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Abstract Background Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by lignocellulose-degrading fungi including Pycnoporus cinnabarinus. We investigated the cellulolytic system of P. cinnabarinus, focusing on the involvement of CDH in the deconstruction of lignocellulosic biomass. Results First, P. cinnabarinus growth conditions were optimized for CDH production. Following growth under cellulolytic conditions, the main components secreted were cellulases, xylanases and CDH. To investigate the contribution of P. cinnabarinus secretome in saccharification processes, the Trichoderma reesei enzymatic cocktail was supplemented with the P. cinnabarinus secretome. A significant enhancement of the degradation of wheat straw was observed with (i) the production of a large amount of gluconic acid, (ii) increased hemicellulose degradation, and (iii) increased overall degradation of the lignocellulosic material. P. cinnabarinus CDH was heterologously expressed in Pichia pastoris to obtain large amounts of pure enzyme. In a bioreactor, the recombinant CDH (rCDH) expression level reached 7800 U/L. rCDH exhibited values of biochemical parameters similar to those of the natural enzyme, and was able to bind cellulose despite the absence of a carbohydrate-binding module (CBM). Following supplementation of purified rCDH to T. reesei enzymatic cocktail, formation of gluconic acid and increased hemicellulose degradation were observed, thus confirming the previous results observed with P. cinnabarinus secretome. Conclusions We demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material.

Abstract Background The retina is a multi-layered sensory tissue that lines the back of the eye and acts at the interface of input light and visual perception. Its main function is to capture photons and convert them into electrical impulses that travel along the optic nerve to the brain where they are turned into images. It consists of neurons, nourishing blood vessels and different cell types, of which neural cells predominate. Defects in any of these cells can lead to a variety of retinal diseases, including age-related macular degeneration, retinitis pigmentosa, Leber congenital amaurosis and glaucoma. Recent progress in genomics and microarray technology provides extensive opportunities to examine alterations in retinal gene expression profiles during development and diseases. However, there is no specific database that deals with retinal gene expression profiling. In this context we have built RETINOBASE, a dedicated microarray database for retina. Description RETINOBASE is a microarray relational database, analysis and visualization system that allows simple yet powerful queries to retrieve information about gene expression in retina. It provides access to gene expression meta-data and offers significant insights into gene networks in retina, resulting in better hypothesis framing for biological problems that can subsequently be tested in the laboratory. Public and proprietary data are automatically analyzed with 3 distinct methods, RMA, dChip and MAS5, then clustered using 2 different K-means and 1 mixture models method. Thus, RETINOBASE provides a framework to compare these methods and to optimize the retinal data analysis. RETINOBASE has three different modules, "Gene Information", "Raw Data System Analysis" and "Fold change system Analysis" that are interconnected in a relational schema, allowing efficient retrieval and cross comparison of data. Currently, RETINOBASE contains datasets from 28 different microarray experiments performed in 5 different model systems: drosophila, zebrafish, rat, mouse and human. The database is supported by a platform that is designed to easily integrate new functionalities and is also frequently updated. Conclusion The results obtained from various biological scenarios can be visualized, compared and downloaded. The results of a case study are presented that highlight the utility of RETINOBASE. Overall, RETINOBASE provides efficient access to the global expression profiling of retinal genes from different organisms under various conditions.

Background: For over a century, scientists have studied host-pathogen interactions between the crayfish plague disease agent Aphanomyces astaci and freshwater crayfish. It has been hypothesised that North American crayfish hosts are disease-resistant due to the long-lasting coevolution with the pathogen. Similarly, the increasing number of latent infections reported in the historically sensitive European crayfish hosts seems to indicate that similar coevolutionary processes are occurring between European crayfish and A. astaci. Our current understanding of these host-pathogen interactions is largely focused on the innate immunity processes in the crayfish haemolymph and cuticle, but the molecular basis of the observed disease-resistance and susceptibility remain unclear. To understand how coevolution is shaping the host's molecular response to the pathogen, susceptible native European noble crayfish and invasive disease-resistant marbled crayfish were challenged with two A. astaci strains of different origin: a haplogroup A strain (introduced to Europe at least 50 years ago, low virulence) and a haplogroup B strain (signal crayfish in lake Tahoe, USA, high virulence). Here, we compare the gene expression profiles of the hepatopancreas, an integrated organ of crayfish immunity and metabolism., Results: We characterised several novel innate immune-related gene groups in both crayfish species. Across all challenge groups, we detected 412 differentially expressed genes (DEGs) in the noble crayfish, and 257 DEGs in the marbled crayfish. In the noble crayfish, a clear immune response was detected to the haplogroup B strain, but not to the haplogroup A strain. In contrast, in the marbled crayfish we detected an immune response to the haplogroup A strain, but not to the haplogroup B strain., Conclusions: We highlight the hepatopancreas as an important hub for the synthesis of immune molecules in the response to A. astaci. A clear distinction between the innate immune response in the marbled crayfish and the noble crayfish is the capability of the marbled crayfish to mobilise a higher variety of innate immune response effectors. With this study we outline that the type and strength of the host immune response to the pathogen is strongly influenced by the coevolutionary history of the crayfish with specific A. astaci strains., (© 2022. The Author(s).)

