Integrative proteomics highlight presynaptic alterations and c-Jun misactivation as convergent pathomechanisms in ALS.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease mainly affecting upper and lower motoneurons. Several functionally heterogeneous genes have been associated with the familial form of this disorder (fALS), depicting an extremely complex pathogenic landscape. This heterogeneity has limited the identification of an effective therapy, and this bleak prognosis will only improve with a greater understanding of convergent disease mechanisms. Recent evidence from human post-mortem material and diverse model systems has highlighted the synapse as a crucial structure actively involved in disease progression, suggesting that synaptic aberrations might represent a shared pathological feature across the ALS spectrum. To test this hypothesis, we performed the first comprehensive analysis of the synaptic proteome from post-mortem spinal cord and human iPSC-derived motoneurons carrying mutations in the major ALS genes. This integrated approach highlighted perturbations in the molecular machinery controlling vesicle release as a shared pathomechanism in ALS. Mechanistically, phosphoproteomic analysis linked the presynaptic vesicular phenotype to an accumulation of cytotoxic protein aggregates and to the pro-apoptotic activation of the transcription factor c-Jun, providing detailed insights into the shared pathobiochemistry in ALS. Notably, sub-chronic treatment of our iPSC-derived motoneurons with the fatty acid docosahexaenoic acid exerted a neuroprotective effect by efficiently rescuing the alterations revealed by our multidisciplinary approach. Together, this study provides strong evidence for the central and convergent role played by the synaptic microenvironment within the ALS spinal cord and highlights a potential therapeutic target that counteracts degeneration in a heterogeneous cohort of human motoneuron cultures.

Transcriptome Profiling Identifies TIGIT as a Marker of T-Cell Exhaustion in Liver Cancer.

BACKGROUND AND AIMS: Programmed death 1 (PD-1) checkpoint inhibition has shown promising results in patients with hepatocellular carcinoma, inducing objective responses in approximately 20% of treated patients. The roles of other coinhibitory molecules and their individual contributions to T-cell dysfunction in liver cancer, however, remain largely elusive. APPROACH AND RESULTS: We performed a comprehensive mRNA profiling of cluster of differentiation 8 (CD8) T cells in a murine model of autochthonous liver cancer by comparing the transcriptome of naive, functional effector, and exhausted, tumor-specific CD8 T cells. Subsequently, we functionally validated the role of identified genes in T-cell exhaustion. Our results reveal a unique transcriptome signature of exhausted T cells and demonstrate that up-regulation of the inhibitory immune receptor T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitor motif domains (TIGIT) represents a hallmark in the process of T-cell exhaustion in liver cancer. Compared to PD-1, expression of TIGIT more reliably identified exhausted CD8 T cells at different stages of their differentiation. In combination with PD-1 inhibition, targeting of TIGIT with antagonistic antibodies resulted in synergistic inhibition of liver cancer growth in immunocompetent mice. Finally, we demonstrate expression of TIGIT on tumor-infiltrating CD8 T cells in tissue samples of patients with hepatocellular carcinoma and intrahepatic cholangiocarcinoma and identify two subsets of patients based on differential expression of TIGIT on tumor-specific T cells. CONCLUSIONS: Our transcriptome analysis provides a valuable resource for the identification of key pathways involved in T-cell exhaustion in patients with liver cancer and identifies TIGIT as a potential target in checkpoint combination therapies.

Whole genome characterization of autochthonous Bos taurus brachyceros and introduced Bos indicus indicus cattle breeds in Cameroon regarding their adaptive phenotypic traits and pathogen resistance.

