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Abstract Background The first week of human pre-embryo development is characterized by the induction of totipotency and then pluripotency. The understanding of this delicate process will have far reaching implication for in vitro fertilization and regenerative medicine. Human mature MII oocytes and embryonic stem (ES) cells are both able to achieve the feat of cell reprogramming towards pluripotency, either by somatic cell nuclear transfer or by cell fusion, respectively. Comparison of the transcriptome of these two cell types may highlight genes that are involved in pluripotency initiation. Results Based on a microarray compendium of 205 samples, we compared the gene expression profile of mature MII oocytes and human ES cells (hESC) to that of somatic tissues. We identified a common oocyte/hESC gene expression profile, which included a strong cell cycle signature, genes associated with pluripotency such as LIN28 and TDGF1, a large chromatin remodelling network (TOP2A, DNMT3B, JARID2, SMARCA5, CBX1, CBX5), 18 different zinc finger transcription factors, including ZNF84, and several still poorly annotated genes such as KLHL7, MRS2, or the Selenophosphate synthetase 1 (SEPHS1). Interestingly, a large set of genes was also found to code for proteins involved in the ubiquitination and proteasome pathway. Upon hESC differentiation into embryoid bodies, the transcription of this pathway declined. In vitro, we observed a selective sensitivity of hESC to the inhibition of the activity of the proteasome. Conclusion These results shed light on the gene networks that are concurrently overexpressed by the two human cell types with somatic cell reprogramming properties.

Abstract Background The huge amount of data generated by DNA chips is a powerful basis to classify various pathologies. However, constant evolution of microarray technology makes it difficult to mix data from different chip types for class prediction of limited sample populations. Affymetrix® technology provides both a quantitative fluorescence signal and a decision (detection call: absent or present) based on signed-rank algorithms applied to several hybridization repeats of each gene, with a per-chip normalization. We developed a new prediction method for class belonging based on the detection call only from recent Affymetrix chip type. Biological data were obtained by hybridization on U133A, U133B and U133Plus 2.0 microarrays of purified normal B cells and cells from three independent groups of multiple myeloma (MM) patients. Results After a call-based data reduction step to filter out non class-discriminative probe sets, the gene list obtained was reduced to a predictor with correction for multiple testing by iterative deletion of probe sets that sequentially improve inter-class comparisons and their significance. The error rate of the method was determined using leave-one-out and 5-fold cross-validation. It was successfully applied to (i) determine a sex predictor with the normal donor group classifying gender with no error in all patient groups except for male MM samples with a Y chromosome deletion, (ii) predict the immunoglobulin light and heavy chains expressed by the malignant myeloma clones of the validation group and (iii) predict sex, light and heavy chain nature for every new patient. Finally, this method was shown powerful when compared to the popular classification method Prediction Analysis of Microarray (PAM). Conclusion This normalization-free method is routinely used for quality control and correction of collection errors in patient reports to clinicians. It can be easily extended to multiple class prediction suitable with clinical groups, and looks particularly promising through international cooperative projects like the "Microarray Quality Control project of US FDA" MAQC as a predictive classifier for diagnostic, prognostic and response to treatment. Finally, it can be used as a powerful tool to mine published data generated on Affymetrix systems and more generally classify samples with binary feature values.

Senescent cells promote progressive tissue degeneration through the establishment of a combined inflammatory and trophic microenvironment. The cellular senescence state has therefore emerged as a central driving mechanism of numerous age-related diseases, including osteoarthritis (OA), the most common rheumatic disease. Senescence hallmarks are detectable in chondrocytes, synoviocytes and sub-chondral bone cells. This study investigates how the senescence-driven microenvironment could impact the cell fate of resident osteoarticular mesenchymal stromal/stem cells (MSCs) that are hence contributing to OA disease progression. For that purpose, we performed a comparative gene expression analysis of MSCs isolated from healthy donors that were in vitro chronically exposed either to interferon-gamma (IFN-γ) or Transforming Growth Factor beta 1 (TGFβ1), two archetypical factors produced by senescent cells. Both treatments reduced MSC self-renewal capacities by upregulating different senescence-driven cycle-dependent kinase inhibitors. Furthermore, a common set of differentially expressed genes was identified in both treated MSCs that was also found enriched in MSCs isolated from OA patients. These findings highlight an imprinting of OA MSCs by the senescent joint microenvironment that changes their matrisome gene expression. Altogether, this research gives new insights into OA etiology and points to new innovative therapeutic opportunities to treat OA patients.

