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The development of vascular networks in microfluidic chips is crucial for the long-term culture of three-dimensional cell aggregates such as spheroids, organoids, tumoroids, or tissue explants. Despite rapid advancement in microvascular network systems and organoid technologies, vascularizing organoids-on-chips remains a challenge in tissue engineering. Most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical set-ups. Considering these constraints, we develop a platform to establish and monitor the formation of endothelial networks around mesenchymal and pancreatic islet spheroids, as well as blood vessel organoids generated from pluripotent stem cells, cultured for up to 30 days on-chip. We show that these networks establish functional connections with the endothelium-rich spheroids and vascular organoids, as they successfully provide intravascular perfusion to these structures. We find that organoid growth, maturation, and function are enhanced when cultured on-chip using our vascularization method. This microphysiological system represents a viable organ-on-chip model to vascularize diverse biological 3D tissues and sets the stage to establish organoid perfusions using advanced microfluidics., (© 2024. The Author(s).)

Unlike Quality by Testing approach, where products were tested only after drug manufacturing, Quality by Design (QbD) is a proactive control quality paradigm, which handles risks from the early development steps. In QbD, regression models built from experimental data are used to predict a risk mapping called Design Space in which the developers can identify values of critical input factors leading to acceptable probabilities to meet the efficacy and safety specifications for the expected product. These empirical models are often limited to quantitative responses. Moreover, in practice the smallness and incompleteness of datasets degrade the quality of predictions. In this study, a Bayesian approach including variable selection, parameter estimation and model quality assessment is proposed and assessed using a real case study devoted to the development of a Cationic Nano-Lipid Structures for siRNA Transfection. Two original model structures are also included to describe both binary and percentage response variables. The results confirm the practical relevance and applicability of the Bayesian implementation of the QbD analysis.

The majority of gene expression studies focus on the search for genes whose mean expression is different between two or more populations of samples in the so-called "differential expression analysis" approach. However, a difference in variance in gene expression may also be biologically and physiologically relevant. In the classical statistical model used to analyze RNA-sequencing (RNA-seq) data, the dispersion, which defines the variance, is only considered as a parameter to be estimated prior to identifying a difference in mean expression between conditions of interest. Here, we propose to evaluate four recently published methods, which detect differences in both the mean and dispersion in RNA-seq data. We thoroughly investigated the performance of these methods on simulated datasets and characterized parameter settings to reliably detect genes with a differential expression dispersion. We applied these methods to The Cancer Genome Atlas datasets. Interestingly, among the genes with an increased expression dispersion in tumors and without a change in mean expression, we identified some key cellular functions, most of which were related to catabolism and were overrepresented in most of the analyzed cancers. In particular, our results highlight autophagy, whose role in cancerogenesis is context-dependent, illustrating the potential of the differential dispersion approach to gain new insights into biological processes and to discover new biomarkers., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Le Priol et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

This is a spectacular moment for genetics to evolve in genome editing, which encompasses the precise alteration of the cellular DNA sequences within various species. One of the most fascinating genome-editing technologies currently available is Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated protein 9 (CRISPR-Cas9), which have integrated deeply into the research field within a short period due to its effectiveness. It became a standard tool utilized in a broad spectrum of biological and therapeutic applications. Furthermore, reliable disease models are required to improve the quality of healthcare. CRISPR-Cas9 has the potential to diversify our knowledge in genetics by generating cellular models, which can mimic various human diseases to better understand the disease consequences and develop new treatments. Precision in genome editing offered by CRISPR-Cas9 is now paving the way for gene therapy to expand in clinical trials to treat several genetic diseases in a wide range of species. This review article will discuss genome-editing tools: CRISPR-Cas9, Zinc Finger Nucleases (ZFNs), and Transcription Activator-Like Effector Nucleases (TALENs). It will also encompass the importance of CRISPR-Cas9 technology in generating cellular disease models for novel therapeutics, its applications in gene therapy, and challenges with novel strategies to enhance its specificity.

