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An important question is what genes govern the differentiation of plant embryos into suspensor and embryo proper regions following fertilization and division of the zygote. We compared embryo proper and suspensor transcriptomes of four plants that vary in embryo morphology within the suspensor region. We determined that genes encoding enzymes in several metabolic pathways leading to the formation of hormones, such as gibberellic acid, and other metabolites are up-regulated in giant scarlet runner bean and common bean suspensors. Genes involved in transport and Golgi body organization are up-regulated within the suspensors of these plants as well, strengthening the view that giant specialized suspensors serve as a hormone factory and a conduit for transferring substances to the developing embryo proper. By contrast, genes controlling transcriptional regulation, development, and cell division are up-regulated primarily within the embryo proper. Transcriptomes from less specialized soybean and Arabidopsis suspensors demonstrated that fewer genes encoding metabolic enzymes and hormones are up-regulated. Genes active in the embryo proper, however, are functionally similar to those active in scarlet runner bean and common bean embryo proper regions. We uncovered a set of suspensor- and embryo proper-specific transcription factors (TFs) that are shared by all embryos irrespective of morphology, suggesting that they are involved in early differentiation processes common to all plants. Chromatin immunoprecipitation sequencing (ChIP-Seq) experiments with scarlet runner bean and soybean WOX9, an up-regulated suspensor TF, gained entry into a regulatory network important for suspensor development irrespective of morphology.

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. [ABSTRACT FROM AUTHOR]

Summary: 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. Significance Statement: For the first time, we show that epidermal chloroplasts are focally deployed towards the host–pathogen interface during infection by the potato blight pathogen Phytophthora infestans, implicating chloroplasts in the focal immune response against a historically and economically important disease. This study explores the spatiotemporal dynamics and changes in morphology of chloroplasts during parasitic invasion, further establishing chloroplasts as key players in the plant immune system. [ABSTRACT FROM AUTHOR]

A limitation of conventional bulk-tissue proteome studies in amyotrophic lateral sclerosis (ALS) is the confounding of motor neuron (MN) signals by admixed non-MN proteins. Here, we leverage laser capture microdissection and nanoPOTS single-cell mass spectrometry-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control tissues. In a follow-up analysis, we examine the impact of stratification of MNs based on cytoplasmic transactive response DNA-binding protein 43 (TDP-43)+ inclusion pathology on the profiles of 2,238 proteins. We report extensive overlap in differentially abundant proteins identified in ALS MNs with or without overt TDP-43 pathology, suggesting early and sustained dysregulation of cellular respiration, mRNA splicing, translation, and vesicular transport in ALS. Together, these data provide insights into proteome-level changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein dynamics in human neurologic diseases. [Display omitted] • Single-cell proteomic analysis of human ALS motor neurons directly captured by LMD • ALS motor neuron deficiencies in splicing, translation, and vesicular transport machinery • Stratification of single-neuron proteomes based on pathologic TDP-43 inclusions • Utility of trace sample proteomics in augmenting the study of human neurological diseases Guise and Misal et al. report the unbiased single-cell proteomic analysis of ALS motor neurons directly captured from human tissues to explore disease-associated protein dynamics across TDP43-pathological strata. This study highlights both important technical challenges accompanying single-cell omics studies and emerging avenues for exploring human disease biology with nanoPOTS. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Expression array data from dozens of laboratories, including our own, show significant changes in expression of many genes in Alzheimer's disease (AD) patients compared with normal controls. These data typically rely on brain homogenates, and information about transcripts specific to microglia and other central nervous system (CNS) cell types, which far outnumber microglia-specific transcripts, is lost. We therefore used single-cell laser capture methods to assess the full range of microglia-specific expression changes that occur in different brain regions (substantia nigra and hippocampus CA1) and disease states (AD, Parkinson's disease, and normal controls). Two novel pathways, neuronal repair and viral processing were identified. Based on KEGG analysis (Kyoto Encyclopedia of Genes and Genomes, a collection of biological pathways), one of the most significant viruses was hepatitis B virus (HBV) (false discovery rate < 0.00000001). Immunohistochemical analysis using HBV-core antibody in HBV-positive control, amnestic mild cognitive impairment, and HBV-positive AD cases show increased HBV immunoreactivity as disease pathology increases. These results are the first, to our knowledge, to show regional differences in human microglia. In addition, these data reveal new functions for microglia and suggest a novel risk factor for AD. [ABSTRACT FROM AUTHOR]

