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Shear stress is known to affect many processes in (patho-) physiology through a complex, multi-molecular mechanism, termed mechanotransduction. The sheer complexity of the process has raised questions how mechanotransduction is regulated. Here, we comprehensively evaluate the literature about the role of small non-coding miRNA in the regulation of mechanotransduction. Regulation of mRNA by miRNA is rather complex, depending not only on the concentration of mRNA to miRNA, but also on the amount of mRNA competing for a single mRNA. The only mechanism to counteract the latter factor is through overarching structures of miRNA. Indeed, two overarching structures are present miRNA families and miRNA clusters, and both will be discussed in details, regarding the latest literature and a previous conducted study focussed on mechanotransduction. Both the literature and our own data support a new hypothesis that miRNA-clusters predominantly regulate mechanotransduction, affecting 65% of signalling pathways. In conclusion, a new and important mode of regulation of mechanotransduction is proposed, based on miRNA clusters. This finding implicates new avenues for treatment of mechanotransduction and atherosclerosis.

Quantitative proteomics/metabolomics investigation of laser-capture-microdissection (LCM) cell populations from clinical cohorts affords precise insights into disease/therapeutic mechanisms, nonetheless high-quality quantification remains a prominent challenge. Here, we devised an LC/MS-based approach allowing parallel, robust global-proteomics and targeted-metabolomics quantification from the same LCM samples, using biopsies from prostate cancer (PCa) patients as the model system. The strategy features: (i) an optimized molecular weight cutoff (MWCO) filter-based separation of proteins and small-molecule fractions with high and consistent recoveries; (ii) microscale derivatization and charge-based enrichment for ultrasensitive quantification of key androgens (LOQ = 5 fg/1k cells) with excellent accuracy/precision; (iii) reproducible/precise proteomics quantification with low-missing-data using a detergent-cocktail-based sample preparation and an IonStar pipeline for reproducible and precise protein quantification with excellent data quality. Key parameters enabling robust/reproducible quantification have been meticulously evaluated and optimized, and the results underscored the importance of surveying quantitative performances against key parameters to facilitate fit-for-purpose method development. As a proof-of-concept, high-quality quantification of the proteome and androgens in LCM samples of PCa patient-matched cancerous and benign epithelial/stromal cells was achieved (N = 16), which suggested distinct androgen distribution patterns across cell types and regions, as well as the dysregulated pathways involved in tumor-stroma crosstalk in PCa pathology. This strategy markedly leverages the scope of quantitative-omics investigations using LCM samples, and combining with IonStar, can be readily adapted to larger-cohort clinical analysis. Moreover, the capacity of parallel proteomics/metabolomics quantification permits precise corroboration of regulatory processes on both protein and small-molecule levels, with decreased batch effect and enhanced utilization of samples.

The molecular pathogenesis of mixed odontogenic tumors has not been established, and understanding their genetic basis could refine their classification and help define molecular markers for diagnostic purposes. Potentially pathogenic mutations in the component tissues of 28 cases of mixed odontogenic tumors were assessed. Laser capture microdissected tissue from 10 ameloblastic fibromas (AF), 4 ameloblastic fibrodentinomas (AFD), 6 ameloblastic fibro-odontomas (AFO), 3 ameloblastic fibrosarcomas (AFS), and 5 odontomas (OD) were screened by next-generation sequencing and results confirmed by TaqMan allele-specific quantitative PCR. BRAF p.V600E mutation in the mesenchymal component was shown in 4 of 10 AF (40%), 2 of 4 AFD (50%), 2 of 6 AFO (33%), and 2 of 3 AFS (67%), whereas all 5 OD were wild type for BRAF p.V600E. Mutation in the epithelial component was only observed in one AF and one AFO. One AFS contained an area of benign AF, and the mesenchymal component of both (AFS and AF) contained BRAF p.V600E, supporting the concept of malignant progression from a benign AF precursor. KDR, TP53, KIT, and PIK3CA single-nucleotide polymorphisms are reported. In conclusion, AF, AFD, AFO, and AFS show BRAF p.V600E in their mesenchymal component, unlike OD, which are BRAF wild type, suggesting that at least a subset of AF, AFD, and AFO are molecularly distinct from OD, and may represent distinct entities and be neoplastic.

