherapy were included. Intraoperatively, two histopathologic samples were obtained a sample of unequivocal tumor tissue (according to anatomical landmarks and the surgeon's visual and tactile impressions) and a small tissue sample obtained using a navigated needle when the surgeon decided to stop the resection. This specimen was considered to be a boundary specimen, where no tumor tissue was apparent. The decision to take the second sample was not influenced by IOUS. The effect of IOUS was analyzed semi-quantitatively. All 25 samples of unequivocal tumor tissue were histopathologically classified as tumor tissue and were hyperechoic on IOUS. Of the boundary specimens, eight were hypoechoic. Only one harbored tumor tissue (P=0.150). Seventeen boundaries were moderately hyperechoic, and these samples contained all possible histological results (i.e., tumor, infiltration, or no tumor). During surgery performed on relapsed, irradiated, high-grade gliomas, IOUS provided a reliable method of navigating towards the core of the tumor. At borders, it did not reliably distinguish between remnants or tumor-free tissue, but hypoechoic areas seldom contained tumor tissue.

A probable risk factor of female breast cancer: study on benign and malignant breast tissue samples.

PubMed

Rehman, Sohaila; Husnain, Syed M

2014-01-01

The study reports enhanced Fe, Cu, and Zn contents in breast tissues, a probable risk factor of breast cancer in females. Forty-one formalin-fixed breast tissues were analyzed using atomic absorption spectrophotometry. Twenty malignant, six adjacent to malignant and 15 benign tissues samples were investigated. The malignant tissues samples were of grade 11 and type invasive ductal carcinoma. The quantitative comparison between the elemental levels measured in the two types of specimen (benign and malignant) tissues (removed after surgery) suggests significant elevation of these metals (Fe, Cu, and Zn) in the malignant tissue. The specimens were collected just after mastectomy of women aged 19 to 59 years from the hospitals of Islamabad and Rawalpindi, Pakistan. Most of the patients belong to urban areas of Pakistan. Findings of study depict that these elements have a promising role in the initiation and development of carcinoma as consistent pattern of elevation for Fe, Cu, and Zn was observed. The results showed the excessive accumulation of Fe (229 ± 121 mg/L) in malignant breast tissue samples of patients (p < 0.05) to that in benign tissues samples (49.1 ± 11.4 mg/L). Findings indicated that excess accumulation of iron in malignant tissues can be a risk factor of breast cancer. In order to validate our method of analysis, certified reference material muscle tissue lyophilized (IAEA) MA-M-2/TM was analyzed for metal studied. Determined concentrations were quite in good agreement with certified levels. Asymmetric concentration distribution for Fe, Cu, and Zn was observed in both malignant and benign tissue samples.

Imaging biological tissues with electrical conductivity contrast below 1 S m−1 by means of magnetoacoustic tomography with magnetic induction

PubMed Central

Hu, Gang; Li, Xu; He, Bin

2010-01-01

Magnetoacoustic tomography with magnetic induction (MAT-MI) is a recently introduced imaging modality for noninvasive electrical impedance imaging, with ultrasound imaging resolution and a contrast reflecting the electrical conductivity properties of tissues. However, previous MAT-MI systems can only image samples that are much more conductive than real human or animal tissues. To image real biological tissue samples, a large-current-carrying coil that can give stronger magnetic stimulations and stronger MAT-MI acoustic signals is employed in this study. The conductivity values of all the tissue samples employed in this study are also directly measured using a well calibrated four-electrode system. The experimental results demonstrated the feasibility to image biological tissues with electrical conductivity contrast below 1.0 S∕m using the MAT-MI technique with safe level of electromagnetic energy applied to tissue samples. PMID:20938494

Imaging biological tissues with electrical conductivity contrast below 1 S m-1 by means of magnetoacoustic tomography with magnetic induction

NASA Astrophysics Data System (ADS)

Hu, Gang; Li, Xu; He, Bin

2010-09-01

Magnetoacoustic tomography with magnetic induction (MAT-MI) is a recently introduced imaging modality for noninvasive electrical impedance imaging, with ultrasound imaging resolution and a contrast reflecting the electrical conductivity properties of tissues. However, previous MAT-MI systems can only image samples that are much more conductive than real human or animal tissues. To image real biological tissue samples, a large-current-carrying coil that can give stronger magnetic stimulations and stronger MAT-MI acoustic signals is employed in this study. The conductivity values of all the tissue samples employed in this study are also directly measured using a well calibrated four-electrode system. The experimental results demonstrated the feasibility to image biological tissues with electrical conductivity contrast below 1.0 S/m using the MAT-MI technique with safe level of electromagnetic energy applied to tissue samples.

76 FR 71315 - Endangered and Threatened Species; Take of Anadromous Fish

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-17

... the fish and would then sample them for biological information (fin tissue and scale samples). They..., measured, weighed, tissue-sampled, and checked for external marks and coded-wire tags depending on the.... Then the researchers would remove and preserve fish body tissues, otoliths, and coded wire tags (from...

Age estimation based on aspartic acid racemization in human sclera.