Human induced pluripotent stem cell (hiPSC)-derived hair-bearing skin organoids offer exciting new possibilities for modeling diseases like epidermolysis bullosa (EB). These inherited diseases affect 1 in 30,000 people worldwide and result from perturbed expression and/or structure of components of the epidermal-dermal junction (EDJ). To establish whether hiPSC-derived skin organoids might be able to capture salient features of EB, it is thus important to characterize their EDJ. Here, we report successful generation of hair-bearing skin organoids from two hiPSC lines that exhibited fully stratified interfollicular epidermis. Using immunofluorescence and electron microscopy, we showed that basal keratinocytes in organoids adhere to laminin-332 and type IV collagen-rich basement membrane via type I hemidesmosomes and integrin β1-based adhesion complexes. Importantly, we demonstrated that EDJs in organoids are almost devoid of type VII collagen, a fibril that mediates anchorage of the epidermis to dermis. This should be considered when using skin organoids for EB modeling., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Complex in vitro models of human immune cells and intestinal mucosa may have a translation-assisting role in the assessment of anti-inflammatory compounds. Chronic inflammation of the gastrointestinal tract is a hallmark of inflammatory bowel diseases (IBD). In both IBD entities, Crohn's disease and ulcerative colitis, impaired immune cell activation and dysfunctional epithelial barrier are the common pathophysiology. Current therapeutic approaches are targeting single immune modulator molecules to stop disease progression and reduce adverse effects. Such molecular targets can be difficult to assess in experimental animal models of colitis, due to the disease complexity and species differences. Previously, a co-culture model based on human epithelial cells and monocytes arranged in a physiological microenvironment was used to mimic inflamed mucosa for toxicological and permeability studies. The leaky gut model described here, a co-culture of Caco-2, THP-1 and MUTZ-3 cells, was used to mimic IBD-related pathophysiology and for combined investigations of permeability and target engagement of two Janus kinase (JAK) inhibitors, tofacitinib (TOFA) and a JAK1-targeting siRNA nanomedicine. The co-culture just before reaching confluency of the epithelium was used to mimic the compromised intestinal barrier. Delivery efficacy and target engagement against JAK1 was quantified via downstream analysis of STAT1 protein phosphorylation after IFN-γ stimulation. Compared to a tight barrier, the leaky gut model showed 92 ± 5% confluence, a barrier function below 200 Ω*cm 2 , and enhanced immune response to bacteria-derived lipopolysaccharides. By confocal microscopy we observed an increased accumulation of siJAK1-nanoparticles within the sub-confluent regions leading to uptake into immune cells near the epithelium. A concentration-dependent downregulation of JAK/STAT pathway was observed for siJAK1-nanoparticles (10 ± 12% to 16 ± 12%), whereas TOFA inhibition was 86 ± 2%, compared to untreated cells. By mimicking the status of severely damaged epithelium, like in IBD, the leaky gut model holds promise as a human in vitro system to evaluate the efficacy of anti-inflammatory drugs and nanomedicines., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The inhibition of neddylation by the preclinical drug MLN4924 represents a new strategy to combat cancer. However, despite being effective against hematologic malignancies, its success in solid tumors, where cell-cell and cell-ECM interactions play essential roles, remains elusive., Methods: Here, we studied the effects of MLN4924 on cell growth, migration and invasion in cultured prostate cancer cells and in disease-relevant prostate tumoroids. Using focused protein profiling, drug and RNAi screening, we analyzed cellular pathways activated by neddylation inhibition., Results: We show that mechanical stress induced by MLN4924 in prostate cancer cells significantly affects the therapeutic outcome. The latter depends on the cell type and involves distinct Rho isoforms. In LNCaP and VCaP cells, the stimulation of RhoA and RhoB by MLN4924 markedly upregulates the level of tight junction proteins at cell-cell contacts, which augments the mechanical strain induced by Rho signaling. This "tight junction stress response" (TJSR) causes the collapse of cell monolayers and a characteristic rupture of cancer spheroids. Notably, TJSR is a major cause of drug-induced apoptosis in these cells. On the other hand, in PC3 cells that underwent partial epithelial-to-mesenchymal transition (EMT), the stimulation of RhoC induces an adverse effect by promoting amoeboid cell scattering and invasion. We identified complementary targets and drugs that allow for the induction of TJSR without stimulating RhoC., Conclusions: Our finding that MLN4924 acts as a mechanotherapeutic opens new ways to improve the efficacy of neddylation inhibition as an anticancer approach., (© 2022. The Author(s).)