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial compounds and associate with the nucleus through tubular extensions called stromules. Although it is well established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and facilitating chloroplast interactions, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1)-mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector-mediated suppression of BAK1-mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant–pathogen interactions. Fil: Savage, Zachary. Imperial College London; Reino Unido Fil: Duggan, Cian. Imperial College London; Reino Unido Fil: Toufexi, Alexia. Imperial College London; Reino Unido Fil: Pandey, Pooja. Imperial College London; Reino Unido Fil: Liang, Yuxi. Imperial College London; Reino Unido Fil: Segretin, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Yuen, Lok Him. Imperial College London; Reino Unido Fil: Gaboriau, David C. A.. Imperial College London; Reino Unido Fil: Leary, Alexandre Y.. Imperial College London; Reino Unido Fil: Tumtas, Yasin. Imperial College London; Reino Unido Fil: Khandare, Virendrasinh. Imperial College London; Reino Unido Fil: Ward, Andrew D.. Science and Technology Facilities Council; Reino Unido Fil: Botchway, Stanley W.. Science and Technology Facilities Council; Reino Unido Fil: Bateman, Benji C.. Science and Technology Facilities Council; Reino Unido Fil: Pan, Indranil. Alan Turing Institute; Reino Unido. Imperial College London; Reino Unido Fil: Schattat, Martin. Martin Luther Universitat Halle-Wittenberg; Alemania Fil: Sparkes, Imogen. University of Bristol; Reino Unido Fil: Bozkurt, Osman Tolga. Imperial College London; Reino Unido

High-resolution spatial transcriptomics identifies previously unknown regulators for barley floral meristem development., Organ development in plants predominantly occurs postembryonically through combinatorial activity of meristems; therefore, meristem and organ fate are intimately connected. Inflorescence morphogenesis in grasses (Poaceae) is complex and relies on a specialized floral meristem, called spikelet meristem, that gives rise to all other floral organs and ultimately the grain. The fate of the spikelet determines reproductive success and contributes toward yield-related traits in cereal crops. Here, we examined the transcriptional landscapes of floral meristems in the temperate crop barley (Hordeum vulgare L.) using RNA-seq of laser capture microdissected tissues from immature, developing floral structures. Our unbiased, high-resolution approach revealed fundamental regulatory networks, previously unknown pathways, and key regulators of barley floral fate and will equally be indispensable for comparative transcriptional studies of grass meristems.

How modification of gene expression generates novel traits is key to understanding the evolutionary process. We investigated the genetic basis for the origin of the piscine gas bladder from lungs of ancestral bony vertebrates. Distinguishing these homologous organs is the direction of budding from the foregut during development; lungs bud ventrally and the gas bladder buds dorsally. We investigated whether this morphological inversion is associated with molecular inversion of conserved genes regulating lung and gasbladder development. Using laser-capture microdissection and RNA-seq, we assayed transcript abundance and compared expression patterns between dorsal and ventral foregut tissues at three developmental stages spanning gasbladder development. Our focal taxon, bowfin (Amia calva), representing the sistergroup to teleosts, is an early diverging ray-finned fish with a gas bladder. We discovered a number of genes with unknown function during lung development that are differentially expressed during gas bladder development and annotated to functions relevant for organ budding. We also identified several known lung-regulatory genes exhibiting inverted dorsoventral expression during gasbladder relative to lung development. Specifically, we found Tbx5 is strongly expressed in the dorsal mesoderm surrounding the gas bladder during bowfin development, and several interacting genes are co-expressed dorsally with Tbx5. In contrast, in mouse and bichir (Polypterus senegalus), the only ray-finned fish that have lungs, Tbx5 is expressed in the ventral lung mesoderm during development. Our data demonstrating dorsoventral inversion of conserved genes suggests that these genes may have contributed to the evolutionary transition between ventral lungs and a dorsal gas bladder in ray-finned fishes.

It is currently unknown how the islet transcriptional pattern changes as glucose metabolism deteriorates and progresses to fulminant type 2 diabetes (T2D). In this study, we hypothesized that islets from donors with elevated HbA1c levels, but not yet diagnosed with T2D, would show signs of cell stress on a transcriptional level. Laser capture microdissection and qPCR arrays including 330 genes related to mitochondria, oxidative stress, or the unfolded protein response were used to extract and analyze islets from organ donors with HbA1c 6.5% (48 mmol/mol)) or established T2D. Principal component analysis and hierarchical clustering based on the expression of all 330 genes displayed no obvious separation of the four different donor groups, indicating that the inter-donor variations were larger than the differences between groups. However, 44 genes were differentially expressed (P < 0.05, false discovery rate