Objectives: Crayfish plague disease, caused by the oomycete pathogen Aphanomyces astaci represents one of the greatest risks for the biodiversity of the freshwater crayfish. This data article covers the de novo transcriptome assembly and annotation data of the noble crayfish and the marbled crayfish challenged with Ap. astaci. Following the controlled infection experiment (Francesconi et al. in Front Ecol Evol, 2021, https://doi.org/10.3389/fevo.2021.647037 ), we conducted a differential gene expression analysis described in (Boštjančić et al. in BMC Genom, 2022, https://doi.org/10.1186/s12864-022-08571-z ) DATA DESCRIPTION: In total, 25 noble crayfish and 30 marbled crayfish were selected. Hepatopancreas tissue was isolated, followed by RNA sequencing using the Illumina NovaSeq 6000 platform. Raw data was checked for quality with FastQC, adapter and quality trimming were conducted using Trimmomatic followed by de novo assembly with Trinity. Assembly quality was assessed with BUSCO, at 93.30% and 93.98% completeness for the noble crayfish and the marbled crayfish, respectively. Transcripts were annotated using the Dammit! pipeline and assigned to KEGG pathways. Respective transcriptome and raw datasets may be reused as the reference transcriptome assemblies for future expression studies., (© 2022. The Author(s).)

The Orthology Benchmark Service (https://orthology.benchmarkservice.org) is the gold standard for orthology inference evaluation, supported and maintained by the Quest for Orthologs consortium. It is an essential resource to compare existing and new methods of orthology inference (the bedrock for many comparative genomics and phylogenetic analysis) over a standard dataset and through common procedures. The Quest for Orthologs Consortium is dedicated to maintaining the resource up to date, through regular updates of the Reference Proteomes and increasingly accessible data through the OpenEBench platform. For this update, we have added a new benchmark based on curated orthology assertion from the Vertebrate Gene Nomenclature Committee, and provided an example meta-analysis of the public predictions present on the platform., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Multiciliogenesis is a complex process that allows the generation of hundreds of motile cilia on the surface of specialized cells, to create fluid flow across epithelial surfaces. Dysfunction of human multiciliated cells is associated with diseases of the brain, airway and reproductive tracts. Despite recent efforts to characterize the transcriptional events responsible for the differentiation of multiciliated cells, a lot of actors remain to be identified. In this work, we capitalize on the ever-growing quantity of high-throughput data to search for new candidate genes involved in multiciliation. After performing a large-scale screening using 10 transcriptomics datasets dedicated to multiciliation, we established a specific evolutionary signature involving Otomorpha fish to use as a criterion to select the most likely targets. Combining both approaches highlighted a list of 114 potential multiciliated candidates. We characterized these genes first by generating protein interaction networks, which showed various clusters of ciliated and multiciliated genes, and then by computing phylogenetic profiles. In the end, we selected 11 poorly characterized genes that seem like particularly promising multiciliated candidates. By combining functional and comparative genomics methods, we developed a novel type of approach to study biological processes and identify new promising candidates linked to that process.