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 picturewinged 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 fastgenerated 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. [ABSTRACT FROM AUTHOR]

For generalist pathogens, host species represent distinct selective environments, providing unique challenges for resource acquisition and defense from host immunity, potentially resulting in hostdependent differences in pathogen fitness. Gene expression modulation should be advantageous, responding optimally to a given host and mitigating the costs of generalism. Batrachochytrium dendrobatidis (Bd), a fungal pathogen of amphibians, shows variability in pathogenicity among isolates, and withinstrain virulence changes rapidly during serial passages through artificial culture. For the first time, we characterize the transcriptomic profile of Bd in vivo, using laser-capture microdissection. Comparison of Bd transcriptomes (strain JEL423) in culture and in two hosts (Atelopus zeteki and Hylomantis lemur), reveals >2000 differentially expressed genes that likely include key Bd defense and host exploitation mechanisms. Variation in Bd transcriptomes from different amphibian hosts demonstrates shifts in pathogen resource allocation. Furthermore, expressed genotype variant frequencies of Bd populations differ between culture and amphibian skin, and among host species, revealing potential mechanisms underlying rapid changes in virulence and the possibility that amphibian community composition shapes Bd evolutionary trajectories. Our results provide new insights into how changes in gene expression and infecting population genotypes can be key to the success of a generalist fungal pathogen. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma growth impacts on the structure and physiology of peritumoral neuronal networks, altering the activity of pyramidal neurons which drives further tumor progression. It is therefore of paramount importance to identify glioma-induced changes in pyramidal neurons, since they represent a key therapeutic target., Methods: We longitudinal monitored visual evoked potentials after the orthotopic implant of murine glioma cells into the mouse occipital cortex. With laser microdissection, we analyzed layer II-III pyramidal neurons molecular profile and with local field potentials recordings we evaluated the propensity to seizures in glioma-bearing animals with respect to control mice., Results: We determine the time course of neuronal dysfunction of glioma-bearing mice and we identify a symptomatic stage, based on the decay of visual response. At that time point, we microdissect layer II-III pyramidal neurons and evaluate the expression of a panel of genes involved in synaptic transmission and neuronal excitability. Compared to the control group, peritumoral neurons show a decrease in the expression of the SNARE complex gene SNAP25 and the alpha1 subunit of the GABA-A receptor. No significant changes are detected in glutamatergic (ie, AMPA or NMDA receptor subunit) markers. Further reduction of GABA-A signaling by delivery of a benzodiazepine inverse agonist, DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) precipitates seizures in 2 mouse models of tumor-bearing mice., Conclusions: These studies reveal novel molecular changes that occur in the principal cells of the tumor-adjacent zone. These modifications may be therapeutically targeted to ameliorate patients' quality of life., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Apatitic mineral of dentin forms within the collagenous matrix (intertubular dentin, ITD) secreted from the odontoblastic processes (OP). Highly calcified mineral (peritubular dentin, PTD) is deposited at the interface between the ITD and each process membrane, creating a tubular system penetrating the dentin that extends from the dentino–enamel junction to the predentin–dentin junction. We focus on determining the composition of the PTD both with regard to its organic matrix and the inorganic phase. A laser capture technique has been adapted for the isolation of the mineralized PTD free from the ITD, and for the analysis of the PTD by SEM, TEM, and energy dispersive spectrometry (EDS), these data were subsequently compared with similar analyses of intact dentin slices containing ITD bounded-PTD annuli. Elemental line scans reveal clearly marked boundaries between ITD, PTD, and OP components, and illustrate the differences in composition, and topographical surface roughness. The organic matrix of the PTD was shown to be sulfur rich, and further antibody labeling showed the sulfated organic component to be chondroitin sulfate B. In this PTD organic matrix the S/Ca and Ca/P ratios were distinctly higher than in the ITD, indicating that polysaccharide bound S supplies the anionic counterion facilitating the formation of the apatitic PTD mineral. [ABSTRACT FROM AUTHOR]