Next-generation sequencing technologies have led to a decreased cost and an increased throughput in genome sequencing. Yet, many genome assemblies based on short sequencing reads have been assembled only to the scaffold level due to the lack of sufficient chromosome mapping information. Traditional ways of mapping scaffolds to chromosomes require a large amount of laboratory work and time to generate genetic and/or physical maps. To address this problem, we conducted a rapid technique which uses laser capture microdissection and enables mapping scaffolds of de novo genome assemblies directly to chromosomes in Hawaiian picture-winged Drosophila. We isolated and sequenced intact chromosome arms from larvae of D. differens. By mapping the reads of each chromosome to the recently assembled scaffolds from 3 Hawaiian picture-winged Drosophila species, at least 67% of the scaffolds were successfully assigned to chromosome arms. Even though the scaffolds are not ordered within a chromosome, the fast-generated chromosome information allows for chromosome-related analyses after genome assembling. We utilize this new information to test the faster-X evolution effect for the first time in these Hawaiian picture-winged Drosophila species.

Laser capture microdissection is a valuable technique in individually isolating single cells whether in tissue networks or deposited from a cell suspension. New developments have enabled coupling of laser capture microdissection with mass spectrometry via liquid vortex capture sampling probe. This enables online metabolic profiling of sectioned cells. Here, we describe the protocol used to deposit, isolate, and individually chemically characterize single Allium cepa and Chlamydomonas reinhardtii cells by laser capture microdissection-liquid vortex capture mass spectrometry.

We developed new tools to build a high-quality chromosomal map of the Komodo dragon (Varanus komodoensis) available for cross-species phylogenomic analyses. First, we isolated chromosomes by flow sorting and determined the chromosome content of each flow karyotype peak by FISH. We then isolated additional Komodo dragon chromosomes by microdissection and amplified chromosome-specific DNA pools. The chromosome-specific DNA pools can be sequenced, assembled, and mapped by next-generation sequencing technology. The chromosome-specific paint probes can be used to investigate karyotype evolution through cross-species chromosome painting. Overall, the set of chromosome-specific DNA pools of V. komodoensis provides new tools for detailed phylogenomic analyses of Varanidae and squamates in general.

As for most human cancer types we now have substantial understanding of the landscape of somatic mutations in breast cancer. However, there is relatively little knowledge of the somatic mutations present in normal breast epithelium. This may potentially be instructive in identifying the earliest stages of breast neoplasia and in understanding why the risk of breast cancer is high compared to other tissues of the body. By sequencing laser capture microdissected elements of normal breast epithelium from healthy women, women with breast cancer and women with breast cancer predisposition we are able to provide the first comprehensive insights into somatic mutagenesis in normal breast and compare these with other normal human tissues. Citation Format: M Stratton. Aging [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP147.

Improved speed and sensitivity of mass spectrometry allow the simultaneous quantification of high numbers of proteins from increasingly smaller quantities of tissue sample. Quantitative data of the proteome is highly valuable for providing unbiased information on, for example, protein expression changes related to disease or identifying related biomarkers. In brain diseases the affected area can be small and pathogenic events can be related to a specific cell type in an otherwise heterogeneous tissue type. An emerging approach dedicated to analyzing this type of samples is laser micro-dissection (LMD) combined with LC-MS/MS into a single workflow. In this chapter, we describe different options for isolating tissue suitable for LC-MS/MS analysis.

We describe a novel method for assessing the “open” or “closed” state of chromatin at selected locations within the genome. This method combines the use of Benzonase, which can digest DNA in the presence of actin, with quantitative polymerase chain reaction to define digested regions. We demonstrate the application of this method in brain homogenates and laser captured cells. We also demonstrate application to selected sites within more than 1 gene and multiple sites within 1 gene. We demonstrate the validity of the method by treating cells with valproate, known to render chromatin more permissive, and by comparison with classical digestion with DNase I in an in vitro preparation. Although we demonstrate the use of this method in brain tissue, we also recognize its applicability to other tissue types.