PubMed

Klumb, Karolin; Matzenauer, Christian; Reckert, Alexandra; Lehmann, Klaus; Ritz-Timme, Stefanie

2016-01-01

Age estimation based on racemization of aspartic acid residues (AAR) in permanent proteins has been established in forensic medicine for years. While dentine is the tissue of choice for this molecular method of age estimation, teeth are not always available which leads to the need to identify other suitable tissues. We examined the suitability of total tissue samples of human sclera for the estimation of age at death. Sixty-five samples of scleral tissue were analyzed. The samples were hydrolyzed and after derivatization, the extent of aspartic acid racemization was determined by gas chromatography. The degree of AAR increased with age. In samples from younger individuals, the correlation of age and D-aspartic acid content was closer than in samples from older individuals. The age-dependent racemization in total tissue samples proves that permanent or at least long-living proteins are present in scleral tissue. The correlation of AAR in human sclera and age at death is close enough to serve as basis for age estimation. However, the precision of age estimation by this method is lower than that of age estimation based on the analysis of dentine which is due to molecular inhomogeneities of total tissue samples of sclera. Nevertheless, the approach may serve as a valuable alternative or addition in exceptional cases.

Fluorescence suppression using wavelength modulated Raman spectroscopy in fiber-probe-based tissue analysis.

PubMed

Praveen, Bavishna B; Ashok, Praveen C; Mazilu, Michael; Riches, Andrew; Herrington, Simon; Dholakia, Kishan

2012-07-01

In the field of biomedical optics, Raman spectroscopy is a powerful tool for probing the chemical composition of biological samples. In particular, fiber Raman probes play a crucial role for in vivo and ex vivo tissue analysis. However, the high-fluorescence background typically contributed by the auto fluorescence from both a tissue sample and the fiber-probe interferes strongly with the relatively weak Raman signal. Here we demonstrate the implementation of wavelength-modulated Raman spectroscopy (WMRS) to suppress the fluorescence background while analyzing tissues using fiber Raman probes. We have observed a significant signal-to-noise ratio enhancement in the Raman bands of bone tissue, which have a relatively high fluorescence background. Implementation of WMRS in fiber-probe-based bone tissue study yielded usable Raman spectra in a relatively short acquisition time (∼30  s), notably without any special sample preparation stage. Finally, we have validated its capability to suppress fluorescence on other tissue samples such as adipose tissue derived from four different species.

Organochlorine compounds in streambed sediment and in biological tissue from streams and their relations to land use, central Arizona

USGS Publications Warehouse

Gebler, Joseph B.

2000-01-01

Streambed-sediment samples from 13 sites and biological-tissue samples from 11 sites in the Gila River Basin in central Arizona were analyzed for 32 organochlorine compounds in streambed sediment and 28 compounds in biological tissue during 1996 as part of the U.S. Geological Survey's National Water-Quality Assessment program. The objectives of the study were to determine the occurrence and distribution of organochlorine compounds and their relation to land use. Sampling sites were categorized on the basis of major land uses in the basin or the source of water in the stream. Because land uses were mixed or had changed over time, some land-use categories were combined. Sites were categorized as forest/rangeland (6), forest/urban (1), urban (4), or agricultural/urban (2). Thirteen organochlorine compounds were detected in streambed-sediment samples, and 10 were detected in tissue samples. The number of compounds found in streambed-sediment samples from individual sites ranged from 0 to 10, and the range for individual tissue samples was 0 to 7. Comparison of the number of detections in streambed-sediment samples to the number of detections in tissue samples from particular sites where both were sampled yielded five instances where more compounds were detected in streambed sediment, six instances where more compounds were detected in tissue, and five instances where the number of detections in streambed sediment and tissue were equal. The frequency of detection of particular compounds for sites where both streambed sediment and tissue were sampled resulted in five compounds being detected more frequently in streambed sediment, five more frequently in tissue, and three compounds that were equally frequent in streambed sediment and in tissue. Few contaminants were detected in samples from the forest/rangeland sites; greater numbers of compounds were detected at the urban sites and at the forest/urban site. The greatest number of compounds and the highest concentrations of many contaminants were detected at agriculture/urban sites. The compound detected most frequently in streambed-sediment and tissue samples was p,p'-DDE. Streambed-sediment guideline values for the protection of aquatic life for p,p'-DDE and total DDT were exceeded at both agricultural/urban sites, The streambed-sediment guideline value for the protection of aquatic life for total chlordane was exceeded at one agricultural/urban site, one urban site, and the forest/urban site. The streambed-sediment guideline value for the protection of aquatic life for total PCB’s was exceeded at one agricultural/urban site. Guideline values for the protection of fish-eating wildlife for total DDT and for toxaphene were exceeded only in samples from the two agricultural/urban sites. The guideline value for the protection of fish-eating wildlife for total PCB’s was equaled or exceeded in samples from two sites—one urban and one agricultural/urban site. Screening values established by the U.S. Environmental Protection Agency for the protection of human health for edible portions of fish were exceeded by total DDT and by toxaphene in fish-tissue samples from both agricultural/urban sites. The human-health criterion for total PCB’s was exceeded in two fish-tissue samples from an agricultural site and from an urban site. Tissue samples analyzed in this study were for whole fish, and thus, concentration data are not entirely comparable to the screening values of the U.S. Environmental Protection Agency. Because these exceedences were an order of magnitude above the criteria, however, it is possible that concentrations in the edible portions of fish from these locations could present a human- health risk. Analyses of samples of edible portions of fish from these locations would be needed to adequately assess the presence or absence of a human-health risk. The similarity of the results of this study to the results of other studies of organochlorine compounds in the environment suggests that there is a correlation between contaminants in sediment and biological-tissue samples and land uses. As with other studies of the occurrence and distribution of organochlorine contaminants in streambed sediments and biological tissue, this study shows that many organochlorine compounds continue to persist in the environment and thus could pose a threat to aquatic life, fish-eating wildlife, and possibly to humans who consume contaminated fish.