Plant reproduction is one key biological process that is very sensitive to heat stress and, as a result, enhanced global warming becomes a serious threat to agriculture. In this work, we have studied the effects of heat on germinated pollen of Arabidopsis thaliana both at the transcriptional and translational level. We have used a high-resolution ribosome profiling technology to provide a comprehensive study of the transcriptome and the translatome of germinated pollen at permissive and restrictive temperatures. We have found significant down-regulation of key membrane transporters required for pollen tube growth by heat, thus uncovering heat-sensitive targets. A subset of the heat-repressed transporters showed coordinated up-regulation with canonical heat-shock genes at permissive conditions. We also found specific regulations at the translational level and we have uncovered the presence of ribosomes on sequences annotated as non-coding. Our results demonstrate that heat impacts mostly on membrane transporters thus explaining the deleterious effects of heat stress on pollen growth. The specific regulations at the translational level and the presence of ribosomes on non-coding RNAs highlights novel regulatory aspects on plant fertilization., (© 2020 John Wiley & Sons Ltd.)

Background: Pediatric high-grade gliomas (pHGGs) are the leading cause of mortality in pediatric neuro-oncology, displaying frequent resistance to standard therapies. Profiling DNA repair and cell cycle gene expression has recently been proposed as a strategy to classify adult glioblastomas. To improve our understanding of the DNA damage response pathways that operate in pHGGs and the vulnerabilities that these pathways might expose, we sought to identify and characterize a specific DNA repair and cell-cycle gene expression signature of pHGGs., Methods: Transcriptomic analyses were performed to identify a DNA repair and cell-cycle gene expression signature able to discriminate pHGGs (n = 6) from low-grade gliomas (n = 10). This signature was compared to related signatures already established. We used the pHGG signature to explore already transcriptomic datasets of DIPGs and sus-tentorial pHGGs. Finally, we examined the expression of key proteins of the pHGG signature in 21 pHGG diagnostic samples and nine paired relapses. Functional inhibition of one DNA repair factor was carried out in four patients who derived H3.3 K27M mutant cell lines., Results: We identified a 28-gene expression signature of DNA repair and cell cycle that clustered pHGGs cohorts, in particular sus-tentorial locations, in two groups. Differential protein expression levels of PARP1 and XRCC1 were associated to TP53 mutations and TOP2A amplification and linked significantly to the more radioresistant pHGGs displaying the worst outcome. Using patient-derived cell lines, we showed that the PARP-1/XRCC1 expression balance might be correlated with resistance to PARP1 inhibition., Conclusion: We provide evidence that PARP1 overexpression, associated to XRCC1 expression, TP53 mutations, and TOP2A amplification, is a new theranostic and potential therapeutic target.

Key Message: Arabidopsis pollen transcriptome analysis revealed new intergenic transcripts of unknown function, many of which are long non-coding RNAs, that may function in pollen-specific processes, including the heat stress response. The male gametophyte is the most heat sensitive of all plant tissues. In recent years, long noncoding RNAs (lncRNAs) have emerged as important components of cellular regulatory networks involved in most biological processes, including response to stress. While examining RNAseq datasets of developing and germinating Arabidopsis thaliana pollen exposed to heat stress (HS), we identified 66 novel and 246 recently annotated intergenic expressed loci (XLOCs) of unknown function, with the majority encoding lncRNAs. Comparison with HS in cauline leaves and other RNAseq experiments indicated that 74% of the 312 XLOCs are pollen-specific, and at least 42% are HS-responsive. Phylogenetic analysis revealed that 96% of the genes evolved recently in Brassicaceae. We found that 50 genes are putative targets of microRNAs and that 30% of the XLOCs contain small open reading frames (ORFs) with homology to protein sequences. Finally, RNAseq of ribosome-protected RNA fragments together with predictions of periodic footprint of the ribosome P-sites indicated that 23 of these ORFs are likely to be translated. Our findings indicate that many of the 312 unknown genes might be functional and play a significant role in pollen biology, including the HS response.

Plant polyamines (PAs) have been assigned a large number of physiological functions with unknown molecular mechanisms in many cases. Among the most abundant and studied polyamines, two of them, namely spermidine (Spd) and thermospermine (Tspm), share some molecular functions related to quality control pathways for tightly regulated mRNAs at the level of translation. In this review, we focus on the roles of Tspm and Spd to facilitate the translation of mRNAs containing upstream ORFs (uORFs), premature stop codons, and ribosome stalling sequences that may block translation, thus preventing their degradation by quality control mechanisms such as the nonsense-mediated decay pathway and possible interactions with other mRNA quality surveillance pathways.