Background: The advent of the NGS revolutionized cancer research by making it possible to study the complexity of cancer using high throughput sequencing methodologies. The current trends are to adapt highthroughput sequencing technologies to the level of small cell populations and even individual cells. In our laboratory, we developed some different methods that would allow us to work optimally with very low amount of material, based on the technique of the SMARTer technology and on the Ion Torrent protocols that we modified carefully, applied to Ion Proton system. Materials and methods: We collected 12 formalin-fixed, paraffin-embedded tissues and 20 laser capture microdissected formalin-fixed, paraffin-embedded samples derived from brain and breast cancer and 13 laser capture microdissected fresh frozen samples derived from mouse brain cells. Results: We developed high performance methods to analyse the whole transcriptomes of our samples, obtaining a very high number of reads (78,186,377 usable reads), perfectly comparable with samples with a large amount of RNA such as samples obtained from cells or fresh tissues. Conclusions: We have found that the combination of SMARTer technology and Ion TargetSeq Exome Enrichment kit, in addition to some improvements to their conventional protocols, provides excellent results with challenging samples.

The intestinal epithelium is a highly structured tissue composed of repeating crypt-villus units. Enterocytes perform the diverse tasks of absorbing a wide range of nutrients while protecting the body from the harsh bacterium-rich environment. It is unknown whether these tasks are spatially zonated along the villus axis. Here, we extracted a large panel of landmark genes characterized by transcriptomics of laser capture microdissected villus segments and utilized it for single-cell spatial reconstruction, uncovering broad zonation of enterocyte function along the villus. We found that enterocytes at villus bottoms express an anti-bacterial gene program in a microbiome-dependent manner. They next shift to sequential expression of carbohydrates, peptides, and fat absorption machineries in distinct villus compartments. Finally, they induce a Cd73 immune-modulatory program at the villus tips. Our approach can be used to uncover zonation patterns in other organs when prior knowledge of landmark genes is lacking.

Proteomics holds the promise of evaluating global changes in protein expression and post-translational modification in response to environmental stimuli. However, difficulties in achieving cellular anatomic resolution and extracting specific types of proteins from cells have limited the efficacy of these techniques. Laser capture microdissection has provided a solution to the problem of anatomical resolution in tissues. New extraction methodologies have expanded the range of proteins identified in subsequent analyses. This review will examine the application of laser capture microdissection to proteomic tissue sampling, and subsequent extraction of these samples for differential expression analysis. Statistical and other quantitative issues important for the analysis of the highly complex datasets generated are also reviewed.

Biomarkers from tissue-based proteomic studies directly contribute to defining disease states as well as promise to improve early detection or provide for further targeted therapeutics. In the clinical setting, tissue samples are preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks for histological examination. However, proteomic analysis of FFPE tissue is complicated due to the high level of covalently cross-linked proteins arising from formalin fixation. To address these challenges, we developed well-based reverse-phase protein array (RPPA). This approach is a robust protein isolation methodology (29.44 ± 7.8 μg per 1 mm(3) of FFPE tissue) paired with a novel on electrochemiluminescence detection system. Protein samples derived from FFPE tissue by means of laser capture dissection, with as few as 500 shots, demonstrate measurable signal differences for different proteins. The lysates coated to the array plate, dried up and vacuum-sealed, remain stable up to 2 months at room temperature. This methodology is directly applicable to FFPE tissue and presents the direct opportunity of addressing hypothesis within clinical trials and well-annotated clinical tissue repositories.

To clarify the correlation of genomic alterations with clinical and histological features, we performed metaphase comparative genomic hybridization analysis on 20 primary cutaneous melanomas, which were obtained by laser capture or manual microdissection, and 16 melanoma cell lines. There were no differences in the average number of aberrations between acral melanomas (AM) and non-AM, although gains of 5q and 11q13 were more frequent (P=0.05) and 10q loss was less frequent (P=0.01) in AM than in non-AM. Although tumor thickness is considered a measurable estimate of clinical expression, there were no differences in the average number of aberrations among 4 groups, classified by thickness of the tumor. While the majority of aberrations were equally distributed among the 4 groups, 6p gains were found only in the thickest tumors. Patients with 6p or 1q gains had a lower overall survival rate than those without them (P=0.0002 or P=0.013). While gains of 1q, 2q, 3p, 3q, 7q, 20p, and 20q were more frequent in the cell lines than in the primary tumors (P

Direct analysis of laser capture microdissected malignant and normal endometrial epithelium using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (MS) was able to detect a number of proteins that are overexpressed in malignant epithelial cells. A total of 16 physiologic and malignant endometrial samples were laser capture microdissected, including four proliferative and four secretory endometria, and eight endometrioid adenocarcinomas. Two of these proteins, at 10,834 and 10,843 Da, likely correspond to calgranulin A and chaperonin 10, two proteins that had previously been identified in endometrioid adenocarcinoma in whole tissue homogenate by MS analysis. Direct analysis by MALDI-MS not only confirms that these proteins are overexpressed in endometrial carcinoma, but also localizes them to the epithelial cells, the expected cancer site.