As opposed to surface marker staining, certain cell types can only be recognized by intracellular markers. Intracellular staining for use in cell sorting remains challenging. Fixation and permeabilization steps for intracellular staining and the presence of RNases notably affect preservation of high-quality mRNA. We report the work required for the optimization of a successful protocol for microarray analysis of intracellular target-sorted, formalin-fixed human bronchial club cells. Cells obtained from differentiated air-liquid interface cultures were stained with the most characteristic intracellular markers for club cell (SCGB1A1 + ) sorting. A benchmarked intracellular staining protocol was carried out before flow cytometry. The primary outcome was the extraction of RNA sufficient quality for microarray analysis as assessed by Bioanalyzer System. Fixation with 4% paraformaldehyde coupled with 0.1% Triton/0.1% saponin permeabilization obtained optimal results for SCGB1A1 staining. Addition of RNase inhibitors throughout the protocol and within the appropriate RNA extraction kit (Formalin-Fixed-Paraffin-Embedded) dramatically improved RNA quality, resulting in samples eligible for microarray analysis. The protocol resulted in successful cell sorting according to specific club cell intracellular marker without using cell surface marker. The protocol also preserved RNA of sufficient quality for subsequent microarray transcriptomic analysis, and we were able to generate transcriptomic signature of club cells., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Platelets are small anucleate cells derived from the fragmentation of megakaryocytes and are involved in different biological processes especially hemostasis, thrombosis, and immune response. Despite their lack of nucleus, platelets contain a reservoir of megakaryocyte-derived RNAs and all the machinery useful for mRNA translation. Interestingly, platelet transcriptome was analyzed in health and diseases and led to the identification of disease-specific molecular signatures. Platelet contamination by leukocytes and erythrocytes during platelet purification is a major problem in transcriptomic analysis and the presence of few contaminants in platelet preparation could strongly alter transcriptome results. Since contaminant impacts on platelet transcriptome remains theoretical, we aimed to determine whether low leukocyte and erythrocyte contamination could cause great or only minor changes in platelet transcriptome. Using microarray technique, we compared the transcriptome of platelets from the same donor, purified by common centrifugation method or using magnetic microbeads to eliminate contaminating cells. We found that platelet transcriptome was greatly altered by contaminants, as the relative amount of 8274 transcripts was different between compared samples. We observed an increase of transcripts related to leukocytes and erythrocytes in platelet purified without microbeads, while platelet specific transcripts were falsely reduced. In conclusion, serious precautions should be taken during platelet purification process for transcriptomic analysis, in order to avoid platelets contamination and result alteration.

AICAR (Acadesine) is a pharmacological precursor of purine nucleotide biosynthesis with anti-tumoral properties. Although recognized as an AMP mimetic activator of the protein kinase AMPK, the AICAR monophosphate derivative ZMP was also shown to mediate AMPK-independent effects. In order to unveil these AMPK-independent functions, we performed a transcriptomic analysis in AMPKα1/α2 double knockout murine embryonic cells. Kinetic analysis of the cellular response to AICAR revealed the up-regulation of the large tumor suppressor kinases (Lats) 1 and 2 transcripts, followed by the repression of numerous genes downstream of the transcriptional regulators Yap1 and Taz. This transcriptional signature, together with the observation of increased levels in phosphorylation of Lats1 and Yap1 proteins, suggested that the Hippo signaling pathway was activated by AICAR. This effect was observed in both fibroblasts and epithelial cells. Knockdown of Lats1/2 prevented the cytoplasmic delocalization of Yap1/Taz proteins in response to AICAR and conferred a higher resistance to the drug. These results indicate that activation of the most downstream steps of the Hippo cascade participates to the antiproliferative effects of AICAR., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) are small noncoding RNAs that attenuate expression of their mRNA targets. Here, we developed a new method and an R package, to easily infer candidate miRNA-mRNA target interactions that could be functional during a given biological process. Using this method, we described, for the first time, a comprehensive integrated analysis of miRNAs and mRNAs during human normal plasma cell differentiation (PCD). Our results reveal 63 miRNAs with significant temporal changes in their expression during normal PCD. We derived a high-confidence network of 295 target relationships comprising 47 miRNAs and 141 targets. These relationships include new examples of miRNAs that appear to coordinately regulate multiple members of critical pathways associated with PCD. Consistent with this, we have experimentally validated a role for the miRNA-30b/c/d-mediated regulation of key PCD factors (IRF4, PRDM1, ELL2 and ARID3A). Furthermore, we found that 24 PCD stage-specific miRNAs are aberrantly overexpressed in multiple myeloma (MM) tumor plasma cells compared to their normal counterpart, suggesting that MM cells frequently acquired expression changes in miRNAs already undergoing dynamic expression modulation during normal PCD. Altogether, our analysis identifies candidate novel key miRNAs regulating networks of significance for normal PCD and malignant plasma cell biology., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