BACKGROUND: African indigenous taurine cattle display unique adaptive traits shaped by husbandry management, regional climate and exposure to endemic pathogens. They are less productive with respect to milk and meat production which has been associated with amongst others, small size, traditional beliefs, husbandry practices, limited feed resources, disease burden and lack of sustained breeding for trait improvement. This resulted in the severe dwindling of their population size rendering them vulnerable to extinction. The Namchi taurine cattle breed is referred to as [Namchi (Doayo)] and shows resistance traits against trypanosome infection and exposure to tick infestation. Nonetheless, the historically later introduced Zebu cattle are the main cattle breeds in Africa today, even though they suffer more from locally prevailing pathogens. By using a whole genome sequencing approach, we sequenced with high depth for the first time the genomes of five cattle breeds from Cameroon in order to provide a valuable genetic resource for future African cattle breeding: the Namchi, an endangered trypano-tolerant taurine breed, the Kapsiki, an indigenous trypano-susceptible taurine breed, and three Zebu (Bos indicus indicus) breeds: Ngaoundere Gudali, White Fulani and Red Fulani. RESULTS: Approximately 167 Gigabases of raw sequencing data were generated for each breed and mapped to the cattle reference genomes ARS-UCD1.2 and UMD3.1.The coverage was 103 to 140-fold when aligning the reads to ARS-UCD1.2 with an average mapping rate of ~ 99%, and 22 to 30-fold when aligning the reads to UMD3.1 with an average mapping rate of ~ 64%. The single nucleotide polymorphisms (SNPs) obtained from analysis using the genome ARS-UCD1.2 were compared with reference genomes of European Bos taurus Holstein, the Asian Bos indicus Brahman, and the African trypanotolerant N'Dama breeds. A total of ~ 100 million (M) SNPs were identified and 7.7 M of those were breed-specific. An approximately 11.1 M constituted of small insertions and deletions. By using only breed-specific non-synonymous variants we identified genes as genetic signatures and associated Gene Ontology (GO) terms that could explain certain cattle-breed specific phenotypes such as increased tolerance against trypanosome parasites in the Namchi breed and heat tolerance in the Kapsiki breed. Phylogenetic analysis grouped, except for Namchi, the Bos taurus breeds Kapsiki, N'Dama and Holstein together while the B. indicus breeds White and Red Fulani, Gudali and Brahman clustered separately. The deviating result for Namchi indicates a hybrid status of the selected animal with a recent introgression of Zebu genes into its genome. CONCLUSIONS: The findings provide the first comprehensive set of genome-wide variant data of the most important Cameroonian cattle breeds. The genomic data shall constitute a foundation for breed amelioration whilst exploiting the heritable traits and support conservation efforts for the endangered local cattle breeds.

Ring1b-dependent epigenetic remodelling is an essential prerequisite for pancreatic carcinogenesis.

BACKGROUND AND AIMS: Besides well-defined genetic alterations, the dedifferentiation of mature acinar cells is an important prerequisite for pancreatic carcinogenesis. Acinar-specific genes controlling cell homeostasis are extensively downregulated during cancer development; however, the underlying mechanisms are poorly understood. Now, we devised a novel in vitro strategy to determine genome-wide dynamics in the epigenetic landscape in pancreatic carcinogenesis. DESIGN: With our in vitro carcinogenic sequence, we performed global gene expression analysis and ChIP sequencing for the histone modifications H3K4me3, H3K27me3 and H2AK119ub. Followed by a comprehensive bioinformatic approach, we captured gene clusters with extensive epigenetic and transcriptional remodelling. Relevance of Ring1b-catalysed H2AK119ub in acinar cell reprogramming was studied in an inducible Ring1b knockout mouse model. CRISPR/Cas9-mediated Ring1b ablation as well as drug-induced Ring1b inhibition were functionally characterised in pancreatic cancer cells. RESULTS: The epigenome is vigorously modified during pancreatic carcinogenesis, defining cellular identity. Particularly, regulatory acinar cell transcription factors are epigenetically silenced by the Ring1b-catalysed histone modification H2AK119ub in acinar-to-ductal metaplasia and pancreatic cancer cells. Ring1b knockout mice showed greatly impaired acinar cell dedifferentiation and pancreatic tumour formation due to a retained expression of acinar differentiation genes. Depletion or drug-induced inhibition of Ring1b promoted tumour cell reprogramming towards a less aggressive phenotype. CONCLUSIONS: Our data provide substantial evidence that the epigenetic silencing of acinar cell fate genes is a mandatory event in the development and progression of pancreatic cancer. Targeting the epigenetic repressor Ring1b could offer new therapeutic options.