An efficient field and laboratory workflow for plant phylotranscriptomic projects1

PubMed Central

Yang, Ya; Moore, Michael J.; Brockington, Samuel F.; Timoneda, Alfonso; Feng, Tao; Marx, Hannah E.; Walker, Joseph F.; Smith, Stephen A.

2017-01-01

Premise of the study: We describe a field and laboratory workflow developed for plant phylotranscriptomic projects that involves cryogenic tissue collection in the field, RNA extraction and quality control, and library preparation. We also make recommendations for sample curation. Methods and Results: A total of 216 frozen tissue samples of Caryophyllales and other angiosperm taxa were collected from the field or botanical gardens. RNA was extracted, stranded mRNA libraries were prepared, and libraries were sequenced on Illumina HiSeq platforms. These included difficult mucilaginous tissues such as those of Cactaceae and Droseraceae. Conclusions: Our workflow is not only cost effective (ca. $270 per sample, as of August 2016, from tissue to reads) and time efficient (less than 50 h for 10–12 samples including all laboratory work and sample curation), but also has proven robust for extraction of difficult samples such as tissues containing high levels of secondary compounds. PMID:28337391

Evaluation of sample holders designed for long-lasting X-ray micro-tomographic scans of ex-vivo soft tissue samples

NASA Astrophysics Data System (ADS)

Dudak, J.; Zemlicka, J.; Krejci, F.; Karch, J.; Patzelt, M.; Zach, P.; Sykora, V.; Mrzilkova, J.

2016-03-01

X-ray microradiography and microtomography are imaging techniques with increasing applicability in the field of biomedical and preclinical research. Application of hybrid pixel detector Timepix enables to obtain very high contrast of low attenuating materials such as soft biological tissue. However X-ray imaging of ex-vivo soft tissue samples is a difficult task due to its structural instability. Ex-vivo biological tissue is prone to fast drying-out which is connected with undesired changes of sample size and shape producing later on artefacts within the tomographic reconstruction. In this work we present the optimization of our Timepix equipped micro-CT system aiming to maintain soft tissue sample in stable condition. Thanks to the suggested approach higher contrast of tomographic reconstructions can be achieved while also large samples that require detector scanning can be easily measured.

Assessment of tissue-specific cortisol activity with regard to degeneration of the suspensory ligaments in horses with pituitary pars intermedia dysfunction.

PubMed

Hofberger, Sina C; Gauff, Felicia; Thaller, Denise; Morgan, Ruth; Keen, John A; Licka, Theresia F

2018-02-01

OBJECTIVE To identify signs of tissue-specific cortisol activity in samples of suspensory ligament (SL) and neck skin tissue from horses with and without pituitary pars intermedia dysfunction (PPID). SAMPLE Suspensory ligament and neck skin tissue samples obtained from 26 euthanized horses with and without PPID. PROCEDURES Tissue samples were collected from 12 horses with and 14 horses without PPID (controls). Two control horses had received treatment with dexamethasone; data from those horses were not used in statistical analyses. The other 12 control horses were classified as old horses (≥ 14 years old) and young horses (≤ 9 years old). Standard histologic staining, staining for proteoglycan accumulation, and immunostaining of SL and neck skin tissue sections for glucocorticoid receptors, insulin, 11β hydroxysteroid dehydrogenase type 1, and 11β hydroxysteroid dehydrogenase type 2 were performed. Findings for horses with PPID were compared with findings for young and old horses without PPID. RESULTS Compared with findings for old and young control horses, there were significantly more cells stained for glucocorticoid receptors in SL samples and for 11 β hydroxysteroid dehydrogenase type 1 in SL and skin tissue samples from horses with PPID. Insulin could not be detected in any of the SL or skin tissue samples. Horses with PPID had evidence of SL degeneration with significantly increased proteoglycan accumulation. Neck skin tissue was found to be significantly thinner in PPID-affected horses than in young control horses. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that tissue-specific dysregulation of cortisol metabolism may contribute to the SL degeneration associated with PPID in horses.

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

USGS Publications Warehouse

Peterson, David A.; Boughton, Gregory K.

2000-01-01

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

Quality control of human tissues--experience from the Indiana University Cancer Center-Lilly Research Labs human tissue bank.

PubMed

Sandusky, George E; Teheny, Katie Heinz; Esterman, Mike; Hanson, Jeff; Williams, Stephen D

2007-01-01

The success of molecular research and its applications in both the clinical and basic research arenas is strongly dependent on the collection, handling, storage, and quality control of fresh human tissue samples. This tissue bank was set up to bank fresh surgically obtained human tissue using a Clinical Annotated Tissue Database (CATD) in order to capture the associated patient clinical data and demographics using a one way patient encryption scheme to protect patient identification. In this study, we determined that high quality of tissue samples is imperative for both genomic and proteomic molecular research. This paper also contains a brief compilation of the literature involved in the patient ethics, patient informed consent, patient de-identification, tissue collection, processing, and storage as well as basic molecular research generated from the tissue bank using good clinical practices. The current applicable rules, regulations, and guidelines for handling human tissues are briefly discussed. More than 6,610 cancer patients have been consented (97% of those that were contacted by the consenter) and 16,800 tissue specimens have been banked from these patients in 9 years. All samples collected in the bank were QC'd by a pathologist. Approximately 1,550 tissue samples have been requested for use in basic, clinical, and/or biomarker cancer research studies. Each tissue aliquot removed from the bank for a research study were evaluated by a second H&E, if the samples passed the QC, they were submitted for genomic and proteomic molecular analysis/study. Approximately 75% of samples evaluated were of high histologic quality and used for research studies. Since 2003, we changed the patient informed consent to allow the tissue bank to gather more patient clinical follow-up information. Ninety two percent of the patients (1,865 patients) signed the new informed consent form and agreed to be re-contacted for follow-up information on their disease state. In addition, eighty five percent of patients (1,584) agreed to be re-contacted to provide a biological fluid sample to be used for biomarker research.