For aerobic human pathogens, adaptation to hypoxia is a critical factor for the establishment of persistent infections, as oxygen availability is low inside the host. The two-component system RegB/A of Brucella suis plays a central role in the control of respiratory systems adapted to oxygen deficiency, and in persistence in vivo . Using an original " in vitro model of persistence" consisting in gradual oxygen depletion, we compared transcriptomes and proteomes of wild-type and Δ regA strains to identify the RegA-regulon potentially involved in the set-up of persistence. Consecutive to oxygen consumption resulting in growth arrest, 12% of the genes in B. suis were potentially controlled directly or indirectly by RegA, among which numerous transcriptional regulators were up-regulated. In contrast, genes or proteins involved in envelope biogenesis and in cellular division were repressed, suggesting a possible role for RegA in the set-up of a non-proliferative persistence state. Importantly, the greatest number of the RegA-repressed genes and proteins, including aceA encoding the functional IsoCitrate Lyase (ICL), were involved in energy production. A potential consequence of this RegA impact may be the slowing-down of the central metabolism as B. suis progressively enters into persistence. Moreover, ICL is an essential determinant of pathogenesis and long-term interactions with the host, as demonstrated by the strict dependence of B. suis on ICL activity for multiplication and persistence during in vivo infection. RegA regulates gene or protein expression of all functional groups, which is why RegA is a key regulator of B. suis in adaptation to oxygen depletion. This function may contribute to the constraint of bacterial growth, typical of chronic infection. Oxygen-dependent activation of two-component systems that control persistence regulons, shared by several aerobic human pathogens, has not been studied in Brucella sp. before. This work therefore contributes significantly to the unraveling of persistence mechanisms in this important zoonotic pathogen.

A role of the transcription factor Krüppel-like factor 4 (KLF4) in the generation of mature plasma cells (PC) is unknown. Indeed, KLF4 is critical in controlling the differentiation of various cell linages, particularly monocytes and epithelial cells. KLF4 is expressed at low levels in pro-B cells and its expression increases as they mature into pre-B cells, resting naïve B cells and memory B cells. We show here that KLF4 is expressed in human bone marrow plasma cells and its function was studied using an in vitro model of differentiation of memory B cells into long lived plasma cells. KLF4 is rapidly lost when memory B cells differentiate into highly cell cycling plasmablasts, poorly cycling early plasma cells and then quiescent long-lived plasma cells. A forced expression of KLF4 in plasmablasts enhances the yield of their differentiation into early plasma cell and long lived plasma cells, by inhibiting apoptosis and upregulating previously unknown plasma cell pathways.

Simplified culture conditions are essential for large-scale drug screening and medical applications of human pluripotent stem cells (hPSCs). However, hPSCs [ie, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (iPSCs) are prone to genomic instability, a phenomenon that is highly influenced by the culture conditions. Enzymatic dissociation, a cornerstone of large-scale hPSC culture systems, has been reported to be deleterious, but the extent and the timeline of the genomic alterations induced by this passaging technique are still unclear. We prospectively monitored three hESC lines that were initially derived and cultured on human feeders and passaged mechanically before switching to enzymatic single-cell passaging. We show that karyotype abnormalities and copy number variations are not restricted to long-term culture, but can occur very rapidly, within five passages after switching hESCs to enzymatic dissociation. Subchromosomal abnormalities preceded or accompanied karyotype abnormalities and were associated with increased occurrence of DNA double-strand breaks. Our results indicate that enzymatic single-cell passaging can be highly deleterious to the hPSC genome, even when used only for a limited period of time. Moreover, hPSC culture techniques should be reappraised by complementing the routine karyotype analysis with more sensitive techniques, such as microarrays, to detect subchromosomal abnormalities.