Rpv3-1 mediated resistance to grapevine downy mildew is associated with specific host transcriptional responses and the accumulation of stilbenes.

BACKGROUND: European grapevine cultivars (Vitis vinifera spp.) are highly susceptible to the downy mildew pathogen Plasmopara viticola. Breeding of resistant V. vinifera cultivars is a promising strategy to reduce the impact of disease management. Most cultivars that have been bred for resistance to downy mildew, rely on resistance mediated by the Rpv3 (Resistance to P. viticola) locus. However, despite the extensive use of this locus, little is known about the mechanism of Rpv3-mediated resistance. RESULTS: In this study, Rpv3-mediated defense responses were investigated in Rpv3+ and Rpv3- grapevine cultivars following inoculation with two distinct P. viticola isolates avrRpv3+ and avrRpv3-, with the latter being able to overcome Rpv3 resistance. Based on comparative microscopic, metabolomic and transcriptomic analyses, our results show that the Rpv3-1-mediated resistance is associated with a defense mechanism that triggers synthesis of fungi-toxic stilbenes and programmed cell death (PCD), resulting in reduced but not suppressed pathogen growth and development. Functional annotation of the encoded protein sequence of genes significantly upregulated during the Rpv3-1-mediated defense response revealed putative roles in pathogen recognition, signal transduction and defense responses. CONCLUSION: This study used histochemical, transcriptomic and metabolomic analyses of Rpv3+ and susceptible cultivars inoculated with avirulent and virulent P. viticola isolates to investigate mechanism underlying the Rpv3-1-mediated resistance response. We demonstrated a strong correlation between the expressions of stilbene biosynthesis related genes, the accumulation of fungi-toxic stilbenes, pathogen growth inhibition and PCD.

The grapevine VvibZIPC22 transcription factor is involved in the regulation of flavonoid biosynthesis.

In grapevine, flavonoids constitute one of the most abundant subgroups of secondary metabolites, influencing the quality, health value, and typicity of wines. Their synthesis in many plant species is mainly regulated at the transcriptional level by modulation of flavonoid pathway genes either by single regulators or by complexes of different regulators. In particular, bZIP and MYB factors interact synergistically in the recognition of light response units present in the promoter of some genes of the pathway, thus mediating light-dependent flavonoid biosynthesis. We recently identified VvibZIPC22, a member of clade C of the grapevine bZIP family, in a quantitative trait locus (QTL) specifically associated with kaemperol content in mature berries. Here, to validate the involvement of this candidate gene in the fine regulation of flavonol biosynthesis, we characterized its function by in vitro and in vivo experiments. A role for this gene in the control of flavonol biosynthesis was indeed confirmed by its highest expression at flowering and during UV light-mediated induction, paralleled by accumulation of the flavonol synthase 1 transcript and flavonol compounds. The overexpression of VvibZIPC22 in tobacco caused a significant increase in several flavonoids in the flower, via induction of general and specific genes of the pathway. In agreement with this evidence, VvibZIPC22 was able to activate the promoters of specific genes of the flavonoid pathway, alone or together with other factors, as revealed by transient reporter assays. These findings, supported by in silico indications, allowed us to propose VvibZIPC22 as a new regulator of flavonoid biosynthesis in grapevine.

The photomorphogenic factors UV-B RECEPTOR 1, ELONGATED HYPOCOTYL 5, and HY5 HOMOLOGUE are part of the UV-B signalling pathway in grapevine and mediate flavonol accumulation in response to the environment.