Large-scale atlas of microarray data reveals the distinct expression landscape of different tissues in Arabidopsis

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

He, Fei; Maslov, Sergei; Yoo, Shinjae

Here, transcriptome datasets from thousands of samples of the model plant Arabidopsis thaliana have been collectively generated by multiple individual labs. Although integration and meta-analysis of these samples has become routine in the plant research community, it is often hampered by the lack of metadata or differences in annotation styles by different labs. In this study, we carefully selected and integrated 6,057 Arabidopsis microarray expression samples from 304 experiments deposited to NCBI GEO. Metadata such as tissue type, growth condition, and developmental stage were manually curated for each sample. We then studied global expression landscape of the integrated dataset andmore » found that samples of the same tissue tend to be more similar to each other than to samples of other tissues, even in different growth conditions or developmental stages. Root has the most distinct transcriptome compared to aerial tissues, but the transcriptome of cultured root is more similar to those of aerial tissues as the former samples lost their cellular identity. Using a simple computational classification method, we showed that the tissue type of a sample can be successfully predicted based on its expression profile, opening the door for automatic metadata extraction and facilitating re-use of plant transcriptome data. As a proof of principle we applied our automated annotation pipeline to 708 RNA-seq samples from public repositories and verified accuracy of our predictions with samples’ metadata provided by authors.« less

Large-scale atlas of microarray data reveals the distinct expression landscape of different tissues in Arabidopsis

DOE PAGES

He, Fei; Maslov, Sergei; Yoo, Shinjae; ...

2016-05-25

Here, transcriptome datasets from thousands of samples of the model plant Arabidopsis thaliana have been collectively generated by multiple individual labs. Although integration and meta-analysis of these samples has become routine in the plant research community, it is often hampered by the lack of metadata or differences in annotation styles by different labs. In this study, we carefully selected and integrated 6,057 Arabidopsis microarray expression samples from 304 experiments deposited to NCBI GEO. Metadata such as tissue type, growth condition, and developmental stage were manually curated for each sample. We then studied global expression landscape of the integrated dataset andmore » found that samples of the same tissue tend to be more similar to each other than to samples of other tissues, even in different growth conditions or developmental stages. Root has the most distinct transcriptome compared to aerial tissues, but the transcriptome of cultured root is more similar to those of aerial tissues as the former samples lost their cellular identity. Using a simple computational classification method, we showed that the tissue type of a sample can be successfully predicted based on its expression profile, opening the door for automatic metadata extraction and facilitating re-use of plant transcriptome data. As a proof of principle we applied our automated annotation pipeline to 708 RNA-seq samples from public repositories and verified accuracy of our predictions with samples’ metadata provided by authors.« less

Identification of testis-specific male contraceptive targets: insights from transcriptional profiling of the cycle of the rat seminiferous epithelium and purified testicular cells.

PubMed

Johnston, Daniel S; Jelinsky, Scott A; Zhi, Yu; Finger, Joshua N; Kopf, Gregory S; Wright, William W

2007-12-01

In an effort to identify novel targets for the development of nonhormonal male contraceptives, genome-wide transcriptional profiling of the rat testis was performed. Specifically, enzymatically purified spermatogonia plus early spermatocyctes, pachytene spermatocytes, round spermatids, and Sertoli cells was analyzed along with microdissected rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX- XI, XII, XIII-XIV of the cycle of the seminiferous epithelium using RAE 230_2.0 microarrays. The combined analysis of these studies identified 16,971 expressed probe sets on the array. How these expression data, combined with additional bioinformatic data analysis and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis, led to the identification of 58 genes that have 1000-fold higher expression transcriptionally in the testis when compared to over 20 other nonreproductive tissues is described. The products of these genes may play important roles in testicular and/or sperm function, and further investigation on their utility as nonhormonal contraceptive targets is warranted. Moreover, these microarray data have been used to expedite the identification of a mutation in RIKEN cDNA 2410004F06 gene as likely being responsible for spermatogenic failure in a line of infertile mice generated by N-ethyl-N-nitrosourea (ENU) mutagenesis. The microarray data and the qRT-PCR data described are available in the Mammalian Reproductive Genetics database (http://mrg.genetics.washington.edu/).

Aging Selectively Modulates Vitamin C Transporter Expression Patterns in the Kidney.