Physical inactivity is an important risk factor in the development of cardiovascular disease. The rostral ventrolateral portion of the medulla (RVLM) is composed of heterogeneous populations of neurons that are involved in the regulation of the cardiovascular system. Because of functional heterogeneity, studying the changes in the gene expression of this specific population of neurons within the RVLM is challenging. In the present study, a fluorescent retrograde tracer was injected into the spinal cord to specifically label bulbospinal RVLM neurons in sedentary and active rats. Laser capture microdissection (LCM) was then employed to collect the fluorescently labeled neurons from sections encompassing the rostrocaudal extent of the RVLM. RNA extracted from the neurons was used in qRT-PCR analysis. Changes in gene expression levels of glutamate and GABA receptor subunits were compared between sedentary and physically active rats. GLUR3 subunit showed a significant negative correlation between total running distance and its relative gene expression in active rats. There were no significant difference in the gene expression of NMDA (NR1, NR2A, NR2B, NR2C and NR2D), AMPA (GLUR1, GLUR2 and GLUR3) and GABA A (GABA A1 and GABA A2 ) receptor subunits. Overall, the present study demonstrates the feasibility of utilizing LCM to investigate the gene expression changes in a specific population of neurons in the RVLM. Correlation studies suggest that physical activity could contribute to neuroplasticity in the RVLM. [ABSTRACT FROM AUTHOR]

Allergen challenges induce airway hyperresponsiveness (AHR) and increased airway smooth muscle (ASM) mass in the sensitized rat. Whether the remodeled ASM changes its phenotype is uncertain. We examined, in sensitized Brown Norway rats, the effects of multiple ovalbumin (Ova) challenges on ASM remodeling and phenotype and the role of the epidermal growth factor receptor (EGFR) in these processes. Rats were sensitized with Ova and challenged three times at 5-day intervals with phosphate-buffered saline or Ova and pretreated with the EGFR inhibitor AG-1478 (5 mg/kg) or its vehicle dimethyl sulfoxide. Ova challenges increased ASM mass in all-sized airways and in large airway mRNA expression of smooth muscle myosin heavy chain (sm-MHC), assessed by laser capture. Myosin light chain kinase and the fast myosin isoform SM-B mRNA expressions were not affected. Ova induced AHR to methacholine, and, based on the constant-phase model, this was largely attributable to the small airways and lung derecruitment at 48 h that recovered by 1 wk. The EGFR ligands amphiregulin and heparin-binding epidermal growth factor (HB-EGF) were increased in bronchoalveolar lavage fluid at 48 h after Ova exposure. AG-1478 inhibited AHR and prevented ASM growth. Epithelial gene expression of EGFR, HB-EGF, matrix metalloproteinase (MMP)-9, Gro-α, and transforming growth factor-β was unaffected by Ova challenges. We conclude that EGFR drives remodeling of ASM, which results from repeated Ova challenge. Furthermore, the latter results in excessive small airway and, to a lesser degree, large airway narrowing to methacholine, and large airway gene expression of contractile protein is conserved. [ABSTRACT FROM AUTHOR]

Abstract: Exposing an animal to light during the normal dark period of its daily cycle induces shifts in the animal''s circadian rhythm of activity. These shifts are preceded by an increase in the expression of an array of immediate early genes in the suprachiasmatic nucleus, the location of the primary circadian clock in the brain. For most of these genes, little is known about the physiological significance of their expression in the SCN. In order to characterize the expression of these genes, laser capture microscopy, and real-time PCR were used to measure the time course of expression of immediate-early genes in the SCN after a 30-min light pulse during the early portion of the night. Most of the measured genes show peak expression shortly after the end of the stimulus and then decline back to baseline after 2h. However, a few genes, including Rrad, Egr3, and Jun, show a more sustained elevation in expression. Analysis of the function of light-induced genes in other cellular systems suggests a possible role for these genes in reducing the SCN to subsequent photic stimuli and in protecting the SCN from excitotoxicity. [Copyright &y& Elsevier]