Cilia (flagella) are important eukaryotic organelles, present in the Last Eukaryotic Common Ancestor, and are involved in cell motility and integration of extracellular signals. Ciliary dysfunction causes a class of genetic diseases, known as ciliopathies, however current knowledge of the underlying mechanisms is still limited and a better characterization of genes is needed. As cilia have been lost independently several times during evolution and they are subject to important functional variation between species, ciliary genes can be investigated through comparative genomics. We performed phylogenetic profiling by predicting orthologs of human protein-coding genes in 100 eukaryotic species. The analysis integrated three independent methods to predict a consensus set of 274 ciliary genes, including 87 new promising candidates. A fine-grained analysis of the phylogenetic profiles allowed a partitioning of ciliary genes into modules with distinct evolutionary histories and ciliary functions (assembly, movement, centriole, etc.) and thus propagation of potential annotations to previously undocumented genes. The cilia/basal body localization was experimentally confirmed for five of these previously unannotated proteins (LRRC23, LRRC34, TEX9, WDR27, and BIVM), validating the relevance of our approach. Furthermore, our multi-level analysis sheds light on the core gene sets retained in gamete-only flagellates or Ecdysozoa for instance. By combining gene-centric and species-oriented analyses, this work reveals new ciliary and ciliopathy gene candidates and provides clues about the evolution of ciliary processes in the eukaryotic domain. Additionally, the positive and negative reference gene sets and the phylogenetic profile of human genes constructed during this study can be exploited in future work., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: The constant and massive increase of biological data offers unprecedented opportunities to decipher the function and evolution of genes and their roles in human diseases. However, the multiplicity of sources and flow of data mean that efficient access to useful information and knowledge production has become a major challenge. This challenge can be addressed by taking inspiration from Web 2.0 and particularly social networks, which are at the forefront of big data exploration and human-data interaction., Objective: MyGeneFriends is a Web platform inspired by social networks, devoted to genetic disease analysis, and organized around three types of proactive agents: genes, humans, and genetic diseases. The aim of this study was to improve exploration and exploitation of biological, postgenomic era big data., Methods: MyGeneFriends leverages conventions popularized by top social networks (Facebook, LinkedIn, etc), such as networks of friends, profile pages, friendship recommendations, affinity scores, news feeds, content recommendation, and data visualization., Results: MyGeneFriends provides simple and intuitive interactions with data through evaluation and visualization of connections (friendships) between genes, humans, and diseases. The platform suggests new friends and publications and allows agents to follow the activity of their friends. It dynamically personalizes information depending on the user's specific interests and provides an efficient way to share information with collaborators. Furthermore, the user's behavior itself generates new information that constitutes an added value integrated in the network, which can be used to discover new connections between biological agents., Conclusions: We have developed MyGeneFriends, a Web platform leveraging conventions from popular social networks to redefine the relationship between humans and biological big data and improve human processing of biomedical data. MyGeneFriends is available at lbgi.fr/mygenefriends., (©Alexis Allot, Kirsley Chennen, Yannis Nevers, Laetitia Poidevin, Arnaud Kress, Raymond Ripp, Julie Dawn Thompson, Olivier Poch, Odile Lecompte. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 16.06.2017.)

CXCL12 has been shown to be involved in colon cancer metastasis, but its expression level and molecular mechanisms regulating its expression remain controversial. We thus evaluated CXCL12 expression in a large cohort of colon adenomas and carcinomas, investigated for an epigenetic mechanism controlling its expression and evaluated the impact of CXCL12 levels on cell migration and tumor growth. CXCL12 expression was measured in human colon adenomas and carcinomas with transcriptome array and RT-qPCR. The promoter methylation was analyzed with whole-genome DNA methylation chips and protein expression by immunohistochemistry. We confirm a reduced expression of CXCL12 in 75% of MSS carcinomas and show that the decrease is an early event as already present in adenomas. The methylome analysis shows that the CXCL12 promoter is methylated in only 30% of microsatellite-stable tumors. In vitro, treatments with HDAC inhibitors, butyrate and valproate restored CXCL12 expression in three colon cell lines, increased acetylation of histone H3 within the CXCL12 promoter and inhibited cell migration. In vivo, valproate diminished (65%) the number of intestinal tumors in APC mutant mice, slowed down xenograft tumor growth concomitant to restored CXCL12 expression. Finally we identified loss of PCAF expression in tumor samples and showed that forced expression of PCAF in colon cancer cell lines restored CXCL12 expression. Thus, reduced PCAF expression may participate to CXCL12 promoter hypoacetylation and its subsequent loss of expression. Our study is of potential clinical interest because agents that promote or maintain histone acetylation through HDAC inhibition and/or HAT stimulation, may help to lower colon adenoma/carcinoma incidence, especially in high-risk families, or could be included in therapeutic protocols to treat advanced colon cancer.