PubMed

Forman, Katherine; Martínez, Fernando; Cifuentes, Manuel; Bertinat, Romina; Salazar, Katterine; Nualart, Francisco

2017-09-01

In the kidney, vitamin C is reabsorbed from the glomerular ultrafiltrate by sodium-vitamin C cotransporter isoform 1 (SVCT1) located in the brush border membrane of the proximal tubules. Although we know that vitamin C levels decrease with age, the adaptive physiological mechanisms used by the kidney for vitamin C reabsorption during aging remain unknown. In this study, we used an animal model of accelerated senescence (SAMP8 mice) to define the morphological alterations and aging-induced changes in the expression of vitamin C transporters in renal tissue. Aging induced significant morphological changes, such as periglomerular lymphocytic infiltrate and glomerular congestion, in the kidneys of SAMP8 mice, although no increase in collagen deposits was observed using 2-photon microscopy analysis and second harmonic generation. The most characteristic histological alteration was the dilation of intracellular spaces in the basolateral region of proximal tubule epithelial cells. Furthermore, a combination of laser microdissection, qRT-PCR, and immunohistochemical analyses allowed us to determine that SVCT1 expression specifically increased in the proximal tubules from the outer strip of the outer medulla (segment S3) and cortex (segment S2) during aging and that these tubules also express GLUT1. We conclude that aging modulates vitamin C transporter expression and that renal over-expression of SVCT1 enhances vitamin C reabsorption in aged animals that may synthesize less vitamin C. J. Cell. Physiol. 232: 2418-2426, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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.

Comparison of gene-expression profiles between diffuse- and intestinal-type gastric cancers using a genome-wide cDNA microarray.

PubMed

Jinawath, Natini; Furukawa, Yoichi; Hasegawa, Suguru; Li, Meihua; Tsunoda, Tatsuhiko; Satoh, Seiji; Yamaguchi, Toshiharu; Imamura, Hiroshi; Inoue, Masatomo; Shiozaki, Hitoshi; Nakamura, Yusuke

2004-09-02

Gastric cancer is the fourth leading cause of cancer-related death in the world. Two histologically distinct types of gastric carcinoma, 'intestinal' and 'diffuse', have different epidemiological and pathophysiological features that suggest different mechanisms of carcinogenesis. A number of studies have investigated intestinal-type gastric cancers at the molecular level, but little is known about mechanisms involved in the diffuse type, which has a more invasive phenotype and poorer prognosis. To clarify the mechanisms that underlie its development and/or progression, we compared the expression profiles of 20 laser-microbeam-microdissected diffuse-type gastric-cancer tissues with corresponding noncancerous mucosae by means of a cDNA microarray containing 23,040 genes. We identified 153 genes that were commonly upregulated and more than 1500 that were commonly downregulated in the tumors. We also identified a number of genes related to tumor progression. Furthermore, comparison of the expression profiles of diffuse-type with those of intestinal-type gastric cancers identified 46 genes that may represent distinct molecular signatures of each histological type. The putative signature of diffuse-type cancer exhibited altered expression of genes related to cell-matrix interaction and extracellular-matrix (ECM) components, whereas that of intestinal-type cancer represented enhancement of cell growth. These data provide insight into different mechanisms underlying gastric carcinogenesis and may also serve as a starting point for identifying novel diagnostic markers and/or therapeutic targets for diffuse-type gastric cancers.

Adaptive Calcified Matrix Response of Dental Pulp to Bacterial Invasion Is Associated with Establishment of a Network of Glial Fibrillary Acidic Protein+/Glutamine Synthetase+ Cells

PubMed Central

Farahani, Ramin M.; Nguyen, Ky-Anh; Simonian, Mary; Hunter, Neil

2010-01-01

We report evidence for anatomical and functional changes of dental pulp in response to bacterial invasion through dentin that parallel responses to noxious stimuli reported in neural crest-derived sensory tissues. Sections of resin-embedded carious adult molar teeth were prepared for immunohistochemistry, in situ hybridization, ultrastructural analysis, and microdissection to extract mRNA for quantitative analyses. In odontoblasts adjacent to the leading edge of bacterial invasion in carious teeth, expression levels of the gene encoding dentin sialo-protein were 16-fold greater than in odontoblasts of healthy teeth, reducing progressively with distance from this site of the carious lesion. In contrast, gene expression for dentin matrix protein-1 by odontoblasts was completely suppressed in carious teeth relative to healthy teeth. These changes in gene expression were related to a gradient of deposited reactionary dentin that displayed a highly modified structure. In carious teeth, interodontoblastic dentin sialo-protein− cells expressing glutamine synthetase (GS) showed up-regulation of glial fibrillary acidic protein (GFAP). These cells extended processes that associated with odontoblasts. Furthermore, connexin 43 established a linkage between adjacent GFAP+/GS+ cells in carious teeth only. These findings indicate an adaptive pulpal response to encroaching caries that includes the deposition of modified, calcified, dentin matrix associated with networks of GFAP+/GS+ interodontoblastic cells. A regulatory role for the networks of GFAP+/GS+ cells is proposed, mediated by the secretion of glutamate to modulate odontoblastic response. PMID:20802180

Can glomerular mRNAs in human type 1 diabetes be used to predict transition from normoalbuminuria to microalbuminuria?