Abstract: The serotonin neurons of the dorsal and medial raphe nuclei project to all areas of the forebrain and play a key role in mood disorders. Hence, any loss or degeneration of serotonin neurons could have profound ramifications. In a monkey model of surgical menopause with hormone replacement and no neural injury, E and P decreased gene expression in the dorsal raphe nucleus of c-jun n-terminal kinase (JNK1) and kynurenine mono-oxygenase (KMO) that promote cell death. In concert, E and P increased gene expression of superoxide dismutase (SOD1), VEGF, and caspase inhibitory proteins that promote cellular resilience in the dorsal raphe nucleus. Subsequently, we showed that ovarian steroids inhibit pivotal genes in the caspase-dependent and caspase-independent pathways in laser-captured serotonin neurons including apoptosis activating factor (Apaf1), apoptosis-inducing factor (AIF) and second mitochondria-derived activator of caspases (Smac/Diablo). SOD1 was also increased specifically in laser-captured serotonin neurons. Examination of protein expression in the dorsal raphe block revealed that JNK1, phosphoJNK1, AIF and the translocation of AIF from the mitochondria to the nucleus decreased with hormone therapy, whereas pivotal execution proteins in the caspase pathway were unchanged. In addition, cyclins A, B, D1 and E were inhibited, which would prevent re-entry into the cell cycle and catastrophic death. These data indicated that in the absence of gross injury to the midbrain, ovarian steroids inhibit the caspase-independent pathway and cell cycle initiation in serotonin neurons. To determine if these molecular actions prevented cellular vulnerability or death, we examined DNA fragmentation in the dorsal raphe nucleus with the TUNEL assay (terminal deoxynucleotidyl transferase nick end labeling). Ovarian steroids significantly decreased the number of TUNEL-positive cells in the dorsal raphe. Moreover, TUNEL staining prominently colocalized with TPH immunostaining, a marker for serotonin neurons. In summary, ovarian steroids increase the cellular resilience of serotonin neurons and may prevent serotonin neuron death in women facing decades of life after menopause. The survival of serotonin neurons would support cognition and mental health. [Copyright &y& Elsevier]

The cuticle of human hair consists of several layers of flat cells that are hardened through their content of cross-linked proteins and protect the hair structure from the environment. Known proteins in the cuticle are the sulphur-rich KAP 5 and KAP10 proteins located in the exocuticle and cross-linked by disulphide bonds. Isopeptide bonds are also present and led to a proposal from amino acid analysis that the surface of cuticle cells also contains keratinocyte cell envelope proteins, loricrin, involucrin and small proline-rich proteins that contribute to the stability of the hair cuticle. Confirmation of that proposal by protein chemical methods is difficult because of the insolubility of the surface membranes. In the previous studies by other authors, involucrin was not detected in the cuticle by in situ hybridization or by immunoelectron microscopy with specific antibodies. An alternative approach was undertaken to determine whether mRNAs encoding keratinocyte envelope proteins are expressed in cuticle cells in the human hair follicle. The study utilized dissection of the cuticle, cortex and inner root sheath layers from follicles by laser capture microscopy. RNA was isolated and subjected to PCR analysis with specific primers to detect expression of mRNAs encoding cell envelope proteins. Their presence in the cuticle was not detected, and it was concluded that the proteins they encode are not produced. The structural consequences including the possibility that KAPs 5 and 10 are the prime components cross-linked by both disulphide and isopeptide bonds are discussed. [ABSTRACT FROM AUTHOR]

The mechanism(s) involved in the regulation of the seasonal-appropriate body weight of the Siberian hamster are currently unknown. We have identified photoperiodically regulated genes including VGF in a sub-region of the arcuate nucleus termed the dorsomedial posterior arcuate (dmpARC). Gene expression changes in this nucleus so far account for a significant number of those reported as photoperiodically regulated and are therefore likely to contribute to seasonal physiological responses of the hamsters. The present study aimed to identify additional genes expressed in the dmpARC regulated by photoperiod that could be involved in regulating the activity of this nucleus with respect to seasonal physiology of the Siberian hamster. Using laser capture microdissection coupled with a microarray analysis and a candidate gene approach, we have identified several photoperiodically regulated genes in the dmpARC that are known to have roles in secretory and intracellular signalling pathways. These include secretogranin (sg) III and SgVI (secretory pathway), melanocortin 3 receptor (MC3-R) and serotonin (5-HT) receptors 2A and 7 (signalling pathway), all of which increase in expression under a short photoperiod. The spatial relationship between receptor signalling and potential secretory pathways was investigated by dual in situ hybridisation, which revealed that 5-HT2A and 5-HT7 receptors are expressed in neurones expressing VGF mRNA and that a sub-population (approximately 40%) of these neurones express MC3-R. These gene expression changes in dmpARC neurones may reflect the functional requirement of these neurones for seasonal physiological responses of the hamster. [ABSTRACT FROM AUTHOR]