Grapevine (Vitis vinifera L.) is a species well known for its adaptation to radiation. However, photomorphogenic factors related to UV-B responses have not been molecularly characterized. We cloned and studied the role of UV-B RECEPTOR (UVR1), ELONGATED HYPOCOTYL 5 (HY5), and HY5 HOMOLOGUE (HYH) from V. vinifera We performed gene functional characterizations, generated co-expression networks, and tested them in different environmental conditions. These genes complemented the Arabidopsis uvr8 and hy5 mutants in morphological and secondary metabolic responses to radiation. We combined microarray and RNA sequencing (RNA-seq) data with promoter inspections to identify HY5 and HYH putative target genes and their DNA binding preferences. Despite sharing a large set of common co-expressed genes, we found different hierarchies for HY5 and HYH depending on the organ and stress condition, reflecting both co-operative and partially redundant roles. New candidate UV-B gene markers were supported by the presence of HY5-binding sites. These included a set of flavonol-related genes that were up-regulated in a HY5 transient expression assay. We irradiated in vitro plantlets and fruits from old potted vines with high and low UV-B exposures and followed the accumulation of flavonols and changes in gene expression in comparison with non-irradiated conditions. UVR1, HY5, and HYH expression varied with organ, developmental stage, and type of radiation. Surprisingly, UVR1 expression was modulated by shading and temperature in berries, but not by UV-B radiation. We propose that the UV-B response machinery favours berry flavonol accumulation through the activation of HY5 and HYH at different developmental stages at both high and low UV-B exposures.

The R2R3-MYB transcription factors MYB14 and MYB15 regulate stilbene biosynthesis in Vitis vinifera.

Plant stilbenes are phytoalexins that accumulate in a small number of plant species, including grapevine (Vitis vinifera), in response to biotic and abiotic stresses and have been implicated in many beneficial effects on human health. In particular, resveratrol, the basic unit of all other complex stilbenes, has received widespread attention because of its cardio-protective, anticarcinogenic, and antioxidant properties. Although stilbene synthases (STSs), the key enzymes responsible for resveratrol biosynthesis, have been isolated and characterized from several plant species, the transcriptional regulation underlying stilbene biosynthesis is unknown. Here, we report the identification and functional characterization of two R2R3-MYB-type transcription factors (TFs) from grapevine, which regulate the stilbene biosynthetic pathway. These TFs, designated MYB14 and MYB15, strongly coexpress with STS genes, both in leaf tissues under biotic and abiotic stress and in the skin and seed of healthy developing berries during maturation. In transient gene reporter assays, MYB14 and MYB15 were demonstrated to specifically activate the promoters of STS genes, and the ectopic expression of MYB15 in grapevine hairy roots resulted in increased STS expression and in the accumulation of glycosylated stilbenes in planta. These results demonstrate the involvement of MYB14 and MYB15 in the transcriptional regulation of stilbene biosynthesis in grapevine.

Transcriptomic Stress Response in Streptococcus mutans following Treatment with a Sublethal Concentration of Chlorhexidine Digluconate.

Despite the widespread use of antiseptics such as chlorhexidine digluconate (CHX) in dental practice and oral care, the risks of potential resistance toward these antimicrobial compounds in oral bacteria have only been highlighted very recently. Since the molecular mechanisms behind antiseptic resistance or adaptation are not entirely clear and the bacterial stress response has not been investigated systematically so far, the aim of the present study was to investigate the transcriptomic stress response in Streptococcus mutans after treatment with CHX using RNA sequencing (RNA-seq). Planktonic cultures of stationary-phase S. mutans were treated with a sublethal dose of CHX (125 µg/mL) for 5 min. After treatment, RNA was extracted, and RNA-seq was performed on an Illumina NextSeq 500. Differentially expressed genes were analyzed and validated by qRT-PCR. Analysis of differential gene expression following pathway analysis revealed a considerable number of genes and pathways significantly up- or downregulated in S. mutans after sublethal treatment with CHX. In summary, the expression of 404 genes was upregulated, and that of 271 genes was downregulated after sublethal CHX treatment. Analysis of differentially expressed genes and significantly regulated pathways showed regulation of genes involved in purine nucleotide synthesis, biofilm formation, transport systems and stress responses. In conclusion, the results show a transcriptomic stress response in S. mutans upon exposure to CHX and offer insight into potential mechanisms that may result in development of resistances.