In S. cerevisiae, replication timing is controlled by epigenetic mechanisms restricting the accessibility of origins to limiting initiation factors. About 30% of these origins are located within repetitive DNA sequences such as the ribosomal DNA (rDNA) array, but their regulation is poorly understood. Here, we have investigated how histone deacetylases (HDACs) control the replication program in budding yeast. This analysis revealed that two HDACs, Rpd3 and Sir2, control replication timing in an opposite manner. Whereas Rpd3 delays initiation at late origins, Sir2 is required for the timely activation of early origins. Moreover, Sir2 represses initiation at rDNA origins, whereas Rpd3 counteracts this effect. Remarkably, deletion of SIR2 restored normal replication in rpd3Δ cells by reactivating rDNA origins. Together, these data indicate that HDACs control the replication timing program in budding yeast by modulating the ability of repeated origins to compete with single-copy origins for limiting initiation factors., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Genetic abnormalities are common in patients with multiple myeloma, and may deregulate gene products involved in tumor survival, proliferation, metabolism and drug resistance. In particular, translocations may result in a high expression of targeted genes (termed spike expression) in tumor cells. We identified spike genes in multiple myeloma cells of patients with newly-diagnosed myeloma and investigated their prognostic value., Design and Methods: Genes with a spike expression in multiple myeloma cells were picked up using box plot probe set signal distribution and two selection filters., Results: In a cohort of 206 newly diagnosed patients with multiple myeloma, 2587 genes/expressed sequence tags with a spike expression were identified. Some spike genes were associated with some transcription factors such as MAF or MMSET and with known recurrent translocations as expected. Spike genes were not associated with increased DNA copy number and for a majority of them, involved unknown mechanisms. Of spiked genes, 36.7% clustered significantly in 149 out of 862 documented chromosome (sub)bands, of which 53 had prognostic value (35 bad, 18 good). Their prognostic value was summarized with a spike band score that delineated 23.8% of patients with a poor median overall survival (27.4 months versus not reached, P<0.001) using the training cohort of 206 patients. The spike band score was independent of other gene expression profiling-based risk scores, t(4;14), or del17p in an independent validation cohort of 345 patients., Conclusions: We present a new approach to identify spike genes and their relationship to patients' survival.

Intracellular deoxyribonucleoside triphosphate (dNTP) pools must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools are associated with increased mutagenesis, genomic instability and tumourigenesis. However, the mechanisms by which altered or imbalanced dNTP pools affect DNA synthesis remain poorly understood. Here, we show that changes in intracellular dNTP levels affect replication dynamics in budding yeast in different ways. Upregulation of the activity of ribonucleotide reductase (RNR) increases elongation, indicating that dNTP pools are limiting for normal DNA replication. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition to a slow-replication mode within minutes after S-phase entry. Upregulation of RNR activity delays this transition and modulates both fork speed and origin usage under replication stress. Interestingly, we also observed that chromosomal instability (CIN) mutants have increased dNTP pools and show enhanced DNA synthesis in the presence of HU. Since upregulation of RNR promotes fork progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.

Maintenance of genome integrity relies on surveillance mechanisms that detect and signal arrested replication forks. Although evidence from budding yeast indicates that the DNA replication checkpoint (DRC) is primarily activated by single-stranded DNA (ssDNA), studies in higher eukaryotes have implicated primer ends in this process. To identify factors that signal primed ssDNA in Saccharomyces cerevisiae, we have screened a collection of checkpoint mutants for their ability to activate the DRC, using the repression of late origins as readout for checkpoint activity. This quantitative analysis reveals that neither RFC(Rad24) and the 9-1-1 clamp nor the alternative clamp loader RFC(Elg1) is required to signal paused forks. In contrast, we found that RFC(Ctf18) is essential for the Mrc1-dependent activation of Rad53 and for the maintenance of paused forks. These data identify RFC(Ctf18) as a key DRC mediator, potentially bridging Mrc1 and primed ssDNA to signal paused forks.

Aging is the primary risk factor of neurodegenerative disorders such as Alzheimer's disease (AD). However, the molecular events occurring during brain aging are extremely complex and still largely unknown. For a better understanding of these age-associated modifications, animal models as close as possible to humans are needed. We thus analyzed the transcriptome of the temporal cortex of the primate Microcebus murinus using human oligonucleotide microarrays (Affymetrix). Gene expression profiles were assessed in the temporal cortex of 6 young adults, 10 healthy old animals and 2 old, "AD-like" animals that presented ß-amyloid plaques and cortical atrophy, which are pathognomonic signs of AD in humans. Gene expression data of the 14,911 genes that were detected in at least 3 samples were analyzed. By SAM (significance analysis of microarrays), we identified 47 genes that discriminated young from healthy old and "AD-like" animals. These findings were confirmed by principal component analysis (PCA). ANOVA of the expression data from the three groups identified 695 genes (including the 47 genes previously identified by SAM and PCA) with significant changes of expression in old and "AD-like" in comparison to young animals. About one third of these genes showed similar changes of expression in healthy aging and in "AD-like" animals, whereas more than two thirds showed opposite changes in these two groups in comparison to young animals. Hierarchical clustering analysis of the 695 markers indicated that each group had distinct expression profiles which characterized each group, especially the "AD-like" group. Functional categorization showed that most of the genes that were up-regulated in healthy old animals and down-regulated in "AD-like" animals belonged to metabolic pathways, particularly protein synthesis. These data suggest the existence of compensatory mechanisms during physiological brain aging that disappear in "AD-like" animals. These results open the way to new exploration of physiological and "AD-like" aging in primates.