PubMed

Adler, Sharon G; Kang, Shin-Wook; Feld, Stella; Cha, Dae Ryong; Barba, Lilly; Striker, Liliane; Striker, Gary; Riser, Bruce L; LaPage, Janine; Nast, Cynthia C

2002-07-01

mRNAs of pathogenetic importance in the development of diabetic nephropathy were measured in subjects with type 1 diabetes to determine whether these might be used to predict progression from normoalbuminuria to microalbuminuria. We proposed that conversion from normoalbuminuria to microalbuminuria would be most likely in subjects whose connective tissue growth factor (CTGF) and collagen mRNAs were above the 95% confidence interval (CI) for live renal donors and within the 95% CI for subjects with abnormal albuminuria. Glomerular CTGF, collagen alpha2(IV), and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were measured in microdissected glomeruli from living renal donors (n = 10), and subjects with normoalbuminuria (n = 12), microalbuminuria (n = 5), and overt proteinuria (n = 6). After 44 +/- 2 months of follow-up, one subject converted from normoalbuminuria to microalbuminuria. Although the data are limited, progression from normoalbuminuria to microalbuminuria occurred in the only normoalbuminuric subject whose mRNA levels were above the live renal donors' 95% CI for CTGF and collagen alpha2(IV) and within the 95% CI of subjects with abnormal albuminuria. No clinical or histopathologic finding distinguished the progressor from the nonprogressors at the time of biopsy. This case report provides proof-of-principle that a panel of glomerular mRNA markers chosen because of their pathogenetic relevance may be useful adjuncts to albuminuria and histology in predicting clinical stability or clinical progression in diabetic nephropathy. Copyright 2002 by the National Kidney Foundation, Inc.

Concordance in diabetic foot ulceration: a cross-sectional study of agreement between wound swabbing and tissue sampling in infected ulcers.

PubMed

Nelson, E Andrea; Wright-Hughes, Alexandra; Brown, Sarah; Lipsky, Benjamin A; Backhouse, Michael; Bhogal, Moninder; Ndosi, Mwidimi; Reynolds, Catherine; Sykes, Gill; Dowson, Christopher; Edmonds, Michael; Vowden, Peter; Jude, Edward B; Dickie, Tom; Nixon, Jane

2016-11-01

There is inadequate evidence to advise clinicians on the relative merits of swabbing versus tissue sampling of infected diabetic foot ulcers (DFUs). To determine (1) concordance between culture results from wound swabs and tissue samples from the same ulcer; (2) whether or not differences in bacterial profiles from swabs and tissue samples are clinically relevant; (3) concordance between results from conventional culture versus polymerase chain reaction (PCR); and (4) prognosis for patients with an infected DFU at 12 months' follow-up. This was a cross-sectional, multicentre study involving patients with diabetes and a foot ulcer that was deemed to be infected by their clinician. Microbiology specimens for culture were taken contemporaneously by swab and by tissue sampling from the same wound. In a substudy, specimens were also processed by PCR. A virtual 'blinded' clinical review compared the appropriateness of patients' initial antibiotic regimens based on the results of swab and tissue specimens. Patients' case notes were reviewed at 12 months to assess prognosis. The main study recruited 400 patients, with 247 patients in the clinical review. There were 12 patients in the PCR study and 299 patients in the prognosis study. Patients' median age was 63 years (range 26-99 years), their diabetes duration was 15 years (range 2 weeks-57 years), and their index ulcer duration was 1.8 months (range 3 days-12 years). Half of the ulcers were neuropathic and the remainder were ischaemic/neuroischaemic. Tissue results reported more than one pathogen in significantly more specimens than swabs {86.1% vs. 70.1% of patients, 15.9% difference [95% confidence interval (CI) 11.8% to 20.1%], McNemar's p -value < 0.0001}. The two sampling techniques reported a difference in the identity of pathogens for 58% of patients. The number of pathogens differed in 50.4% of patients. In the clinical review study, clinicians agreed on the need for a change in therapy for 73.3% of patients (considering swab and tissue results separately), but significantly more tissue than swab samples required a change in therapy. Compared with traditional culture, the PCR technique reported additional pathogens for both swab and tissue samples in six (50%) patients and reported the same pathogens in four (33.3%) patients and different pathogens in two (16.7%) patients. The estimated healing rate was 44.5% (95% CI 38.9% to 50.1%). At 12 months post sampling, 45 (15.1%) patients had died, 52 (17.4%) patients had a lower-extremity ipsilateral amputation and 18 (6.0%) patients had revascularisation surgery. We did not investigate the potential impact of microbiological information on care. We cannot determine if the improved information yield from tissue sampling is attributable to sample collection, sample handling, processing or reporting. Tissue sampling reported both more pathogens and more organisms overall than swabbing. Both techniques missed some organisms, with tissue sampling missing fewer than swabbing. Results from tissue sampling more frequently led to a (virtual) recommended change in therapy. Long-term prognosis for patients with an infected foot ulcer was poor. Research is needed to determine the effect of sampling/processing techniques on clinical outcomes and antibiotic stewardship. The National Institute for Health Research Health Technology Assessment programme.

Extraction efficiency and implications for absolute quantitation of propranolol in mouse brain, liver and kidney thin tissue sections using droplet-based liquid microjunction surface sampling-HPLC ESI-MS/MS