Toward Systematic Understanding of Flower Bud Induction in Apple: A Multi-Omics Approach.

The induction of flower buds in apple (Malus × domestica Borkh.) is tightly connected to biennial bearing, which is characterized by alternating years with high (ON) and low or no (OFF) crop loads. In order to study this irregular cropping behavior, spur buds from ON- and OFF-trees of the biennial-bearing cultivar 'Fuji' and the regular bearing cultivar 'Gala' were collected. First, the time of flower bud initiation was precisely determined for both cultivars by histological analysis. Moreover, for a systematic understanding of flower bud induction in apple, the physiological and molecular mechanisms within the bud tissue were evaluated over four weeks prior to flower bud initiation by employing a multi-omics approach, including RNA sequencing, proteomic and metabolic profiling. Gene and protein enrichment analysis detected physiological pathways promoting and inhibiting early flower bud development. Metabolic profiles from the cropping treatments revealed a greater abundance of thiamine, chlorogenic acid, and an adenine derivative in spur buds from OFF-trees, whereas tryptophan was more abundant in the buds collected from ON-trees. Cultivar comparison indicated that chlorogenic acid was more abundant in 'Gala' than in 'Fuji' spur buds, whereas the opposite effect was found for tryptophan. Genes controlling tryptophan biosynthesis were not affected by ON- and OFF-treatments, but genes assigned to the metabolism of tryptophan into indoleacetate were differentially expressed between cultivars and treatments. The multi-omics approach permitted analyzing complex plant metabolic processes involved in early flower bud development and more specifically presumably in flower bud induction by tracing some pathways from gene to product level.

The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in developing grape berries.

Flavonols are important ultraviolet light protectants in many plants and contribute substantially to the quality and health-promoting effects of fruits and derived plant products. To study the regulation of flavonol synthesis in fruit, we isolated and characterized the grapevine (Vitis vinifera 'Shiraz') R2R3-MYB transcription factor VvMYBF1. Transient reporter assays established VvMYBF1 to be a specific activator of flavonol synthase1 (VvFLS1) and several other promoters of grapevine and Arabidopsis (Arabidopsis thaliana) genes involved in flavonol synthesis. Expression of VvMYBF1 in the Arabidopsis mutant myb12 resulted in complementation of its flavonol-deficient phenotype and confirmed the function of VvMYBF1 as a transcriptional regulator of flavonol synthesis. Transcript analysis of VvMYBF1 throughout grape berry development revealed its expression during flowering and in skins of ripening berries, which correlates with the accumulation of flavonols and expression of VvFLS1. In addition to its developmental regulation, VvMYBF1 expression was light inducible, implicating VvMYBF1 in the control of VvFLS1 transcription. Sequence analysis of VvMYBF1 and VvFLS1 indicated conserved putative light regulatory units in promoters of both genes from different cultivars. By analysis of the VvMYBF1 amino acid sequence, we identified the previously described SG7 domain and an additional sequence motif conserved in several plant MYB factors. The described motifs have been used to identify MYB transcription factors from other plant species putatively involved in the regulation of flavonol biosynthesis. To our knowledge, this is the first functional characterization of a light-inducible MYB transcription factor controlling flavonol synthesis in fruit.

Contribution of mTOR and PTEN to Radioresistance in Sporadic and NF2-Associated Vestibular Schwannomas: A Microarray and Pathway Analysis.