OBJECTIVE To investigate the feasibility of transcript profiling in diagnostic formalin-fixed and paraffin-embedded (FFPE) biopsies for prostate cancer. MATERIALS AND METHODS Laser-capture microdissection (LCM) was used to microdissect glandular epithelium as well as stromal tissue in archival prostate needle biopsies. Optimized RNA extraction, reverse transcription and real-time PCR (QPCR) protocols were used to detect transcript expression. RNA degradation effects were assessed using hydrolysed cell line RNA and matched xenograft FFPE and frozen tumours. RESULTS LCM and RNA extraction was achieved in all biopsies from a pilot cohort of five patients. cDNA produced was successfully used to detect expression of glyceraldehyde-3-phosphate dehydrogenase, RPL13, prostate-specific antigen, vimentin, inhibitor of differentiation/DNA binding 1 (Id-1) and polycomb group protein enhancer of zeste homolog 2 (EZH2) transcripts. In the cell line and xenograft models, we investigated the effect of RNA degradation on transcript quantification by QPCR. In both models normalization of transcript quantity with a housekeeping gene resulted in restored expression in all degraded samples to within a 50% difference of control samples. Using an extended cohort of 29 biopsies, we tested application in detecting differences in EZH2 and Id-1 expression between malignant and benign epithelium. The results confirmed that our technique was capable of quantifying significant differences in expression between malignant and benign epithelium consistent with the reported trends. CONCLUSION This study reports the use of standard FFPE needle biopsies for transcript profiling and supports the concept of molecular prognostic studies in tissue acquired at diagnosis in prostate cancer. [ABSTRACT FROM AUTHOR]

Mice lacking CD2-associated protein (CD2AP−/−) develop glomerular lesions resembling human focal segmental glomerulosclerosis (FSGS) between 3–4 weeks of age and die approximately 2 weeks later from massive proteinuria and renal failure. The mechanisms involved in the glomerular injury in this model are unclear. In this study, we used laser capture microdissection (LCM) and real-time PCR, and examined expression of TGF-ß isoforms in CD2AP−/− mice at the level of isolated glomeruli. Total RNA yield from cryosections of 30 glomeruli was 10.71 ng (SD, 5.45) in CD2AP+/+ group ( n =7), and 4.20 ng (SD, 2.04) in CD2AP−/− group ( n =8), p =0.008. Expression of TGF-ß1 mRNA was increased 1.5-fold in the whole kidney ( p =0.030), and twofold in isolated CD2AP−/− glomeruli ( p =0.026). Whole kidney mRNA of TGF-ß receptor I (RI) and II (RII) was not different in CD2AP−/− and CD2AP+/+ animals, but it was increased in CD2AP−/− glomerular samples by 4.38-fold ( p =0.001) and 11.37-fold ( p =0.0163), respectively. By using LCM we confirmed increased glomerular expression levels of TGF-ß isoforms previously described by our group in glomeruli isolated by sieving in CD2AP KO mice and underscored the importance of local factors in the development of glomerulosclerosis. [ABSTRACT FROM AUTHOR]