Background: Hematopoietic cells are endowed with very specific biological functions, including cell motility and immune response. These specific functions are dramatically altered during hematopoietic cell differentiation, whereby undifferentiated hematopoietic stem and progenitor cells (HSPC) residing in bone marrow differentiate into platelets, red blood cells and immune cells that exit into the blood stream and eventually move into lymphoid organs or inflamed tissues. The contribution of alternative splicing (AS) to these functions has long been minimized due to incomplete knowledge on AS events in hematopoietic cells., Principal Findings: Using Human Exon ST 1.0 microarrays, the entire exome expression profile of immature CD34+ HSPC and mature whole blood cells was mapped, compared to a collection of solid tissues and made freely available as an online exome expression atlas (Amazonia Exon! : http://amazonia.transcriptome.eu/exon.php). At a whole transcript level, HSPC strongly expressed EREG and the pluripotency marker DPPA4. Using a differential splicing index scheme (dsi), a list of 849 transcripts differentially expressed between hematopoietic cells and solid tissues was computed, that included NEDD9 and CD74. Some of these genes also underwent alternative splicing events during hematopoietic differentiation, such as INPP4B, PTPLA or COMMD6, with varied contribution of CD3+ T cells, CD19+ B cells, CD14+ or CD15+ myelomonocytic populations. Strikingly, these genes were significantly enriched for genes involved in cell motility, cell adhesion, response to wounding and immune processes., Conclusion: The relevance and the precision provided by this exon expression map highlights the contribution of alternative splicing to key feature of blood cells differentiation and function.

Background: Formation of blood vessels requires the concerted regulation of an unknown number of genes in a spatial-, time- and dosage-dependent manner. Determining genes, which drive vascular maturation is crucial for the identification of new therapeutic targets against pathological angiogenesis., Methodology/principal Findings: [corrected] We accessed global gene regulation throughout maturation of the chick chorio-allantoic membrane (CAM), a highly vascularized tissue, using pan genomic microarrays. Seven percent of analyzed genes showed a significant change in expression (>2-fold, FDR<5%) with a peak occurring from E7 to E10, when key morphogenetic and angiogenic genes such as BMP4, SMO, HOXA3, EPAS1 and FGFR2 were upregulated, reflecting the state of an activated endothelium. At later stages, a general decrease in gene expression occurs, including genes encoding mitotic factors or angiogenic mediators such as CYR61, EPAS1, MDK and MYC. We identified putative human orthologs for 77% of significantly regulated genes and determined endothelial cell enrichment for 20% of the orthologs in silico. Vascular expression of several genes including ENC1, FSTL1, JAM2, LDB2, LIMS1, PARVB, PDE3A, PRCP, PTRF and ST6GAL1 was demonstrated by in situ hybridization. Up to 9% of the CAM genes were also overexpressed in human organs with related functions, such as placenta and lung or the thyroid. 21-66% of CAM genes enriched in endothelial cells were deregulated in several human cancer types (P<.0001). Interfering with PARVB (encoding parvin, beta) function profoundly changed human endothelial cell shape, motility and tubulogenesis, suggesting an important role of this gene in the angiogenic process., Conclusions/significance: Our study underlines the complexity of gene regulation in a highly vascularized organ during development. We identified a restricted number of novel genes enriched in the endothelium of different species and tissues, which may play crucial roles in normal and pathological angiogenesis.

Topoisomerase I (Top1) is a key enzyme in functioning at the interface between DNA replication, transcription and mRNA maturation. Here, we show that Top1 suppresses genomic instability in mammalian cells by preventing a conflict between transcription and DNA replication. Using DNA combing and ChIP (chromatin immunoprecipitation)-on-chip, we found that Top1-deficient cells accumulate stalled replication forks and chromosome breaks in S phase, and that breaks occur preferentially at gene-rich regions of the genome. Notably, these phenotypes were suppressed by preventing the formation of RNA-DNA hybrids (R-loops) during transcription. Moreover, these defects could be mimicked by depletion of the splicing factor ASF/SF2 (alternative splicing factor/splicing factor 2), which interacts functionally with Top1. Taken together, these data indicate that Top1 prevents replication fork collapse by suppressing the formation of R-loops in an ASF/SF2-dependent manner. We propose that interference between replication and transcription represents a major source of spontaneous replication stress, which could drive genomic instability during the early stages of tumorigenesis.