The use of radiation treatment has increased for both sporadic and neurofibromatosis type 2 (NF2)-associated vestibular schwannoma (VS). However, there are a subset of radioresistant tumors and systemic treatments that are seldom used in these patients. We investigated molecular alterations after radiation in three NF2-associated and five sporadically operated recurrent VS after primary irradiation. We compared these findings with 49 non-irradiated (36 sporadic and 13 NF2-associated) VS through gene-expression profiling and pathway analysis. Furthermore, we stained the key molecules of the distinct pathway by immunohistochemistry. A total of 195 differentially expressed genes in sporadic and NF2-related comparisons showed significant differences based on the criteria of p value < 0.05 and a two-fold change. These genes were involved in pathways that are known to be altered upon irradiation (e.g., mammalian target of rapamycin (mTOR), phosphatase and tensin homolog (PTEN) and vascular endothelial growth factor (VEGF) signaling). We observed a combined downregulation of PTEN signaling and an upregulation of mTOR signaling in progressive NF2-associated VS after irradiation. Immunostainings with mTOR and PTEN antibodies confirmed the respective molecular alterations. Taken together, mTOR inhibition might be a promising therapeutic strategy in NF2-associated VS progress after irradiation.

Experimental glioma with high bHLH expression harbor increased replicative stress and are sensitive toward ATR inhibition.

BACKGROUND: The overexpression of (basic)helix-loop-helix ((b)HLH) transcription factors (TFs) is frequent in malignant glioma. We investigated molecular effects upon disruption of the (b)HLH network by a dominant-negative variant of the E47 protein (dnE47). Our goal was to identify novel molecular subgroup-specific therapeutic strategies. METHODS: Glioma cell lines LN229, LNZ308, and GS-2/GS-9 were lentivirally transduced. Functional characterization included immunocytochemistry, immunoblots, cytotoxic, and clonogenic survival assays in vitro, and latency until neurological symptoms in vivo. Results of cap analysis gene expression and RNA-sequencing were further validated by immunoblot, flow cytometry, and functional assays in vitro. RESULTS: The induction of dnE47-RFP led to cytoplasmic sequestration of (b)HLH TFs and antiglioma activity in vitro and in vivo. Downstream molecular events, ie, alterations in transcription start site usage and in the transcriptome revealed enrichment of cancer-relevant pathways, particularly of the DNA damage response (DDR) pathway. Pharmacologic validation of this result using ataxia telangiectasia and Rad3 related (ATR) inhibition led to a significantly enhanced early and late apoptotic effect compared with temozolomide alone. CONCLUSIONS: Gliomas overexpressing (b)HLH TFs are sensitive toward inhibition of the ATR kinase. The combination of ATR inhibition plus temozolomide or radiation therapy in this molecular subgroup are warranted.

The Draft Whole-Genome Sequence of the Antibiotic Producer Empedobacter haloabium ATCC 31962 Provides Indications for Its Taxonomic Reclassification.

Strain ATCC 31962 was formerly taxonomically classified as Empedobacter haloabium and reported to be the producer of the lipopeptide antibiotic empedopeptin. Here, we report the draft genome sequence of ATCC 31962, which encodes regions that suggest a distinct biosynthetic capacity and suggests its taxonomic reclassification.

Multi-omics discovery of exome-derived neoantigens in hepatocellular carcinoma.

