Gut-derived Metabolites Influence Neurodevelopmental Gene Expression and Wnt Signalling Events in a Germ-free Zebrafish Model

Background : Small molecule metabolites produced by the microbiome are known to be neuroactive and are capable of directly impacting the brain and central nervous system, yet there is little data on the contribution of these metabolites to the earliest stages of neural development and neural gene expression. Here, we explore the impact of deriving zebrafish embryos in the absence of microbes on early neural development as well as investigate whether any potential changes can be rescued with treatment of metabolites derived from the zebrafish gut microbiota. Results : Overall, we did not observe any gross morphological changes between treatments but did observe a significant decrease in neural gene expression in embryos raised germ-free, which was rescued with the addition of zebrafish metabolites. Specifically, we identified 354 genes significantly down regulated in germ-free embryos compared to conventionally raised embryos via RNA-Seq analysis. Of these, 42 were rescued with a single treatment of zebrafish gut-derived metabolites to germ-free embryos. Gene ontology analysis revealed that these genes are involved in prominent neurodevelopmental pathways including transcriptional regulation and Wnt signalling. Consistent with the ontology analysis, we found alterations in the development of Wnt dependent events which was rescued in the germ-free embryos treated with metabolites. Conclusions : These findings demonstrate that gut-derived metabolites are in part responsible for regulating critical signalling pathways in the brain, especially during neural development.

Meta-Analysis and Bioinformatics Detection of Susceptibility Genes in Diabetic Nephropathy

The latest meta-analysis of genome-wide linkage studies (GWLS) identified nine cytogenetic locations suggestive of a linkage with diabetic nephropathy (DN) due to type 1 diabetes mellitus (T1DM) and seven locations due to type 2 diabetes mellitus (T2DM). In order to gain biological insight about the functional role of the genes located in these regions and to prioritize the most significant genetic loci for further research, we conducted a gene ontology analysis with an over representation test for the functional annotation of the protein coding genes. Protein analysis through evolutionary relationships (PANTHER) version 16.0 software and Cytoscape with the relevant plugins were used for the gene ontology analysis, and the overrepresentation test and STRING database were used for the construction of the protein network. The findings of the over-representation test highlight the contribution of immune related molecules like immunoglobulins, cytokines, and chemokines with regard to the most overrepresented protein classes, whereas the most enriched signaling pathways include the VEGF signaling pathway, the Cadherin pathway, the Wnt pathway, the angiogenesis pathway, the p38 MAPK pathway, and the EGF receptor signaling pathway. The common section of T1DM and T2DM results include the significant over representation of immune related molecules, and the Cadherin and Wnt signaling pathways that could constitute potential therapeutic targets for the treatment of DN, irrespective of the type of diabetes.

tRForest: a novel random forest-based algorithm for tRNA-derived fragment target prediction

tRNA fragments (tRFs) are small RNAs comparable to the size and function of miRNAs. tRFs are generally Dicer independent, are found associated with Ago, and can repress expression of genes post-transcriptionally. Given that this expands the repertoire of small RNAs capable of post-transcriptional gene expression, it is important to predict tRF targets with confidence. Some attempts have been made to predict tRF targets, but are limited in the scope of tRF classes used in prediction or limited in feature selection. We hypothesized that established miRNA target prediction features applied to tRFs through a random forest machine learning algorithm will immensely improve tRF target prediction. Using this approach, we show significant improvements in tRF target prediction for all classes of tRFs and validate our predictions in two independent cell lines. Finally, Gene Ontology analysis suggests that among the tRFs conserved between mice and humans, the predicted targets are enriched significantly in neuronal function, and we show this specifically for tRF-3009a. These improvements to tRF target prediction further our understanding of tRF function broadly across species and provide avenues for testing novel roles for tRFs in biology. We have created a publicly available website for the targets of tRFs predicted by tRForest.

Influence of Risk Factors for Male Infertility on Sperm Protein Composition

Male infertility is a common health problem that can be influenced by a host of lifestyle risk factors such as environment, nutrition, smoking, stress, and endocrine disruptors. These effects have been largely demonstrated on sperm parameters (e.g., motility, numeration, vitality, DNA integrity). In addition, several studies showed the deregulation of sperm proteins in relation to some of these factors. This review inventories the literature related to the identification of sperm proteins showing abundance variations in response to the four risk factors for male infertility that are the most investigated in this context: obesity, diabetes, tobacco smoking, and exposure to bisphenol-A (BPA). First, we provide an overview of the techniques used to identify deregulated proteins. Then, we summarise the main results obtained in the different studies and provide a compiled list of deregulated proteins in relation to each risk factor. Gene ontology analysis of these deregulated proteins shows that oxidative stress and immune and inflammatory responses are common mechanisms involved in sperm alterations encountered in relation to the risk factors.