Ultrashort pulses of X-rays from a free-electron laser capture the spatiotemporal evolution of spins in the different crystallographic directions of a complex magnetic material.

Introduction: Molecular characteristics of breast tumors play an important role in determining patients’ survival outcome. Here, we report preliminary findings of proteogenomic profiling of 50 breast tumors using RNA-Seq and mass-spectrometry (MS) based proteomic technologies. An additional 60 tumors are being analyzed, including WGS for all samples. We are also collecting patient survival outcome data. Methods: Cases used in this study were drawn from the Clinical Breast Care Project, where patients were consented using an IRB-approved protocol. A total of 50 breast tumors were selected and processed by laser capture microdissection (LCM). This cohort includes 36 Caucasian Americans (CA) and 8 African Americans (AA), and the age of the patients is 57 ± 13 years. Protein and RNA were extracted using the Illustra triplePrep kit, which isolates DNA from the same cells as well. Quantitative global proteomics and phosphoproteomics analyses were performed using isobaric TMT 6-plex labeling with the “universal reference” strategy and IMAC enrichment of phosphopeptides. Mass spectrometry data were acquired using a Q-Exactive instrument and analyzed using Proteome Discoverer with Byonic node. Phosphopeptide abundance was normalized to abundance measurements of the parent protein for all of the phosphorylation analyses. Phosphoproteomic data was also searched for the presence of O-GlcNAc modifications. RNA-Seq analyses were done on Illumina HiSeq and the data were analyzed using GSNAP. Results: There were 19 Luminal A, 7 Luminal B, 8 HER2-enriched, and 16 basal-like subtypes based on the PAM50 algorithm. In the global proteomics data, we were able to quantitate >8600 proteins. Unsupervised clustering on the highly varying proteins across the samples resulted in two primary clusters, with one being luminal-enriched. The other cluster contains a basal-like tumor sub-cluster and a sub-cluster of mixed subtypes. Differential protein expression analyses between the two primary clusters confirmed known markers (e.g., overexpression of KRT8/KRT18 in luminal-enriched cluster). The luminal-enriched cluster is primarily CA with post-menopausal status. A similar search of the phosphoproteomic data yielded quantitation of >12500 phosphopeptides. Unsupervised clustering of the phosphoproteins resulted in four primary groups, with one being basal-enriched and another being luminal-enriched. We also observed >50 overexpressed phosphopeptides. While some of these phosphosites have been previously reported (e.g., on RANBP2), other phosphosites appeared to be novel (e.g., on IRF2BP2). Conclusion: Analysis of LCM breast tumors using proteogenomic technologies resulted in basal- and luminal-enriched clusters, thus enabling us to study protein and phosphopeptide markers across multiple platforms. The views expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S. Government. Citation Format: Viswanadham Sridhara, Tao Liu, Marina A. Gritsenko, Lori A. Sturtz, Albert J. Kovatich, Vladislav A. Petyuk, Brenda Deyarmin, Jason E. McDermott, Anil K. Shukla, Ronald J. Moore, Matthew E. Monroe, Bobbie-Jo M. Webb-Robertson, Jeffrey A. Hooke, Leigh Fantacone-Campbell, Praveen Kumar Raj Kumar, Leonid Kvecher, Jianfang Liu, Jennifer Kane, Jennifer Melley, Stella Somiari, Joji Iida, Stephen C. Benz, Justin Golovato, Shahrooz Rabizadeh, Patrick Soon-Shiong, Richard D. Smith, Richard J. Mural, Craig D. Shriver, Hai Hu, Karin D. Rodland. Integrated proteogenomic analysis of laser capture microdissected breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 213. doi:10.1158/1538-7445.AM2017-213

A basic process to investigate biomolecular systems is observing and manipulating single molecules within their native context. While many recent advances in super-resolution microscopy methods (e.g. STORM, PALM, PAINT) have allowed observation of molecular architecture down to the scale individual components (∼5 nm), there has not been a parallel development of manipulation methods at a comparable scale. Current light-based manipulation techniques, including laser capture micro-dissection or focused laser crosslinking, are still diffraction-limited, and therefore incapable of selective targeting below a ∼200 nm sized area.