BACKGROUND: Although mutated HLA ligands are considered ideal cancer-specific immunotherapy targets, evidence for their presentation is lacking in hepatocellular carcinomas (HCCs). Employing a unique multi-omics approach comprising a neoepitope identification pipeline, we assessed exome-derived mutations naturally presented as HLA class I ligands in HCCs. METHODS: In-depth multi-omics analyses included whole exome and transcriptome sequencing to define individual patient-specific search spaces of neoepitope candidates. Evidence for the natural presentation of mutated HLA ligands was investigated through an in silico pipeline integrating proteome and HLA ligandome profiling data. RESULTS: The approach was successfully validated in a state-of-the-art dataset from malignant melanoma, and despite multi-omics evidence for somatic mutations, mutated naturally presented HLA ligands remained elusive in HCCs. An analysis of extensive cancer datasets confirmed fundamental differences of tumor mutational burden in HCC and malignant melanoma, challenging the notion that exome-derived mutations contribute relevantly to the expectable neoepitope pool in malignancies with only few mutations. CONCLUSIONS: This study suggests that exome-derived mutated HLA ligands appear to be rarely presented in HCCs, inter alia resulting from a low mutational burden as compared to other malignancies such as malignant melanoma. Our results therefore demand widening the target scope for personalized immunotherapy beyond this limited range of mutated neoepitopes, particularly for malignancies with similar or lower mutational burden.

qPortal: A platform for data-driven biomedical research.

Modern biomedical research aims at drawing biological conclusions from large, highly complex biological datasets. It has become common practice to make extensive use of high-throughput technologies that produce big amounts of heterogeneous data. In addition to the ever-improving accuracy, methods are getting faster and cheaper, resulting in a steadily increasing need for scalable data management and easily accessible means of analysis. We present qPortal, a platform providing users with an intuitive way to manage and analyze quantitative biological data. The backend leverages a variety of concepts and technologies, such as relational databases, data stores, data models and means of data transfer, as well as front-end solutions to give users access to data management and easy-to-use analysis options. Users are empowered to conduct their experiments from the experimental design to the visualization of their results through the platform. Here, we illustrate the feature-rich portal by simulating a biomedical study based on publically available data. We demonstrate the software's strength in supporting the entire project life cycle. The software supports the project design and registration, empowers users to do all-digital project management and finally provides means to perform analysis. We compare our approach to Galaxy, one of the most widely used scientific workflow and analysis platforms in computational biology. Application of both systems to a small case study shows the differences between a data-driven approach (qPortal) and a workflow-driven approach (Galaxy). qPortal, a one-stop-shop solution for biomedical projects offers up-to-date analysis pipelines, quality control workflows, and visualization tools. Through intensive user interactions, appropriate data models have been developed. These models build the foundation of our biological data management system and provide possibilities to annotate data, query metadata for statistics and future re-analysis on high-performance computing systems via coupling of workflow management systems. Integration of project and data management as well as workflow resources in one place present clear advantages over existing solutions.

New roles for nuclear EGFR in regulating the stability and translation of mRNAs associated with VEGF signaling.

Cell membrane-associated epidermal growth factor receptor (EGFR) translocates into a perinuclear/nuclear location upon stimulation, where it complexes with mRNAs. Treatment with radiation and cisplatin decreases the amounts of mRNAs present within this complex. Gene array analyses of mRNAs in complex with immunoprecipitated nEGFR revealed significant enrichment of different mRNA species compared to the control immunoprecipitation. Functional annotation with help of DAVID Gene Ontology Analysis identified under other terms the HIF-1A/VEGF signaling pathway as one of the top scoring KEGG pathways. RT-PCR and western blots revealed the radiation-induced expression of mRNAs and proteins involved in HIF-1A/VEGF signaling. Simultaneously, the levels of the corresponding validated miRNAs within the complex containing nEGFR and mRNAs were decreased. This finding argues that an mRNA/miRNA/nEGFR complex regulates protein expression. Indeed, we detected the GW182, AGO2, PABPC1 and cNOT1 proteins, which belong to the deadenylase complex, in a complex with nuclear EGFR. Erlotinib-mediated inhibition of EGFR kinase reduced the radiation-induced increase in mRNA expression. In this context, erlotinib reduced AGO2 phosphorylation by the EGFR kinase at residue Y393, which was associated with increased cNOT1 deadenylase activity and reduced mRNA stability. To prove the roles of miRNAs in this context, we transfected cells with an inhibitor of Hsa-mir-1180p5, which targets the NFATC4 mRNA, an mRNA associated with VEGF signaling, or pretreated cells with erlotinib. Indeed, Hsa-mir-1180p5 knockdown increased and the erlotinib treatment decreased the expression of the NFATC4 protein. The expression of the NFATC4 protein controlled the cloning efficiency and radiosensitivity of A549 and FaDu tumor cells. Thus, this study is the first to show that a membrane-located tyrosine kinase receptor, such as EGFR, is internalized to a nuclear/perinuclear location upon exposure to stress and modulates the stability and translation of miRNA-selected mRNAs. This mechanism enables cells to directly express proteins in response to EGFR activation and may contribute to treatment resistance in EGFR-overexpressing tumors.