Aims : The bland histology of odontogenic keratocyst (OKC) belies its capacity for aggressive behaviour. Genetic alterations of OKC have not been well studied. We examined the frequency and pattern of allelic imbalance on five different chromosome regions from 15 patients with OKC. Methods and results : Laser-assisted microdissection was performed on formalin-fixed paraffin-embedded tissue. Polymerase chain reaction analysis of extracted DNA targeted five polymorphic DNA markers (D3S1285, D9S161, D11S1316, D13S290, and TP53) representing chromosome regions 3p14, 9p21, 11q23, 13q12.1 and 17p13, respectively. All 15 cases of OKC were informative at a minimum of three of five loci, with 11 informative on all five loci. Twelve of 15 cases (80%) demonstrated loss of heterozygosity (LOH). Seven cases (47%) showed LOH at more than two DNA loci. The frequency of LOH was 5/11 (45%) at D3S1285, 3/15 (20%) at D9S161, 4/14 (29%) at D11S1316, 8/14 (57%) at D13S290 and 3/15 (20%) at TP53. Conclusions : The majority of OKCs harbour chromosomal abnormalities. This finding supports the supposition that OKCs are neoplastic. Furthermore, OKCs harbour allelic loss at some of the same loci identified in squamous cell carcinoma. This may aid in explaining the rare occurrence of squamous cell carcinoma arising in OKC. [ABSTRACT FROM AUTHOR]

Because of the paucity of available tissue, little information has previously been available regarding the gene expression profiles of primary melanomas. To understand the molecular basis of melanoma progression, we compared the gene expression profiles of a series of nevi, primary melanomas, and melanoma metastases. We found that metastatic melanomas exhibit two dichotomous patterns of gene expression, which unexpectedly reflect gene expression differences already apparent in comparing laser-capture microdissected radial and vertical phases of a large primary melanoma. Unsupervised hierarchical clustering accurately separated nevi and primary melanomas. Multiclass significance analysis of microarrays comparing normal skin, nevi, primary melanomas, and the two types of metastatic melanoma identified 2,602 transcripts that significantly correlated with sample class. These results suggest that melanoma pathogenesis can be understood as a series of distinct molecular events. The gene expression signatures identified here provide the basis for developing new diagnostics and targeting therapies for patients with malignant melanoma. [ABSTRACT FROM AUTHOR]

Although the gene expression patterns during the development of diabetic nephropathy have been studied in both rodent models and humans, only a small portion of the mRNAs expressed in the mesangium or in glomerular cells has been characterized. In the present study we report larger groups of transcripts displaying significant expression modulation in glomerular cells obtained from the early phase of diabetic nephropathy. Methods: We used 12-week-old female db/db mice, a rodent model of type 2 diabetes, and their non-diabetic db/m litter-mates. Glomerular cells were obtained from the kidneys of mice by laser capture microdissection. Preparation of cRNA and target hybridization were performed according to the Affymetrix GeneChip Eukaryotic Small Sample Target Labeling Assay Protocol (Version II). The gene expression profile was determined by the mouse Expression Set 430A GeneChip. Results: By comparison between db/m and db/db mice, 649 probes that increased in expression with the induction of diabetes and 340 probes that decreased in diabetic kidneys were identified. Although some of these genes have previously been shown to play an important role in diabetic nephropathy, the large majority of them have never been demonstrated to be regulated during the development of nephropathy. Conclusions: Although the precise involvement of these genes in diabetic nephropathy remains to be clarified, the data presented here will aid in the identification of genes that play a significant role in this pathological condition. [ABSTRACT FROM AUTHOR]

With completion of the human genome sequence, it is now possible to study the expression of the entire human gene complement of ∼30,000-35,000 genes. To accomplish this goal, microarrays have become the leading methodology for the analysis of global gene expression. Improvements in technology have increased the sensitivity of microarrays to the point where it is feasible to study gene expression in a small number of cells and even at the single cell level. A summary of developments in the area of expression profiling in single cells will be described, and the rationale for these types of studies will be presented. In addition, from a biologist's point of view, some bioinformatic challenges of expression analysis of single cells will be discussed. [ABSTRACT FROM AUTHOR]

It is frequently useful to determine the methylation state of samples containing limited amounts of DNA such as from embryos, or from fixed tissue samples in which DNA is degraded or difficult to isolate. By modification of the standard protocols for DNA preparation and bisulfite treatment, it is possible to obtain DNA methylation sequence data for such samples. We present methods for bisulfite treatment of embryos, fixed sections, and samples obtained by laser capture microdissection, and discuss the additional experimental considerations required when working with small numbers of cells or degraded DNA samples. [Copyright &y& Elsevier]