Background and Aims: Inflammatory bowel diseases are highly debilitating conditions that require constant monitoring and life-long medication. Current treatments are focused on systemic administration of immunomodulatory drugs, but they have a broad range of undesirable side-effects. RNA interference is a highly specific endogenous mechanism that regulates the expression of the gene at the transcript level, which can be repurposed using exogenous short interfering RNA [siRNA] to repress expression of the target gene. While siRNA therapeutics can offer an alternative to existing therapies, with a high specificity critical for chronically administrated drugs, evidence of their potency compared to chemical kinase inhibitors used in clinics is still lacking in alleviating an adverse inflammatory response., Methods: We provide a framework to select highly specific siRNA, with a focus on two kinases strongly involved in pro-inflammatory diseases, namely JAK1 and JAK3. Using western-blot, real-time quantitative PCR and large-scale analysis, we assessed the specificity profile of these siRNA drugs and compared their efficacy to the most recent and promising kinase inhibitors for Janus kinases [Jakinibs], tofacitinib and filgotinib., Results: siRNA drugs can reach higher efficiency and selectivity at lower doses [5 pM vs 1 µM] than Jakinibs. Moreover, JAK silencing lasted up to 11 days, even with 6 h pulse transfection., Conclusions: The siRNA-based drugs developed hold the potential to develop more potent therapeutics for chronic inflammatory diseases., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

The extension of islet transplantation to a wider number of Type 1 diabetic patients is compromised by the scarcity of donors, the reduced ex vivo survival of pancreatic islets and the use of immunosuppressive treatments. Islets of Langerhans isolated from brain-dead donors are currently the only cell source for transplantation. Thus, it is crucial to find an alternative and an abundant source of functional insulin secreting cells not only for clinical use but also for the development of research dedicated to the screening of drugs and to the development of new therapeutic targets. Several groups around the world, including ours, develop 3D culture models as Langerhanoids that closely mimick human pancreatic islets physiology. In this review, we describe recent advances to mimic the pancreatic niche (extracellular matrix, vascularization, microfluidics) allowing better functionality of Langerhanoids., (© 2022 médecine/sciences – Inserm.)

Androgen receptor (AR) signalling is a key prostate cancer (PC) driver, even in advanced 'castrate-resistant' disease (CRPC). To systematically identify microRNAs (miRs) modulating AR activity in lethal disease, hormone-responsive and -resistant PC cells expressing a luciferase-based AR reporter were transfected with a miR inhibitor library; 78 inhibitors significantly altered AR activity. Upon validation, miR-346, miR-361-3p and miR-197 inhibitors markedly reduced AR transcriptional activity, mRNA and protein levels, increased apoptosis, reduced proliferation, repressed EMT, and inhibited PC migration and invasion, demonstrating additive effects with AR inhibition. Corresponding miRs increased AR activity through a novel and anti-dogmatic mechanism of direct association with AR 6.9 kb 3'UTR and transcript stabilisation. In addition, miR-346 and miR-361-3p modulation altered levels of constitutively active AR variants, and inhibited variant-driven PC cell proliferation, so may contribute to persistent AR signalling in CRPC in the absence of circulating androgens. Pathway analysis of AGO-PAR-CLIP-identified miR targets revealed roles in DNA replication and repair, cell cycle, signal transduction and immune function. Silencing these targets, including tumour suppressors ARHGDIA and TAGLN2, phenocopied miR effects, demonstrating physiological relevance. MiR-346 additionally upregulated the oncogene, YWHAZ, which correlated with grade, biochemical relapse and metastasis in patients. These AR-modulatory miRs and targets correlated with AR activity in patient biopsies, and were elevated in response to long-term enzalutamide treatment of patient-derived CRPC xenografts. In summary, we identified miRs that modulate AR activity in PC and CRPC, via novel mechanisms, and may represent novel PC therapeutic targets.

Inhibition of protein degradation by blocking Cullin-RING E3 ligases (CRLs) is a new approach in cancer therapy though of unknown risk because CRL inhibition may stabilize both oncoproteins and tumor suppressors. Probing CRLs in prostate cancer cells revealed a remarkable plasticity of cells with TMPRSS2-ERG translocation. CRL suppression by chemical inhibition or knockdown of RING component RBX1 led to reversible G0/G1 cell cycle arrest that prevented cell apoptosis. Conversely, complete blocking of CRLs at a higher inhibitor dose-induced cytotoxicity that was amplified by knockdown of CRL regulator Cand1. We analyzed cell signaling to understand how varying degrees of CRL inhibition translated to distinct cell fates. Both tumor suppressor and oncogenic cell signaling pathways and transcriptional activities were affected, with pro-metastatic Wnt/β-catenin as the most upregulated. Suppression of the NF-κB pathway contributed to anti-apoptotic effect, and androgen receptor (AR) and ERG played decisive, though opposite, roles: AR was involved in protective quiescence, whereas ERG promoted apoptosis. These data define AR-ERG interaction as a key plasticity and survival determinant in prostate cancer and suggest supplementary treatments that may overcome drug resistance mechanisms regulated by AR-ERG interaction.