Analysis of the leaf metabolome in Arabidopsis thaliana mutation accumulation lines reveals association of pleiotropy and fitness consequences

Understanding the mechanisms by which mutations affect fitness and the distribution of mutational effects are central goals in evolutionary biology. Mutation accumulation (MA) lines have long been an important tool for understanding the effect of new mutations on fitness, phenotypic variation, and mutational parameters. However, there is a clear gap in predicting the effect of specific new mutations to their effects on fitness. Here, we complete gene ontology analysis and metabolomics experiments on Arabidopsis thaliana MA lines to determine how spontaneous mutations directly affect global metabolic output in lines that have measured fitness consequences. For these analyses, we compared three lines with relative fitness consistently higher than the unmutated progenitor and three lines with lower relative fitness as measured in four different field trials. In a gene ontology analysis, we find that the high fitness lines were significantly enriched in mutations in or near genes with transcription regulator activity. We also find that although they do not have an average difference in the number of mutations, low fitness lines have significantly more metabolic subpathways disrupted than high fitness lines. Taken together, these results suggest that the effect of a new mutation on fitness depends less on the specific metabolic pathways disrupted and more on the pleiotropic effects of those mutations, and that organisms can explore a considerable amount of physiological space with only a few mutations.

Hells Promotes Transcription in Lymphoma By Resolving Topological Conflicts and By Preventing DNA Damage

T cell lymphomas (TCLs) are a rare and heterogeneous group of non Hodgkin Lymphoma with aggressive courses and prognoses. TCLs are characterized by genomic instability and massive transcriptional activity that is required to sustain cancer cell proliferation. Indeed, high rate of transcription increases DNA topological tension resulting in accumulation of DNA-RNA hybrid structures called R-Loops, in which the nascent RNA pairs with its DNA-template, favouring structural alterations and DNA-lesions. DNA helicases are a class of enzyme which major function is to alleviate DNA topological stresses by solving R-Loops and protecting DNA from damage. DNA helicases also facilitate RNA-PolymeraseII (RNAPII) progression along the gene body during transcription thus being the major determinant of gene expression. We recently identified the DNA-helicase HELLS - a member of the SWI/SNF2 family- as a new genetic vulnerability of TLCs. HELLS orchestrates a transcriptional program essential to the survival and proliferation of TCLs and its genetic ablation profoundly impairs mitosis and cell proliferation. To understand how HELLS manages and coordinates transcription and to define the HELLS-associated gene expression program in TCLs, we performed RNA-seq in TLBR-2 after the depletion of HELLS (HELLS KD). Gene expression profiling showed that HELLS KD affects the expression of 728 genes (413 downregulated and 315 upregulated genes). These genes were mainly involved in the regulation of cytoskeleton, cell cycle, cytokinesis, chromatin remodelling and DNA repair as indicated by a gene ontology analysis. Next, to evaluate the distribution of chromatin markers after HELLS KD, we performed Chromatin Immunoprecipitation (ChIP) sequencing against active (H3K4me3) and repressive (H3K9me3) histone markers. Depletion of HELLS results in a modest but significant change in the H3K4me3 level at promoters and distal intergenic regions. Differential analysis identified 1,571 bound sites corresponding to 1,278 genes deregulated after HELLS KD. Notably, 59/1,278 genes resulted concordantly deregulated in RNA-seq analysis. Accordingly with the role of HELLS in T lymphocytes development, gene ontology analysis on identified 59 genes reveals enrichment in T cell activation, T-helper 17 differentiation and lineage commitment processes. Instead, no significant changes in H3K9me3 level at examined regulatory regions were observed after HELLS KD, suggesting that HELLS does not modify the distribution of this marker. To deeply study the transcriptional function of HELLS, we investigated its ability to promote transcription by solving DNA topological tension. By performing S9.6 antibody staining on a panel of TCL cells, we showed that loss of HELLS leads to an accumulation of R-Loops that co-localized with the active form of RNAPII (S2-CTD), suggesting that HELLS alleviates RNAPII stall upon collision with R-loops during elongation. Co-immunoprecipitation (Co-IP) experiments showed that HELLS interacts with RNAPII and Chromatin Immunoprecipitation (ChIP) assays in TLBR-2 cells demonstrating that the loss of HELLS leads to changes in the distribution of active form of RNAPII that accumulates on transcriptional starting sites of selective target genes. By immunofluorescence staining, we detected that the decreased RNAPII activity after HELLS KD was associated with a significant decrease in the incorporation of 5-ethynyl uridine (EU) into nascent RNA confirming that transcription process is attenuated across all transcriptome. As R-loops accumulation and persistence is strictly associated with DNA damage, we assessed by immunofluorescences, the level of yH2AX (marker of DNA-damage) upon depletion of HELLS. We observed a significant increase in the intensity of nuclear yH2AX signal and a formation of yH2AX foci in TCL HELLS KD cell lines. Noticeably, accumulating yH2AX foci were observed in correspondence of R-loops and in co-localization with the active form RNAPII (S2-CTD). Collectively, our results indicate that HELLS plays an essential function in supporting TCLs progression promoting gene expression by resolving DNA topological conflicts, easing RNAPII progression and protecting DNA from damaging events simultaneously. These key functions qualify HELLS as a new dependency of TCLs and therefore as potential vulnerability of these lymphomas. Disclosures No relevant conflicts of interest to declare.