The survival of malignant plasma cells is a key event in disease occurrence, progression and chemoresistance. Using DNA-microarrays, we analysed the expression of genes coding for 58 proteins linked with extrinsic and intrinsic apoptotic pathways, caspases and inhibitor of apoptosis proteins. We considered six memory B cells (MBC), seven plasmablasts (PPC), seven bone marrow plasma cells (BMPC) and purified myeloma cells (MMC) from 92 newly-diagnosed patients. Forty out of the 58 probe sets enabled the separation of MBC, PPC and BMPC in three homogeneous clusters, characterized by an elevated expression of TNFRSF10A, TNFRSF10B, BCL2A1, CASP8, CASP9 and PMAIP1 genes for MBC, of FAS, FADD, AIFM1, BIRC5, CASP CASP2, CASP3 and CASP6 for PPC and of BCL2, MCL1, BID, BIRC3 and XIAP for BMPC. Thus, B cell differentiation was associated with change of expression of pro-apoptotic and anti-apoptotic genes. Regarding MMC, the major finding was TRAIL upregulation that might be counteracted by a high osteoprotegerin production by BM stromal cells and a decreased expression of FAS, APAF1 and BNIP3 compared to normal BMPC. Out of the 40 genes, CASP2 and BIRC5 expression in MMC had adverse prognosis in two independent series of previously-untreated patients.

Background: The first week of human pre-embryo development is characterized by the induction of totipotency and then pluripotency. The understanding of this delicate process will have far reaching implication for in vitro fertilization and regenerative medicine. Human mature MII oocytes and embryonic stem (ES) cells are both able to achieve the feat of cell reprogramming towards pluripotency, either by somatic cell nuclear transfer or by cell fusion, respectively. Comparison of the transcriptome of these two cell types may highlight genes that are involved in pluripotency initiation., Results: Based on a microarray compendium of 205 samples, we compared the gene expression profile of mature MII oocytes and human ES cells (hESC) to that of somatic tissues. We identified a common oocyte/hESC gene expression profile, which included a strong cell cycle signature, genes associated with pluripotency such as LIN28 and TDGF1, a large chromatin remodelling network (TOP2A, DNMT3B, JARID2, SMARCA5, CBX1, CBX5), 18 different zinc finger transcription factors, including ZNF84, and several still poorly annotated genes such as KLHL7, MRS2, or the Selenophosphate synthetase 1 (SEPHS1). Interestingly, a large set of genes was also found to code for proteins involved in the ubiquitination and proteasome pathway. Upon hESC differentiation into embryoid bodies, the transcription of this pathway declined. In vitro, we observed a selective sensitivity of hESC to the inhibition of the activity of the proteasome., Conclusion: These results shed light on the gene networks that are concurrently overexpressed by the two human cell types with somatic cell reprogramming properties.

Identification of new criteria for embryo quality is required to improve the clinical outcome of in vitro fertilization. The aim of this study was to determine the gene expression profile of cumulus cells (CC) surrounding the oocyte as biomarkers for embryo potential and to identify genes to be used as prognostic indicators of successful pregnancy. CC from single oocytes were analysed using DNA microarrays. Gene expression profiles of CC surrounding the oocyte associated with good embryonic quality and pregnancy outcome were computed. We observed that CC issued from oocytes that developed into embryos with a good morphology had differing gene expression profile according to the pregnancy outcome of the embryo. We demonstrated that the expression of BCL2L11, PCK1 and NFIB in CC is significantly correlated with embryo potential and successful pregnancy. These results were confirmed by quantitative RT-PCR. The gene expression profiling of human CC correlates with embryo potential and pregnancy outcome. BCL2L11, PCK1 and NFIB genes are proposed as biomarkers for predicting pregnancy. Our findings suggest a non-invasive approach, offering a new potential strategy for competent embryo selection. This approach should be validated in single-embryo transfer programmes.

Background: The huge amount of data generated by DNA chips is a powerful basis to classify various pathologies. However, constant evolution of microarray technology makes it difficult to mix data from different chip types for class prediction of limited sample populations. Affymetrix(R) technology provides both a quantitative fluorescence signal and a decision (detection call: absent or present) based on signed-rank algorithms applied to several hybridization repeats of each gene, with a per-chip normalization. We developed a new prediction method for class belonging based on the detection call only from recent Affymetrix chip type. Biological data were obtained by hybridization on U133A, U133B and U133Plus 2.0 microarrays of purified normal B cells and cells from three independent groups of multiple myeloma (MM) patients., Results: After a call-based data reduction step to filter out non class-discriminative probe sets, the gene list obtained was reduced to a predictor with correction for multiple testing by iterative deletion of probe sets that sequentially improve inter-class comparisons and their significance. The error rate of the method was determined using leave-one-out and 5-fold cross-validation. It was successfully applied to (i) determine a sex predictor with the normal donor group classifying gender with no error in all patient groups except for male MM samples with a Y chromosome deletion, (ii) predict the immunoglobulin light and heavy chains expressed by the malignant myeloma clones of the validation group and (iii) predict sex, light and heavy chain nature for every new patient. Finally, this method was shown powerful when compared to the popular classification method Prediction Analysis of Microarray (PAM)., Conclusion: This normalization-free method is routinely used for quality control and correction of collection errors in patient reports to clinicians. It can be easily extended to multiple class prediction suitable with clinical groups, and looks particularly promising through international cooperative projects like the "Microarray Quality Control project of US FDA" MAQC as a predictive classifier for diagnostic, prognostic and response to treatment. Finally, it can be used as a powerful tool to mine published data generated on Affymetrix systems and more generally classify samples with binary feature values.