Due to immunosuppression, transplant patients are more susceptible to infections, particularly viruses. The strengths of EM detection include the facts that it is rapid, non-invasive or minimally invasive, does not require probes, and can detect many different viruses. A limitation is that is it not as sensitive as PCR, but this can be an advantage in instances where endogenous viruses in healthy individuals may be detected by exquisitely sensitive tests. Additionally, concentration methods, such as ultracentrifugation, ultrafiltration, or immunoaggregation can increase the chances of finding them by EM in fluids, and localization of focal pathology in tissues can be enhanced by semithin sections, on-slide embedment of H&E-stained sections, confocal microscopy, and laser capture.

When analyzing alcohol's effects on the brain, researchers often want to look at small clusters of cells that can be studied in isolation from the surrounding brain tissue rather than at the entire brain or larger brain areas. This implies that relatively small numbers of cells have to be retrieved from the brain and studied in culture or subjected to biochemical analyses. The challenge then becomes how to isolate small numbers of cells from a specific brain region without including unwanted cells. One approach to solving this problem is to use a technology known as laser-assisted microdissection (LMD). This article reviews some of the principles of LMD and its use in alcohol research.

Tooth development is a multi-stage process that is regulated by complex molecular events and the minipig is a good model for tooth development research. However, the expression patterns of homeobox genes during tooth development of the minipig are still unclear. In this work, we utilized the Chinese experimental minipig as a model to examine expression patterns of homeobox genes in different teeth of the minipig embryo at E40. Laser capture microdissection was used to collect teeth samples for tissue sections. We partially cloned and sequenced homeobox genes Barx1, Msx1, Dlx1, Pax9, Pitx2 and developmental marker gene Bmp4. Real-time PCR was used to analyze expression patterns of these genes in different teeth. The results showed that Msx1, Pitx2 and Bmp4 were relatively highly expressed in the third premolar during the cap stage, while Barx1, Dlx1 and Pax9 were relatively highly expressed in the fourth premolar during the early bell stage. Key words: Tooth development, homeobox genes, minipig embryo.

The expression pattern of estrogen receptor (ER) isoforms in normal and tumor thyroid tissues is still controversial and poor defined, therefore, a more detailed study of the distribution of these molecules is needed. Most discrepancies might be due to the methods utilized. We studied the expression of ER isoforms in human papillary thyroid carcinoma (PTC), in fine-needle aspiration biopsy-derived specimens, and in cells, using more accurate techniques, such as laser-capture microdissection, real-time quantitative PCR, and Western blot. Laser-capture microdissection allowed us to isolate homogeneous cell populations from human PTC surgical samples. Tumor, peritumor, or normal host tissue of the same sample were separately dissected and analyzed by RT-PCR and Western blot. Estrogen receptor-α mRNA was more expressed in cancer-microdissected cells from human PTC, as compared with microdissected cells obtained from surrounding normal host tissue (450 vs 12, P = 0.001). A similar pattern was observed with Western blot for the ER-α protein. By contrast, ER-β mRNA expression was not detected among the microdissected tissue fractions. Fine-needle aspiration biopsy-derived specimens showed a similar expression pattern to ER. Moreover, human PTC cell line BCPAP and cancer stem cells from PTC, analyzed under hypoxic conditions, showed a hypoxia-driven increase in ER-α expression. In conclusion, ER-α might have an important role in human PTC, and its overexpression can be studied in routine needle aspirate as a possible marker of malignancy. (Cancer Sci 2011; 102: 1921–1927)

An Optimized Protocol for First Strand cDNA Synthesis from Laser Capture Microdissected Tissue