MicroRNAs (miRNAs) are short, noncoding RNAs involved in the regulation of several processes associated with inflammatory diseases and infection. Bacterial infection modulates miRNA expression to subvert any innate immune response. In this study we analyzed, using microarray analysis, the bacterial modulation of miRNAs in bone marrow-derived macrophages (BMMs) in which activity was induced by infection with Porphyromonas gingivalis The expression of several miRNAs was modulated 3 h postinfection (at a multiplicity of infection of 25). A bioinformatic analysis was performed to further identify pathways related to the innate immune host response under the influence of selected miRNAs. To assess the effects of the miRNAs identified on cytokine secretion (tumor necrosis factor alpha [TNF-α] and interleukin-10 [IL-10]), BMMs were transfected with selected miRNA mimics and inhibitors. Transfection with mmu-miR-155 and mmu-miR-2137 did not modify TNF-α secretion, while their inhibitors increased it. Inhibitors of mmu-miR-2137 and mmu-miR-7674 increased the secretion of the anti-inflammatory factor IL-10. In P. gingivalis -infected BMMs, mmu-miR-155-5p significantly decreased TNF-α secretion while inhibitor of mmu-miR-2137 increased IL-10 secretion. In vivo , in a mouse model of P. gingivalis -induced calvarial bone resorption, injection of mmu-miR-155-5p or anti-mmu-miR-2137 reduced the size of the lesion significantly. Furthermore, anti-mmu-miR-2137 significantly reduced inflammatory cell infiltration, osteoclast activity, and bone loss. Bioinformatic analysis demonstrated that pathways related to cytokine- and chemokine-related pathways but also osteoclast differentiation may be involved in the effects observed. This study contributes further to our understanding of P. gingivalis -induced modulation of miRNAs and their physiological effects. It highlights the potential therapeutic merits of targeting mmu-miR-155-5p and mmu-miR-2137 to control inflammation induced by P. gingivalis infection., (Copyright © 2017 American Society for Microbiology.)

To investigate the complexity of alternative splicing in the retina, we sequenced and analyzed a total of 115,706 clones from normalized cDNA libraries from mouse neural retina (66,217) and rat retinal pigmented epithelium (49,489). Based upon clustering the cDNAs and mapping them with their respective genomes, the estimated numbers of genes were 9,134 for the mouse neural retina and 12,050 for the rat retinal pigmented epithelium libraries. This unique collection of retinal of messenger RNAs is maintained and accessible through a web-base server to the whole community of retinal biologists for further functional characterization. The analysis revealed 3,248 and 3,202 alternative splice events for mouse neural retina and rat retinal pigmented epithelium, respectively. We focused on transcription factors involved in vision. Among the six candidates suitable for functional analysis, we selected Otx2S, a novel variant of the Otx2 gene with a deletion within the homeodomain sequence. Otx2S is expressed in both the neural retina and retinal pigmented epithelium, and encodes a protein that is targeted to the nucleus. OTX2S exerts transdominant activity on the tyrosinase promoter when tested in the physiological environment of primary RPE cells. By overexpressing OTX2S in primary RPE cells using an adeno associated viral vector, we identified 10 genes whose expression is positively regulated by OTX2S. We find that OTX2S is able to bind to the chromatin at the promoter of the retinal dehydrogenase 10 (RDH10) gene.

Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies.

An in situ hybridization (ISH) study was performed on 2000 murine genes representing around 10% of the protein-coding genes present in the mouse genome using data generated by the EURExpress consortium. This study was carried out in 25 tissues of late gestation embryos (E14.5), with a special emphasis on the developing ear and on five distinct developing sensory organs, including the cochlea, the vestibular receptors, the sensory retina, the olfactory organ, and the vibrissae follicles. The results obtained from an analysis of more than 11,000 micrographs have been integrated in a newly developed knowledgebase, called ImAnno. In addition to managing the multilevel micrograph annotations performed by human experts, ImAnno provides public access to various integrated databases and tools. Thus, it facilitates the analysis of complex ISH gene expression patterns, as well as functional annotation and interaction of gene sets. It also provides direct links to human pathways and diseases. Hierarchical clustering of expression patterns in the 25 tissues revealed three main branches corresponding to tissues with common functions and/or embryonic origins. To illustrate the integrative power of ImAnno, we explored the expression, function and disease traits of the sensory epithelia of the five presumptive sensory organs. The study identified 623 genes (out of 2000) concomitantly expressed in the five embryonic epithelia, among which many (∼12%) were involved in human disorders. Finally, various multilevel interaction networks were characterized, highlighting differential functional enrichments of directly or indirectly interacting genes. These analyses exemplify an under-represention of "sensory" functions in the sensory gene set suggests that E14.5 is a pivotal stage between the developmental stage and the functional phase that will be fully reached only after birth.