Transcriptome-Wide Identification of Novel UV-B- and Light Modulated Flavonol Pathway Genes Controlled by VviMYBF1.

Flavonols constitute a group of flavonoids with important photoprotective roles in plants. In addition, flavonol content and composition greatly influences fruit quality. We previously demonstrated that the grapevine R2R3-MYB transcription factor (TF) VviMYBF1 promotes flavonol accumulation by inducing the expression of flavonol synthase (VviFLS1/VviFLS4), a key step of the initial flavonol pathway. Despite this, gene networks underlying flavonol modification in grapevine including both structural and regulatory genes remain poorly understood. In order to identify flavonol modifying genes and TFs acting downstream of VviMYBF1 a microarray-based transcriptome analysis was performed on grapevine hairy roots ectopically expressing VviMYBF1 or a Green Fluorescent Protein as control. VviFLS1 was induced in VviMYBF1 transgenic roots and glycosylated flavonols accumulated significantly compared with control lines. Among the differentially expressed genes, potential flavonol-modifying enzymes with predicted rhamnosyltransferase (e.g., RhaT1) or glycosyltransferase (e.g., GT3) activities were identified. In addition, important TFs of the MYB and bZIP families such as the proanthocyanidin regulator VviMYBPA1 and the UV-B light responsive HY5 homolog VviHYH were significantly altered in their expression pattern by overexpression of VviMYBF1. Co-temporal expression analysis demonstrated positive correlation of VviMYBF1 with VviFLS1, VviGT3, and VviRhaT1 during berry development and in fruits ripened with different light and UV-B radiation conditions at field. These results show that VviMYBF1 overexpression led to the identification of novel genes of the flavonol pathway and that the flavonol modifying machinery can be influenced by agricultural practices to optimize flavonol composition in grapes.

A Nexus Consisting of Beta-Catenin and Stat3 Attenuates BRAF Inhibitor Efficacy and Mediates Acquired Resistance to Vemurafenib.

Acquired resistance to second generation BRAF inhibitors (BRAFis), like vemurafenib is limiting the benefits of long term targeted therapy for patients with malignant melanomas that harbor BRAF V600 mutations. Since many resistance mechanisms have been described, most of them causing a hyperactivation of the MAPK- or PI3K/AKT signaling pathways, one potential strategy to overcome BRAFi resistance in melanoma cells would be to target important common signaling nodes. Known factors that cause secondary resistance include the overexpression of receptor tyrosine kinases (RTKs), alternative splicing of BRAF or the occurrence of novel mutations in MEK1 or NRAS. In this study we show that ß-catenin is stabilized and translocated to the nucleus in approximately half of the melanomas that were analyzed and which developed secondary resistance towards BRAFi. We further demonstrate that ß-catenin is involved in the mediation of resistance towards vemurafenib in vitro and in vivo. Unexpectedly, ß-catenin acts mainly independent of the TCF/LEF dependent canonical Wnt-signaling pathway in resistance development, which partly explains previous contradictory results about the role of ß-catenin in melanoma progression and therapy resistance. We further demonstrate that ß-catenin interacts with Stat3 after chronic vemurafenib treatment and both together cooperate in the acquisition and maintenance of resistance towards BRAFi.