Modularity, feedback control, functional redundancy and bowtie architecture have been proposed as key factors that confer robustness to complex biological systems. MicroRNAs (miRNAs) are highly conserved but functionally dispensable. These antinomic properties suggest that miRNAs fine-tune gene expression rather than act as genetic switches. We synthesize published and unpublished data and hypothesize that miRNA pluripotentiality acts to buffer gene expression, while miRNA degeneracy tunes the expression of targets, thus providing robustness to gene expression networks. Furthermore, we propose a Lavallière-tie architecture by integrating signal transduction, miRNAs and protein expression data to model complex gene expression networks.

Extensive efforts have been made to understand the molecular actors that control epithelial cell fate. Although pieces of information have been obtained from single-gene function investigations, the entire picture of the molecular mechanisms involved in the regulation of epithelial homeostasis is still mysterious. Growing data indicate that gene networks rather than single "master" genes dictate cell fate. In an attempt to characterize such gene networks, we have been investigating the human keratinocyte proliferation and differentiation genes that act downstream of the transcription factor p63, a major regulator of epidermal homeostasis. We identified two networks: the cell cycle network that controls cell proliferation and the keratinocyte cell fate network. Through further analysis of the existing data on epithelial tumorigenesis and induced pluripotent stem cells, we propose a wind rose model of cell fate that is based on a balance between these two different networks that ultimately control human keratinocyte fate and epidermal homeostasis.

Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design.

To analyze the phenotypic consequences of perturbing mammalian cells with drugs, there is an increasing need for systematic cell-based assays in an HTS format. Cell microarrays provide an attractive solution as they offer more than a simple miniaturization and mechanization of conventional microtiter plates. While standard monolayer two-dimensional culture conditions are poor mimics of the cellular environment in situ, microfabricated systems enable three-dimensional organotypic cell cultures and have the potential to provide biological insight not achievable before. This article compares different cell microarray formats and evaluates their potential use in the drug discovery process.

B cell receptor (BCR) signaling contributes to the pathogenesis of B cell malignancies, and most B cell lymphomas depend on BCR signals for survival. Identification of genes that restrain BCR-mediated proliferation is therefore an important goal toward improving the therapy of B cell lymphoma. Here, we identify Ptger4 as a negative feedback regulator of proliferation in response to BCR signals and show that its encoded EP4 receptor is a principal molecule conveying the growth-suppressive effect of prostaglandin E2 (PGE2). Stable knockdown of Ptger4 in B cell lymphoma markedly accelerated tumor spread in mice, whereas Ptger4 overexpression yielded significant protection. Mechanistically, we show that the intrinsic activity of Ptger4 and PGE2-EP4 signaling target a similar set of activating genes, and find Ptger4 to be significantly down-regulated in human B cell lymphoma. We postulate that Ptger4 functions in B cells as a candidate tumor suppressor whose activity is regulated by PGE2 in the microenvironment. These findings suggest that targeting EP4 receptor for prostaglandin may present a novel strategy for treatment of B cell malignancies.

To compare the responses of the different lymphocyte subtypes after an exposure of whole blood to low doses of ionizing radiation, we examined variations in gene expression in different lymphocyte subpopulations using microarray technology. Blood samples from five healthy donors were independently exposed to 0 (sham irradiation), 0.05 and 0.5 Gy of ionizing radiation. Three and 24 h after exposure, CD56+, CD4+ and CD8+ cells were negatively isolated. RNA from each set of experimental conditions was competitively hybridized on 25k oligonucleotide microarrays. Modifications of gene expression were measured after both intervals and in all cell types. Twenty-four hours after exposure to 0.5 Gy, we observed an induction of the expression of BAX, PCNA, GADD45, DDB2 and CDKN1A. However, the numbers of modulated genes greatly differed between cell types. In particular, 3 h after exposure to doses as low as 0.05 Gy, the number of down-modulated genes was 10 times greater for CD4+ cells than for all other cell types. Moreover, most of these repressed genes were taking part in the cell processes of protein biosynthesis and oxidative phosphorylation. The results suggest that several biological pathways in CD4+ cells could be sensitive to low doses of radiation. Therefore, specifically studying CD4+ cells could help to understand the mechanisms involved in low-dose response and allow their detection.