The genome of the coprophilous fungus Podospora anserina harbors a large and highly diverse set of putative lignocellulose-acting enzymes. In this study, we investigated the enzymatic diversity of a broad range of P. anserina secretomes induced by various carbon sources (dextrin, glucose, xylose, arabinose, lactose, cellobiose, saccharose, Avicel, Solka-floc, birchwood xylan, wheat straw, maize bran, and sugar beet pulp (SBP)). Compared with the Trichoderma reesei enzymatic cocktail, P. anserina secretomes displayed similar cellulase, xylanase, and pectinase activities and greater arabinofuranosidase, arabinanase, and galactanase activities. The secretomes were further tested for their capacity to supplement a T. reesei cocktail. Four of them improved significantly the saccharification yield of steam-exploded wheat straw up to 48 %. Fine analysis of the P. anserina secretomes produced with Avicel and SBP using proteomics revealed a large array of CAZymes with a high number of GH6 and GH7 cellulases, CE1 esterases, GH43 arabinofuranosidases, and AA1 laccase-like multicopper oxidases. Moreover, a preponderance of AA9 (formerly GH61) was exclusively produced in the SBP condition. This study brings additional insights into the P. anserina enzymatic machinery and will facilitate the selection of promising targets for the development of future biorefineries.

Summary: We present PARSEC (PAtteRn Search and Contextualization), a new open source platform for guided discovery, allowing localization and biological characterization of short genomic sites in entire eukaryotic genomes. PARSEC can search for a sequence or a degenerated pattern. The retrieved set of genomic sites can be characterized in terms of (i) conservation in model organisms, (ii) genomic context (proximity to genes) and (iii) function of neighboring genes. These modules allow the user to explore, visualize, filter and extract biological knowledge from a set of short genomic regions such as transcription factor binding sites., Availability: Web site implemented in Java, JavaScript and C++, with all major browsers supported. Freely available at lbgi.fr/parsec. Source code is freely available at sourceforge.net/projects/genomicparsec.

The ascomycete Podospora anserina is a coprophilous fungus that grows at late stages on droppings of herbivores. Its genome encodes a large diversity of carbohydrate-active enzymes. Among them, four genes encode glycoside hydrolases from family 6 (GH6), the members of which comprise putative endoglucanases and exoglucanases, some of them exerting important functions for biomass degradation in fungi. Therefore, this family was selected for functional analysis. Three of the enzymes, P. anserina Cel6A (PaCel6A), PaCel6B, and PaCel6C, were functionally expressed in the yeast Pichia pastoris. All three GH6 enzymes hydrolyzed crystalline and amorphous cellulose but were inactive on hydroxyethyl cellulose, mannan, galactomannan, xyloglucan, arabinoxylan, arabinan, xylan, and pectin. PaCel6A had a catalytic efficiency on cellotetraose comparable to that of Trichoderma reesei Cel6A (TrCel6A), but PaCel6B and PaCel6C were clearly less efficient. PaCel6A was the enzyme with the highest stability at 45°C, while PaCel6C was the least stable enzyme, losing more than 50% of its activity after incubation at temperatures above 30°C for 24 h. In contrast to TrCel6A, all three studied P. anserina GH6 cellulases were stable over a wide range of pHs and conserved high activity at pH values of up to 9. Each enzyme displayed a distinct substrate and product profile, highlighting different modes of action, with PaCel6A being the enzyme most similar to TrCel6A. PaCel6B was the only enzyme with higher specific activity on carboxymethylcellulose (CMC) than on Avicel and showed lower processivity than the others. Structural modeling predicts an open catalytic cleft, suggesting that PaCel6B is an endoglucanase.