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

Kertesz, Vilmos; Weiskittel, Taylor M.; Vavek, Marissa

Currently, absolute quantitation aspects of droplet-based surface sampling for thin tissue analysis using a fully automated autosampler/HPLC-ESI-MS/MS system are not fully evaluated. Knowledge of extraction efficiency and its reproducibility is required to judge the potential of the method for absolute quantitation of analytes from thin tissue sections. Methods: Adjacent thin tissue sections of propranolol dosed mouse brain (10- μm-thick), kidney (10- μm-thick) and liver (8-, 10-, 16- and 24- μm-thick) were obtained. Absolute concentration of propranolol was determined in tissue punches from serial sections using standard bulk tissue extraction protocols and subsequent HPLC separations and tandem mass spectrometric analysis. Thesemore » values were used to determine propranolol extraction efficiency from the tissues with the droplet-based surface sampling approach. Results: Extraction efficiency of propranolol using 10- μm-thick brain, kidney and liver thin tissues using droplet-based surface sampling varied between ~45-63%. Extraction efficiency decreased from ~65% to ~36% with liver thickness increasing from 8 μm to 24 μm. Randomly selecting half of the samples as standards, precision and accuracy of propranolol concentrations obtained for the other half of samples as quality control metrics were determined. Resulting precision ( ±15%) and accuracy ( ±3%) values, respectively, were within acceptable limits. In conclusion, comparative quantitation of adjacent mouse thin tissue sections of different organs and of various thicknesses by droplet-based surface sampling and by bulk extraction of tissue punches showed that extraction efficiency was incomplete using the former method, and that it depended on the organ and tissue thickness. However, once extraction efficiency was determined and applied, the droplet-based approach provided the required quantitation accuracy and precision for assay validations. Furthermore, this means that once the extraction efficiency was calibrated for a given tissue type and drug, the droplet-based approach provides a non-labor intensive and high-throughput means to acquire spatially resolved quantitative analysis of multiple samples of the same type.« less

Extraction efficiency and implications for absolute quantitation of propranolol in mouse brain, liver and kidney thin tissue sections using droplet-based liquid microjunction surface sampling-HPLC ESI-MS/MS

DOE PAGES

Kertesz, Vilmos; Weiskittel, Taylor M.; Vavek, Marissa; ...

2016-06-22

Currently, absolute quantitation aspects of droplet-based surface sampling for thin tissue analysis using a fully automated autosampler/HPLC-ESI-MS/MS system are not fully evaluated. Knowledge of extraction efficiency and its reproducibility is required to judge the potential of the method for absolute quantitation of analytes from thin tissue sections. Methods: Adjacent thin tissue sections of propranolol dosed mouse brain (10- μm-thick), kidney (10- μm-thick) and liver (8-, 10-, 16- and 24- μm-thick) were obtained. Absolute concentration of propranolol was determined in tissue punches from serial sections using standard bulk tissue extraction protocols and subsequent HPLC separations and tandem mass spectrometric analysis. Thesemore » values were used to determine propranolol extraction efficiency from the tissues with the droplet-based surface sampling approach. Results: Extraction efficiency of propranolol using 10- μm-thick brain, kidney and liver thin tissues using droplet-based surface sampling varied between ~45-63%. Extraction efficiency decreased from ~65% to ~36% with liver thickness increasing from 8 μm to 24 μm. Randomly selecting half of the samples as standards, precision and accuracy of propranolol concentrations obtained for the other half of samples as quality control metrics were determined. Resulting precision ( ±15%) and accuracy ( ±3%) values, respectively, were within acceptable limits. In conclusion, comparative quantitation of adjacent mouse thin tissue sections of different organs and of various thicknesses by droplet-based surface sampling and by bulk extraction of tissue punches showed that extraction efficiency was incomplete using the former method, and that it depended on the organ and tissue thickness. However, once extraction efficiency was determined and applied, the droplet-based approach provided the required quantitation accuracy and precision for assay validations. Furthermore, this means that once the extraction efficiency was calibrated for a given tissue type and drug, the droplet-based approach provides a non-labor intensive and high-throughput means to acquire spatially resolved quantitative analysis of multiple samples of the same type.« less

Simulation-assisted design of microfluidic sample traps for optimal trapping and culture of non-adherent single cells, tissues, and spheroids.

PubMed

Rousset, Nassim; Monet, Frédéric; Gervais, Thomas

2017-03-21

This work focuses on modelling design and operation of "microfluidic sample traps" (MSTs). MSTs regroup a widely used class of microdevices that incorporate wells, recesses or chambers adjacent to a channel to individually trap, culture and/or release submicroliter 3D tissue samples ranging from simple cell aggregates and spheroids, to ex vivo tissue samples and other submillimetre-scale tissue models. Numerous MST designs employing various trapping mechanisms have been proposed in the literature, spurring the development of 3D tissue models for drug discovery and personalized medicine. Yet, there lacks a general framework to optimize trapping stability, trapping time, shear stress, and sample metabolism. Herein, the effects of hydrodynamics and diffusion-reaction on tissue viability and device operation are investigated using analytical and finite element methods with systematic parametric sweeps over independent design variables chosen to correspond to the four design degrees of freedom. Combining different results, we show that, for a spherical tissue of diameter d < 500 μm, the simplest, closest to optimal trap shape is a cube of dimensions w equal to twice the tissue diameter: w = 2d. Furthermore, to sustain tissues without perfusion, available medium volume per trap needs to be 100× the tissue volume to ensure optimal metabolism for at least 24 hours.

A novel semi-quantitative method for measuring tissue bleeding.