Debate persists regarding the role of Desulfovibrio subspecies in ulcerative colitis. Combined microscopic and molecular techniques enable this issue to be investigated by allowing precise enumeration of specific bacterial species within the colonic mucous gel. The aim of this study was to combine laser capture microdissection and quantitative polymerase chain reaction to determine Desulfovibrio copy number in crypt-associated mucous gel in health and in acute and chronic ulcerative colitis.Colonic mucosal biopsies were harvested from healthy controls (n = 19) and patients with acute (n = 10) or chronic (n = 10) ulcerative colitis. Crypt-associated mucous gel was obtained by laser capture microdissection throughout the colon. Pan-bacterial 16S rRNA and Desulfovibrio copy number/mm were obtained by polymerase chain reaction at each locus. Bacterial copy numbers were interrogated for correlation with location and disease activity. Data were evaluated using a combination of ordinary linear methods and linear mixed-effects models to cater for multiple interactions.Desulfovibrio positivity was significantly increased in acute and chronic ulcerative colitis at multiple levels within the colon, and after normalization with total bacterial signal, the relative Desulfovibrio load was increased in acute colitis compared with controls. Desulfovibrio counts did not significantly correlate with age, disease duration, or disease activity but interlevel correlations were found in adjacent colonic segments in the healthy control and chronic ulcerative colitis groups.The presence of Desulfovibrio subspecies is increased in ulcerative colitis and the data presented suggest that these bacteria represent an increased percentage of the colonic microbiome in acute ulcerative colitis.

Both the nucellar projection (NP) and endosperm transfer cells (ETC) of the developing barley grain (harvested 8 days after flowering) were isolated by laser capture micro-dissection combined with pressure catapulting. Protein extracts were analyzed by nanoUPLC separation combined with ESI-Q-TOF mass spectrometry. The majority of the ∼160 proteins identified were involved in translation, protein synthesis, or protein destination. The NP proteome was enriched for stress defense molecules, while proteins involved in assimilate transport and the mobilization of nutrients were common to both the NP and the ETC. The combined qualitative and quantitative protein profiling allowed for the identification of several proteins showing tissue specificity in their expression, which underlines the distinct biological functions of these two tissues within the developing barley grain.

EuroKUP (Urine and Kidney Proteomics www.eurokup.org) is a COST (European Cooperation in the field of Scientific and Technical research: www.cost.esf.org Action fostering amulti-disciplinary network of investigators from 25 countries and focusing on facilitating translational proteomic research in kidney diseases. Four Working Groups focusing respectively on defining clinically important research questions in kidney diseases, kidney tissue proteomics, urine proteomics and bioinformatics have been generated. The EuroKUP members had their second combined Working Group and Management Committee (MC) meeting in Nafplio, Greece from March 29 to 30, 2009. This report summarizes the main presentations, discussions and agreed action points during this meeting. These refer to the design of collaborative projects and clinical center networks for specific kidney diseases establishment of guidelines for kidney tissue proteomics analysis by laser-based imaging- and laser capture microdissection-MS development and characterization of a "standard" urine specimen to be used for assessment of platform capability and data comparability in clinical proteomics applications definition of statistical requirements in biomarker discovery studies and development of a specialized kidney and urine ontology. Various training activities are planned involving training schools on laser capture microdissection- and imaging-MS, workshops on ontologies as well as short-term travel grants for junior investigators. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. 3 1017 1022 Cited By :8

We assessed proteomic patterns in breast cancer using MALDI MS and laser capture microdissected cells. Protein and peptide expression in invasive mammary carcinoma versus normal mammary epithelium and estrogen-receptor positive versus estrogen-receptor negative tumors were compared. Biomarker candidates were identified by statistical analysis and classifiers were developed and validated in blinded test sets. Several of the m/ z features used in the classifiers were identified by LC-MS/MS and two were confirmed by immunohistochemistry.

Successful collection of tissue samples for molecular analysis requires critical considerations. We describe here our procedure for tissue specimen collection for proteomic purposes with emphasis on the most important steps, including timing issues and the procedures for immediate freezing, storage, and microdissection of the cells of interest or "tissue targets" and the lysates for protein isolation for SELDI, MALDI, and 2DGE applications. The pathologist is at the cornerstone of this process and is an invaluable collaborator. In most institutions, pathologists are responsible for "tissue custody," and they closely supervise the tissue bank. In addition, they are optimally trained in histopathology in order to they assist investigators to correlate tissue morphology with molecular findings. In recent years, the advent of the laser capture microscope, a tool ideally designed for pathologists, has tremendously facilitated the efficiency of collecting tissue targets for molecular analysis.