Background: The core promoter is the region flanking the transcription start site (TSS) that directs formation of the pre-initiation complex. Core promoters have been studied intensively in mammals and yeast, but not in more diverse eukaryotes. Here we investigate core promoters in oomycetes, a group within the Stramenopile kingdom that includes important plant and animal pathogens. Prior studies of a small collection of genes proposed that oomycete core promoters contain a 16 to 19 nt motif bearing an Initiator-like sequence (INR) flanked by a novel sequence named FPR, but this has not been extended to whole-genome analysis., Results: We used expectation maximization to find over-represented motifs near TSSs of Phytophthora infestans, the potato blight pathogen. The motifs corresponded to INR, FPR, and a new element found about 25 nt downstream of the TSS called DPEP. TATA boxes were not detected. Assays of DPEP function by mutagenesis were consistent with its role as a core motif. Genome-wide searches found a well-conserved combined INR+FPR in only about 13% of genes after correcting for false discovery, which contradicted prior reports that INR and FPR are found together in most genes. INR or FPR were found alone near TSSs in 18% and 7% of genes, respectively. Promoters lacking the motifs had pyrimidine-rich regions near the TSS. The combined INR+FPR motif was linked to higher than average mRNA levels, developmentally-regulated transcription, and functions related to plant infection, while DPEP and FPR were over-represented in constitutively-expressed genes. The INR, FPR, and combined INR+FPR motifs were detected in other oomycetes including Hyaloperonospora arabidopsidis, Phytophthora sojae, Pythium ultimum, and Saprolegnia parasitica, while DPEP was found in all but S. parasitica. Only INR seemed present in a non-oomycete stramenopile., Conclusions: The absence of a TATA box and presence of novel motifs show that the oomycete core promoter is diverged from that of model systems, and likely explains the lack of activity of non-oomycete promoters in Phytophthora transformants. The association of the INR+FPR motif with developmentally-regulated genes shows that oomycete core elements influence stage-specific transcription in addition to regulating formation of the pre-initiation complex.

The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnabarinus on cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference between PaGH61A and PaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination of PaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties of PaGH61A and PaGH61B and their effect on the interaction with CDH are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

A major challenge in the post-genomic era is a better understanding of how human genetic alterations involved in disease affect the gene products. The KD4v (Comprehensible Knowledge Discovery System for Missense Variant) server allows to characterize and predict the phenotypic effects (deleterious/neutral) of missense variants. The server provides a set of rules learned by Induction Logic Programming (ILP) on a set of missense variants described by conservation, physico-chemical, functional and 3D structure predicates. These rules are interpretable by non-expert humans and are used to accurately predict the deleterious/neutral status of an unknown mutation. The web server is available at http://decrypthon.igbmc.fr/kd4v.

Recent genome-wide chromatin immunoprecipitation coupled high throughput sequencing (ChIP-seq) analyses performed in various eukaryotic organisms, analysed RNA Polymerase II (Pol II) pausing around the transcription start sites of genes. In this study we have further investigated genome-wide binding of Pol II downstream of the 3' end of the annotated genes (EAGs) by ChIP-seq in human cells. At almost all expressed genes we observed Pol II occupancy downstream of the EAGs suggesting that Pol II pausing 3' from the transcription units is a rather common phenomenon. Downstream of EAGs Pol II transcripts can also be detected by global run-on and sequencing, suggesting the presence of functionally active Pol II. Based on Pol II occupancy downstream of EAGs we could distinguish distinct clusters of Pol II pause patterns. On core histone genes, coding for non-polyadenylated transcripts, Pol II occupancy is quickly dropping after the EAG. In contrast, on genes, whose transcripts undergo polyA tail addition [poly(A)(+)], Pol II occupancy downstream of the EAGs can be detected up to 4-6 kb. Inhibition of polyadenylation significantly increased Pol II occupancy downstream of EAGs at poly(A)(+) genes, but not at the EAGs of core histone genes. The differential genome-wide Pol II occupancy profiles 3' of the EAGs have also been confirmed in mouse embryonic stem (mES) cells, indicating that Pol II pauses genome-wide downstream of the EAGs in mammalian cells. Moreover, in mES cells the sharp drop of Pol II signal at the EAG of core histone genes seems to be independent of the phosphorylation status of the C-terminal domain of the large subunit of Pol II. Thus, our study uncovers a potential link between different mRNA 3' end processing mechanisms and consequent Pol II transcription termination processes.