PubMed

Vukcevic, G; Volarevic, V; Raicevic, S; Tanaskovic, I; Milicic, B; Vulovic, T; Arsenijevic, S

2014-03-01

In this study, we describe a new semi-quantitative method for measuring the extent of bleeding in pathohistological tissue samples. To test our novel method, we recruited 120 female patients in their first trimester of pregnancy and divided them into three groups of 40. Group I was the control group, in which no dilation was applied. Group II was an experimental group, in which dilation was performed using classical mechanical dilators. Group III was also an experimental group, in which dilation was performed using a hydraulic dilator. Tissue samples were taken from the patients' cervical canals using a Novak's probe via energetic single-step curettage prior to any dilation in Group I and after dilation in Groups II and III. After the tissue samples were prepared, light microscopy was used to obtain microphotographs at 100x magnification. The surfaces affected by bleeding were measured in the microphotographs using the Autodesk AutoCAD 2009 program and its "polylines" function. The lines were used to mark the area around the entire sample (marked A) and to create "polyline" areas around each bleeding area on the sample (marked B). The percentage of the total area affected by bleeding was calculated using the formula: N = Bt x 100 / At where N is the percentage (%) of the tissue sample surface affected by bleeding, At (A total) is the sum of the surfaces of all of the tissue samples and Bt (B total) is the sum of all the surfaces affected by bleeding in all of the tissue samples. This novel semi-quantitative method utilizes the Autodesk AutoCAD 2009 program, which is simple to use and widely available, thereby offering a new, objective and precise approach to estimate the extent of bleeding in tissue samples.

High-throughput simultaneous analysis of RNA, protein, and lipid biomarkers in heterogeneous tissue samples.

PubMed

Reiser, Vladimír; Smith, Ryan C; Xue, Jiyan; Kurtz, Marc M; Liu, Rong; Legrand, Cheryl; He, Xuanmin; Yu, Xiang; Wong, Peggy; Hinchcliffe, John S; Tanen, Michael R; Lazar, Gloria; Zieba, Renata; Ichetovkin, Marina; Chen, Zhu; O'Neill, Edward A; Tanaka, Wesley K; Marton, Matthew J; Liao, Jason; Morris, Mark; Hailman, Eric; Tokiwa, George Y; Plump, Andrew S

2011-11-01

With expanding biomarker discovery efforts and increasing costs of drug development, it is critical to maximize the value of mass-limited clinical samples. The main limitation of available methods is the inability to isolate and analyze, from a single sample, molecules requiring incompatible extraction methods. Thus, we developed a novel semiautomated method for tissue processing and tissue milling and division (TMAD). We used a SilverHawk atherectomy catheter to collect atherosclerotic plaques from patients requiring peripheral atherectomy. Tissue preservation by flash freezing was compared with immersion in RNAlater®, and tissue grinding by traditional mortar and pestle was compared with TMAD. Comparators were protein, RNA, and lipid yield and quality. Reproducibility of analyte yield from aliquots of the same tissue sample processed by TMAD was also measured. The quantity and quality of biomarkers extracted from tissue prepared by TMAD was at least as good as that extracted from tissue stored and prepared by traditional means. TMAD enabled parallel analysis of gene expression (quantitative reverse-transcription PCR, microarray), protein composition (ELISA), and lipid content (biochemical assay) from as little as 20 mg of tissue. The mean correlation was r = 0.97 in molecular composition (RNA, protein, or lipid) between aliquots of individual samples generated by TMAD. We also demonstrated that it is feasible to use TMAD in a large-scale clinical study setting. The TMAD methodology described here enables semiautomated, high-throughput sampling of small amounts of heterogeneous tissue specimens by multiple analytical techniques with generally improved quality of recovered biomolecules.

A qualitative signature for early diagnosis of hepatocellular carcinoma based on relative expression orderings.

PubMed

Ao, Lu; Zhang, Zimei; Guan, Qingzhou; Guo, Yating; Guo, You; Zhang, Jiahui; Lv, Xingwei; Huang, Haiyan; Zhang, Huarong; Wang, Xianlong; Guo, Zheng

2018-04-23

Currently, using biopsy specimens to confirm suspicious liver lesions of early hepatocellular carcinoma are not entirely reliable because of insufficient sampling amount and inaccurate sampling location. It is necessary to develop a signature to aid early hepatocellular carcinoma diagnosis using biopsy specimens even when the sampling location is inaccurate. Based on the within-sample relative expression orderings of gene pairs, we identified a simple qualitative signature to distinguish both hepatocellular carcinoma and adjacent non-tumour tissues from cirrhosis tissues of non-hepatocellular carcinoma patients. A signature consisting of 19 gene pairs was identified in the training data sets and validated in 2 large collections of samples from biopsy and surgical resection specimens. For biopsy specimens, 95.7% of 141 hepatocellular carcinoma tissues and all (100%) of 108 cirrhosis tissues of non-hepatocellular carcinoma patients were correctly classified. Especially, all (100%) of 60 hepatocellular carcinoma adjacent normal tissues and 77.5% of 80 hepatocellular carcinoma adjacent cirrhosis tissues were classified to hepatocellular carcinoma. For surgical resection specimens, 99.7% of 733 hepatocellular carcinoma specimens were correctly classified to hepatocellular carcinoma, while 96.1% of 254 hepatocellular carcinoma adjacent cirrhosis tissues and 95.9% of 538 hepatocellular carcinoma adjacent normal tissues were classified to hepatocellular carcinoma. In contrast, 17.0% of 47 cirrhosis from non-hepatocellular carcinoma patients waiting for liver transplantation were classified to hepatocellular carcinoma, indicating that some patients with long-lasting cirrhosis could have already gained hepatocellular carcinoma characteristics. The signature can distinguish both hepatocellular carcinoma tissues and tumour-adjacent tissues from cirrhosis tissues of non-hepatocellular carcinoma patients even using inaccurately sampled biopsy specimens, which can aid early diagnosis of hepatocellular carcinoma. © 2018 The Authors. Liver International Published by John Wiley & Sons Ltd.

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

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.