The heparan sulfate (HS) proteoglycan, syndecan-1, plays a major role in multiple myeloma (MM) by concentrating heparin-binding growth factors on the surface of MM cells (MMCs). Using Affymetrix microarrays and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we show that the gene encoding heparanase (HPSE), an enzyme that cleaves HS chains, is expressed by 11 of 19 myeloma cell lines (HMCLs). In HSPE(pos) HMCLs, syndecan-1 gene expression and production of soluble syndecan-1, unlike expression of membrane syndecan-1, were significantly increased. Knockdown of HPSE by siRNA resulted in a decrease of syndecan-1 gene expression and soluble syndecan-1 production without affecting membrane syndecan-1 expression. Thus, HPSE influences expression and shedding of syndecan-1. Contrary to HMCLs, HPSE is expressed in only 4 of 39 primary MMC samples, whereas it is expressed in 36 of 39 bone marrow (BM) microenvironment samples. In the latter, HPSE is expressed at a median level in polymorphonuclear cells and T cells; it is highly expressed in monocytes and osteoclasts. Affymetrix data were validated at the protein level, both on HMCLs and patient samples. We report for the first time that a gene's expression mainly in the BM environment (ie, HSPE) is associated with a shorter event-free survival of patients with newly diagnosed myeloma treated with high-dose chemotherapy and stem cell transplantation. Our study suggests that clinical inhibitors of HPSE could be beneficial for patients with MM.

Microarray technology provides a unique opportunity to examine gene expression patterns in human embryonic stem cells (hESCs). We performed a meta-analysis of 38 original studies reporting on the transcriptome of hESCs. We determined that 1,076 genes were found to be overexpressed in hESCs by at least three studies when compared to differentiated cell types, thus composing a "consensus hESC gene list." Only one gene was reported by all studies: the homeodomain transcription factor POU5F1/OCT3/4. The list comprised other genes critical for pluripotency such as the transcription factors NANOG and SOX2, and the growth factors TDGF1/CRIPTO and Galanin. We show that CD24 and SEMA6A, two cell surface protein-coding genes from the top of the consensus hESC gene list, display a strong and specific membrane protein expression on hESCs. Moreover, CD24 labeling permits the purification by flow cytometry of hESCs cocultured on human fibroblasts. The consensus hESC gene list also included the FZD7 WNT receptor, the G protein-coupled receptor GPR19, and the HELLS helicase, which could play an important role in hESCs biology. Conversely, we identified 783 genes downregulated in hESCs and reported in at least three studies. This "consensus differentiation gene list" included the IL6ST/GP130 LIF receptor. We created an online hESC expression atlas, http://amazonia.montp.inserm.fr, to provide an easy access to this public transcriptome dataset. Expression histograms comparing hESCs to a broad collection of fetal and adult tissues can be retrieved with this web tool for more than 15,000 genes.

Cancer-testis (CT) Ags are expressed in testis and malignant tumors but rarely in nongametogenic tissues. Due to this pattern, they represent attractive targets for cancer vaccination approaches. The aims of the present study are: 1) to assess the expression of CT genes on a pangenomic base in multiple myeloma (MM); 2) to assess the prognosis value of CT gene expression; and 3) to provide selection strategies for CT Ags in clinical vaccination trials. We report the expression pattern of CT genes in purified MM cells (MMC) of 64 patients with newly diagnosed MM and12 patients with monoclonal gammopathy of unknown significance, in normal plasma cell and B cell samples, and in 20 MMC lines. Of the 46 CT genes interrogated by the Affymetrix HG-U133 set arrays, 35 are expressed in the MMC of at least one patient. Of these, 25 are located on chromosome X. The expression of six CT genes is associated with a shorter event-free survival. The MMC of 98% of the patients express at least one CT gene, 86% at least two, and 70% at least three CT genes. By using a set of 10 CT genes including KM-HN-1, MAGE-C1, MAGE-A3/6/12, MAGE-A5, MORC, DDX43, SPACA3, SSX-4, GAGE-1-8, and MAGE-C2, a combination of at least three CT genes-desirable for circumventing tumor escape mechanisms-is obtained in the MMC of 67% of the patients. Provided that the immunogenicity of the products of these 10 CT genes is confirmed, gene expression profiling could be useful in identifying which CT Ags could be used to vaccinate a given patient.