Rod-derived cone viability factor (RdCVF) is produced by the Nxnl1 gene that codes for a second polypeptide, RdCVFL, by alternative splicing. Although the role of RdCVF in promoting cone survival has been described, the implication of RdCVFL, a putative thioredoxin enzyme, in the protection of photoreceptors is presently unknown. Using a proteomics approach we identified 90 proteins interacting with RdCVFL including the microtubule-binding protein TAU. We demonstrate that the level of phosphorylation of TAU is increased in the retina of the Nxnl1(-/-) mice as it is hyperphosphorylated in the brain of patients suffering from Alzheimer disease, presumably in some cases through oxidative stress. Using a cell-based assay, we show that RdCVFL inhibits TAU phosphorylation. In vitro, RdCVFL protects TAU from oxidative damage. Photooxidative stress is implicated in retinal degeneration, particularly in retinitis pigmentosa, where it is considered to be a contributor to secondary cone death. The functional interaction between RdCVFL and TAU described here is the first characterization of the RdCVFL signaling pathway involved in neuronal cell death mediated by oxidative stress.

Background: The retina is a multi-layered sensory tissue that lines the back of the eye and acts at the interface of input light and visual perception. Its main function is to capture photons and convert them into electrical impulses that travel along the optic nerve to the brain where they are turned into images. It consists of neurons, nourishing blood vessels and different cell types, of which neural cells predominate. Defects in any of these cells can lead to a variety of retinal diseases, including age-related macular degeneration, retinitis pigmentosa, Leber congenital amaurosis and glaucoma. Recent progress in genomics and microarray technology provides extensive opportunities to examine alterations in retinal gene expression profiles during development and diseases. However, there is no specific database that deals with retinal gene expression profiling. In this context we have built RETINOBASE, a dedicated microarray database for retina., Description: RETINOBASE is a microarray relational database, analysis and visualization system that allows simple yet powerful queries to retrieve information about gene expression in retina. It provides access to gene expression meta-data and offers significant insights into gene networks in retina, resulting in better hypothesis framing for biological problems that can subsequently be tested in the laboratory. Public and proprietary data are automatically analyzed with 3 distinct methods, RMA, dChip and MAS5, then clustered using 2 different K-means and 1 mixture models method. Thus, RETINOBASE provides a framework to compare these methods and to optimize the retinal data analysis. RETINOBASE has three different modules, "Gene Information", "Raw Data System Analysis" and "Fold change system Analysis" that are interconnected in a relational schema, allowing efficient retrieval and cross comparison of data. Currently, RETINOBASE contains datasets from 28 different microarray experiments performed in 5 different model systems: drosophila, zebrafish, rat, mouse and human. The database is supported by a platform that is designed to easily integrate new functionalities and is also frequently updated., Conclusion: The results obtained from various biological scenarios can be visualized, compared and downloaded. The results of a case study are presented that highlight the utility of RETINOBASE. Overall, RETINOBASE provides efficient access to the global expression profiling of retinal genes from different organisms under various conditions.

Background: DNA ligases are required for DNA strand joining in all forms of cellular life. NAD+-dependent DNA ligases are found primarily in eubacteria but also in some eukaryotic viruses, bacteriophage and archaea. Among the archaeal NAD+-dependent DNA ligases is the LigN enzyme of the halophilic euryarchaeon Haloferax volcanii, the gene for which was apparently acquired by Hfx. volcanii through lateral gene transfer (LGT) from a halophilic eubacterium. Genetic studies show that the LGT-acquired LigN enzyme shares an essential function with the native Hfx. volcanii ATP-dependent DNA ligase protein LigA., Results: To characterise the enzymatic properties of the LigN protein, wild-type and three mutant forms of the LigN protein were separately expressed in recombinant form in E.coli and purified to apparent homogeneity by immobilised metal ion affinity chromatography (IMAC). Non-isotopic DNA ligase activity assays using lambda DNA restriction fragments with 12 bp cos cohesive ends were used to show that LigN activity was dependent on addition of divalent cations and salt. No activity was detected in the absence of KCl, whereas maximum activity could be detected at 3.2 M KCl, close to the intracellular KCl concentration of Hfx. volcanii cells., Conclusion: LigN is unique amongst characterised DNA ligase enzymes in displaying maximal DNA strand joining activity at high (> 3 M) salt levels. As such the LigN enzyme has potential both as a novel tool for biotechnology and as a model enzyme for studying the adaptation of proteins to high intracellular salt levels.