Background Identifying the influence formalin fixation has on RNA integrity and recovery from clinical tissue specimens is integral to determining the utility of using archival tissue blocks in future molecular studies. For clinical material, the current gold standard is unfixed tissue that has been snap frozen. Fixed and frozen tissue however, both require laser capture microdissection to select for a specific cell population to study. The recent development of a sampling method capable of obtaining a viable, enriched cell population represents an alternative option in procuring cells from clinical material for molecular research purposes. The expression profiles of cells obtained by using this procurement approach, in conjunction with the profiles from cells laser capture microdissected from frozen tissue sections, were compared to the expression profiles from formalin fixed cells to determine the influence fixation has on expression profiles in clinical material. Methods Triplicate samples of non-neoplastic colonic epithelial cells were recovered from a hemicolectomy specimen using three different procurement methods from the same originating site: 1) an exfoliation and enrichment strategy 2) laser capture microdissection from formalin fixed tissue and 3) laser capture microdissection from frozen tissue. Parameters currently in use to assess RNA integrity were utilized to assess the quality of recovered RNA. Additionally, an expression microarray was performed on each sample to assess the influence each procurement technique had on RNA recovery and degradation. Results The exfoliation/enrichment strategy was quantitatively and qualitatively superior to tissue that was formalin fixed. Fixation negatively influenced the expression profile of the formalin fixed group compared to both the frozen and exfoliated/enrichment groups. Conclusion The exfoliation/enrichment technique represents a superior alternative in tissue procurement and RNA recovery relative to formalin fixed tissue. None of the deleterious effects associated with formalin fixation are encountered in the exfoliated/enriched samples because of the absence of its use in this protocol. The exfoliation/enrichment technique also represents an economical alternative that will yield comparable results to cells enriched by laser capture microdissection from frozen tissue sections.

Laser Capture Microdissection (LCM) is a technique providing a rapid and reliable method to procure purified cell populations, either as multiple single cells or a group of cells, from a heterogenic tissue sample allowing a targeted approach to genomic profiling. When analyzing gene expression profiles from a group of cells, the average profile may not be a true representation of the many different profiles that could exist even in a pure cell population (e.g., in different states of growth, differentiation, or activation). As a result, the transcriptional variability of individual cells and any insight into the relationship between specific genes in single cells are lost. To fully understand the complexity of tissue and cellular heterogeneity, it is necessary to measure molecular signatures at the single cell resolution. Here we developed a workflow for gene expression profiling of a single LCM cell that can also be applied to small number of LCM cells. The ArcturusXT™ LCM system was used to harvest single cells from frozen human tumor tissue sample which were then processed for quantitative RT-PCR (qRT-PCR) gene expression analysis. Citation Format: Shirley Chu. Gene expression profiling of a single laser capture microdissected (LCM) cell. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3471. doi:10.1158/1538-7445.AM2014-3471

Multiple myeloma is characterized by the growth of myeloma cells in bone and the development of bone disease. This is mediated by increased bone resorption and suppressed bone formation. The mechanism responsible for the inhibition of bone formation is poorly understood. The wnt antagonist Dkk1 has been implicated, but is not abnormally expressed in all patients, suggesting other molecules may also contribute. We sought to identify candidate molecules in experimental models of myeloma using laser-capture micro-dissection (LCMD) and gene array analysis. Injection of 5T2MM myeloma cells into C57BLKalwRij mice results in bone disease characterised by increased resorption, decreased bone formation, reduced bone volume and bone lesions. In contrast, 5T33MM cells have no effect on resorption, did not cause bone loss, or promote bone lesions. mRNA isolated from LCMD areas of the tibia infiltrated with 5T2MM, 5T33MM, or naïve bones, was subjected to micro-array analysis (Affymetrix). Data was analyses using PartekGenomic Suite software. Genes that define myeloma cells, including CD138 and immunoglobulin genes were up-regulated in 5T2MM and 5T33MM bones. Genes implicated in promoting bone resorption, including MIP1?, RANKL, IL-1, and HGF were rarely up-regulated more than 2-fold in 5T2MM compared to 5T33MM or naïve mice. Analysis of genes implicated in regulating bone formation, including Dkk1, sFRP1, 2 and 3, IL3, and IL7 was not significantly up-regulated. In contrast, a panel of genes, not previously implicated in myeloma bone disease, was highly upregulated in 5T2MM bearing mice when compared to 5T33MM bearing, or naïve, mice. These included periostin and osteoglycin. Quantitative RT-PCR confirmed that these genes were up-regulated. Co-culture of myeloma cells with osteoblasts induced contactdependent expression of periostin in myeloma cells. Periostin also induced Dkk1 expression in osteoblasts. This study has identified new genes that may play an important role in myeloma bone disease and represent potential new therapeutic targets.