Background: The understanding of the mechanisms regulating human oocyte maturation is still rudimentary. We have identified transcripts differentially expressed between immature and mature oocytes and cumulus cells., Methods: Using oligonucleotide microarrays, genome-wide gene expression was studied in pooled immature and mature oocytes or cumulus cells from patients who underwent IVF., Results: In addition to known genes, such as DAZL, BMP15 or GDF9, oocytes up-regulated 1514 genes. We show that PTTG3 and AURKC are respectively the securin and the Aurora kinase preferentially expressed during oocyte meiosis. Strikingly, oocytes overexpressed previously unreported growth factors such as TNFSF13/APRIL, FGF9, FGF14 and IL4 and transcription factors including OTX2, SOX15 and SOX30. Conversely, cumulus cells, in addition to known genes such as LHCGR or BMPR2, overexpressed cell-to-cell signalling genes including TNFSF11/RANKL, numerous complement components, semaphorins (SEMA3A, SEMA6A and SEMA6D) and CD genes such as CD200. We also identified 52 genes progressively increasing during oocyte maturation, including CDC25A and SOCS7., Conclusion: The identification of genes that were up- and down-regulated during oocyte maturation greatly improves our understanding of oocyte biology and will provide new markers that signal viable and competent oocytes. Furthermore, genes found expressed in cumulus cells are potential markers of granulosa cell tumours.

B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) have been shown to promote multiple myeloma (MM) cell growth. We show that the main site of production for BAFF and APRIL is the bone marrow (BM) environment, and that production is mainly by monocytes and neutrophils. In addition, osteoclasts produce very high levels of APRIL, unlike BM stromal cells. Myeloma cells (MMCs) express TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor), the receptor of BAFF/APRIL, at varying levels. TACI expression is a good indicator of a BAFF-binding receptor. Expression data of purified MMCs from 65 newly diagnosed patients have been generated using Affymetrix microarrays and were analyzed by supervised clustering of groups with higher (TACI(hi)) versus lower (TACI(lo)) TACI expression levels. Patients in the TACI(lo) group had clinical parameters associated with bad prognosis. A set of 659 genes was differentially expressed between TACI(hi) and TACI(lo) MMCs. This set makes it possible to efficiently classify TACI(hi) and TACI(lo) MMCs in an independent cohort of 40 patients. TACI(hi) MMCs displayed a mature plasma cell gene signature, indicating dependence on the BM environment. In contrast, the TACI(lo) group had a gene signature of plasmablasts, suggesting an attenuated dependence on the BM environment. Taken together, our findings suggest using gene expression profiling to identify the group of patients who might benefit most from treatment with BAFF/APRIL inhibitors.

The DNA microarray technology enables the identification of the large number of genes involved in the complex deregulation of cell homeostasis taking place in cancer. Using Affymetrix microarrays, we have compared the gene expression profiles of highly purified malignant plasma cells from nine patients with multiple myeloma (MM) and eight myeloma cell lines to those of highly purified nonmalignant plasma cells (eight samples) obtained by in vitro differentiation of peripheral blood B cells. Two unsupervised clustering algorithms classified these 25 samples into two distinct clusters: a malignant plasma cell cluster and a normal plasma cell cluster. Two hundred and fifty genes were significantly up-regulated and 159 down-regulated in malignant plasma samples compared to normal plasma samples. For some of these genes, an overexpression or downregulation of the encoded protein was confirmed (cyclin D1, c-myc, BMI-1, cystatin c, SPARC, RB). Two genes overexpressed in myeloma cells (ABL and cystathionine beta synthase) code for enzymes that could be a therapeutic target with specific drugs. These data provide a new insight into the understanding of myeloma disease and prefigure that the development of DNA microarray could help to develop an 'à la carte' treatment in cancer disease.

During neurogenesis in Drosophila, groups of equipotential, neurally competent cells choose between epidermal and neural fates. Notch, a phylogenetically conserved transmembrane protein, may act as a receptor in a lateral signalling pathway in which a single neural precursor is chosen from each group and the neural fate of the other cells is inhibited, causing them to differentiate into epidermis. Possible intracellular transduction events mediating signals from Notch are, however, unknown. shaggy is also required for the lateral signal and encodes serine/threonine protein kinases with homology to the glycogen synthase kinase-3 (GSK-3) enzymes that act in signal transduction pathways in vertebrates. We report here that, in transgenic flies, GSK-3 beta can substitute for shaggy, and we also present a study of epistatic relationships between shaggy and gain and loss of function alleles of Notch. The results indicate that shaggy/GSK-3 is part of a signalling pathway downstream of Notch.