The cell is a very complex system and although molecular biology allowed spectacular progresses, biologists are currently trying to tackle complexity at the system level, to unravel cell mysteries. The inference of transcriptional regulatory networks, the characterization of their topology, their dynamic, their role in major cell functions is at the heart of this strategy. We are proposing a systemic approach, based on integration of large scale data such as expression profiling, ChIP on chip analysis and high throughput RNA interference using siRNA microarrays to infer these networks and characterize associated phenotypes. Ultimately, the characterization of the properties of these networks should impact our understanding of biology and offer potential applications in medicine and pharmacology.

As more genomes are sequenced, challenge of rapidly unraveling the functions of genes was faced. To that end, cell microarrays have been recently described that permit transfection of thousands of nucleic acids in parallel and enable the analysis of phenotypic consequences of such perturbations. As many parameters can influence the efficiency of transfection and consequently protein expression or extinction, some important features in manufacturing cell microarrays for functional exploration of genomes were described.

Two collections of oligonucleotides have been designed for preparing pangenomic human and mouse microarrays. A total of 148,993 and 121,703 oligonucleotides were designed against human and mouse transcripts. Quality scores were created in order to select 25,342 human and 24,109 mouse oligonucleotides. They correspond to: (i) a BLAST-specificity score; (ii) the number of expressed sequence tags matching each probe; (iii) the distance to the 3' end of the target mRNA. Scores were also used to compare in silico the two microarrays with commercial microarrays. The sets described here, called RNG/MRC collections, appear at least as specific and sensitive as those from the commercial platforms. The RNG/MRC collections have now been used by an Anglo-French consortium to distribute more than 3500 microarrays to the academic community. Ad hoc identification of tissue-specific transcripts and a approximately 80% correlation with hybridizations performed on Affymetrix GeneChiptrade mark suggest that the RNG/MRC microarrays perform well. This work provides a comprehensive open resource for investigators working on human and mouse transcriptomes, as well as a generic method to generate new microarray collections in other organisms. All information related to these probes, as well as additional information about commercial microarrays have been stored in a freely-accessible database called MEDIANTE.

Hydroxyurea (HU) is a specific inhibitor of ribonucleotide reductase and thus impairs dNTP synthesis and DNA replication. The long-term transcriptional response of yeast cells to hydroxyurea was investigated using DNA microarrays containing all yeast coding sequences. We show that the redox-responsive Yap regulon and the iron-mobilization Aft regulon are activated in yeast cells treated with HU. Yap1 accumulates in the nucleus in response to HU, but HU activation of the Yap regulon was only partially dependent on Yap1 and yap1Delta mutants were not hypersensitive to HU. In contrast, deletion of the AFT1 and AFT2 transcription factor genes blocked the HU activation of a subset of the Aft regulon and the aft1Delta aft2Delta double mutant was hypersensitive to HU in an iron-suppressible manner. These results highlight the importance of the redox and iron mobilization regulons in the cellular response to HU.

Our knowledge of the molecular mechanisms that regulate hematopoiesis in physiologic and pathologic conditions is limited. Using a molecular approach based on cDNA microarrays, we demonstrated the emergence of an alternative pathway for mature bone marrow cell recovery after the programmed and reversible eradication of CD41+ cells in transgenic mice expressing a conditional toxigene targeted by the platelet alphaIIb promoter. The expression profile of the newly produced CD41+ cells showed high levels of transcripts encoding Ezh2, TdT, Rag2, and various immunoglobulin (Ig) heavy chains. In this context, we identified and characterized a novel population of Lin-Sca-1hi c-Kit- cells, with a lymphoid-like expression pattern, potentially involved in the reconstitution process. Our study revealed novel transcriptional cross talk between myeloid and lymphoid lineages and identified gene expression modifications that occur in vivo under these particular stress conditions, opening important prospects for therapeutic applications.

We performed a microarray study on human differentiated HaCaT keratinocytes exposed to ionizing radiation (2 or 10 Gy). At 3 h after exposure, more than 150 known and unknown genes were found regulated in irradiated HaCaT keratinocytes. Among the genes regulated at 3 h, those involved in cell energy metabolism appeared to be the most abundant and the most responsive. Two mitochondrial ATP-synthases and several other genes involved in energy producing pathways, such as glucose metabolism, were induced, whereas many genes from energy requiring pathways were shut down. These changes in energy metabolism were confirmed both in normal primary keratinocytes and in HaCaT keratinocytes by RT-PCR and proteins studies. Moreover, measures of intracellular ATP revealed a 50% increase in keratinocytes immediately after irradiation, supporting an energy procurement response. The overall results indicate that irradiation induces an immediate burst of ATP that seems to be a general response of human differentiated keratinocytes to the radiation stress. This article contains Supplementary Material available at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/v95.html, ((c) 2005 Wiley-Liss, Inc.)

Skin is the organ most exposed to various environmental aggressors, including ionizing radiation. Low-dose and low-dose-rate exposures to gamma rays account for most occupational, medical or environmental irradiations. To examine whether this type of exposure triggers specific molecular responses, cultured primary keratinocytes isolated from adult normal skin were irradiated with single acute doses of 1 cGy or 2 Gy. DNA microarrays containing 10,500 probes were used to assess transcriptional changes over a time course between 3 and 72 h postirradiation. Keratinocytes were studied at a differentiated stage to mimic the response of cells from the suprabasal layers of the epidermis. A major finding of this study was the identification of an important number of low-dose-specific genes (140), most of which were modulated at 48 h. Clustering analysis also revealed low-dose-specific profiles. One of these clusters (17 known genes) was further analyzed using Gibbs sampling algorithm, which led to the identification of 7 putative promoter sequences. These results show for the first time that low-dose ionizing radiation is able to induce specific transcriptional responses in human keratinocytes. Our findings support the potential usefulness of microarrays in biological dosimetry studies after low-dose exposures.

With the complete sequencing of the human genome, research priorities have shifted from the identification of genes to the elucidation of their function. Methods currently used by scientists to characterize gene function, such as knock-out mice, are based upon loss of protein function and analysis of the resulting phenotypes to infer a potential role for the protein under scrutiny. Until now, these methods have been successful but time consuming and only a few genes at a time could be analyzed. Cell microarrays allow to simultaneously transfect thousands of different nucleic acid molecules, RNA or DNA, into adherent cells. It is then possible to analyze a large pallet of resulting phenotypes in clusters of transfected cells. We are currently manufacturing cell microarrays with collections of full-length cDNA cloned in expression vectors (gain of function analyses) or siRNA (loss of function studies) to unravel function of genes involved in differentiation and proliferation of human cells. Although there are still some technological difficulties to overcome, the potential for cell microarrays to speed up functional exploration of genomes is very promising.

The knowledge of the mechanism of keratinocyte differentiation in culture is still uncompleted. The emergence of new technologies, such as cDNA microarrays or 2D electrophoresis followed by mass spectrometry analysis, has allowed the identification of genes and proteins expressed in biological processes in keratinocytes. Here, we report a genome wide analysis of proliferating versus differentiated human HaCaT keratinocytes. We found that genes and proteins which take part in the cell cycle control, carbohydrate metabolism, cell auto-immunity, adhesion and cytokine signal transduction pathways were regulated in differentiated HaCaT keratinocytes. In addition, we identified seven proteins and 33 transcripts that had not been previously described as differentially expressed in proliferating versus differentiated HaCaT cells. Furthermore, some of these transcripts or proteins were similarly regulated in human primary keratinocytes and in human epidermis. The present study opens new areas of investigation in the comprehension of keratinocyte differentiation., ((c) 2004 Wiley-Liss, Inc.)

Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 microg usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes.

As more genomes are sequenced, we are facing the challenge of rapidly unraveling the functions of genes. To that end, cell microarrays have recently been described that transfect thousands of nucleic acids in parallel and can be used to analyze the phenotypic consequences of such perturbations. As many parameters can influence the efficacy of transfection in such a format, we describe some important features in manufacturing cell microarrays that may improve reliability and efficiency of both plasmid DNA and siRNA transfection. We have also developed image analysis software that allows automatic detection of cell clusters, quantification of transfection efficiency and levels of expression/extinction of genes. Along with cell microarrays, this bioinformatic tool should expedite functional exploration of the human genome.

RNA polymerase III (Pol III) transcribes a large set of genes encoding small untranslated RNAs like tRNAs, 5S rRNA, U6 snRNA or RPR1 RNA. To get a global view of class III (Pol III-transcribed) genes, the distribution of essential components of Pol III, TFIIIC and TFIIIB was mapped across the yeast genome. During active growth, most class III genes and few additional loci were targeted by TFIIIC, TFIIIB and Pol III, indicating that they were transcriptionally active. SNR52, which encodes a snoRNA, was identified as a new class III gene. During the late growth phase, TFIIIC remained bound to most class III genes while the recruitment of Pol III and, to a lesser extent, of TFIIIB was down regulated. This study fixes a reasonable upper bound to the number of class III genes in yeast and points to a global regulation at the level of Pol III and TFIIIB recruitment.