Correspondence of aCGH and long-read genome assembly for detection of copy number differences: A proof-of-concept with cichlid genomes

Copy number variation is an important source of genetic variation, yet data are often lacking due to technical limitations for detection given the current genome assemblies. Our goal is to demonstrate the extent to which an array-based platform (aCGH) can identify genomic loci that are collapsed in genome assemblies that were built with short-read technology. Taking advantage of two cichlid species for which genome assemblies based on Illumina and PacBio are available, we show that inter-species aCGH log2 hybridization ratios correlate more strongly with inferred copy number differences based on PacBio-built genome assemblies than based on Illumina-built genome assemblies. With regard to inter-species copy number differences of specific genes identified by each platform, the set identified by aCGH intersects to a greater extent with the set identified by PacBio than with the set identified by Illumina. Gene function, according to Gene Ontology analysis, did not substantially differ among platforms, and platforms converged on functions associated with adaptive phenotypes. The results of the current study further demonstrate that aCGH is an effective platform for identifying copy number variable sequences, particularly those collapsed in short read genome assemblies.

Stress Granules Involved in Formation, Progression and Metastasis of Cancer: A Scoping Review

The assembly of stress granules (SGs) is a well-known cellular strategy for reducing stress-related damage and promoting cell survival. SGs have become important players in human health, in addition to their fundamental role in the stress response. The critical role of SGs in cancer cells in formation, progression, and metastasis makes sense. Recent researchers have found that several SG components play a role in tumorigenesis and cancer metastasis via tumor-associated signaling pathways and other mechanisms. Gene-ontology analysis revealed the role of these protein components in the structure of SGs. Involvement in the translation process, regulation of mRNA stability, and action in both the cytoplasm and nucleus are among the main features of SG proteins. The present scoping review aimed to consider all studies on the effect of SGs on cancer formation, proliferation, and metastasis and performed based on a six-stage methodology structure and the PRISMA guideline. A systematic search of seven databases for qualified articles was conducted before July 2021. Publications were screened, and quantitative and qualitative analysis was performed on the extracted data. Go analysis was performed on seventy-one SGs protein components. Remarkably G3BP1, TIA1, TIAR, and YB1 have the largest share among the proteins considered in the studies. Altogether, this scoping review tries to demonstrate and provide a comprehensive summary of the role of SGs in the formation, progression, and metastasis of cancer by reviewing all studies.

Dorso-ventral heterogeneity in tracheal basal stem cells

The tracheal basal cells (BCs) function as stem cells to maintain the epithelium in steady state and repair it after injury. The airway is surrounded by cartilage ventrolaterally and smooth muscle dorsally. Lineage tracing using Krt5-CreER shows dorsal BCs produce more, larger, clones than ventral BCs. Large clones were found between cartilage and smooth muscle where subpopulation of dorsal BCs exists. Three-dimensional organoid culture of BCs demonstrated that dorsal BCs show higher colony forming efficacy to ventral BCs. Gene ontology analysis revealed that genes expressed in dorsal BCs are enriched in wound healing while ventral BCs are enriched in response to external stimulus and immune response. Significantly, ventral BCs express Myostatin, which inhibits the growth of smooth muscle cells, and HGF, which facilitates cartilage repair. The results support the hypothesis that BCs from the dorso-ventral airways have intrinsic molecular and behavioural differences relevant to their in vivo function.

Polyubiquitin gene Ubb is required for upregulation of Piwi protein level during mouse testis development

Testis development, including early embryonic gonad formation and late postnatal spermatogenesis, is essential for the reproduction of higher metazoans to generate fertile gametes, called sperm. We have previously reported that the polyubiquitin gene Ubb is required for fertility in both male and female mice. In particular, the Ubb-null male mice showed an azoospermia phenotype due to arrest of spermatogenesis at the pachytene stage. Here, we analyzed the whole testis proteome at postnatal day 20 to define the molecular mediators of the male-infertility phenotype caused by Ubb knockout. From the identified proteome, 564 proteins were significantly and differentially expressed in Ubb-knockout testes and, among these, 36 downregulated proteins were involved at different stages of spermatogenesis. We also found that levels of piRNA metabolic process-related proteins, including Piwil2 and Tdrd1, were downregulated in Ubb-null testes through functional gene ontology analysis. Further, protein–protein interaction mapping revealed that 24 testis development-related proteins, including Hsp90aa1, Eef1a1, and Pabpc1, were directly influenced by the depletion of ubiquitin. In addition, the reduced mRNA levels of these proteins were observed in Ubb-knockout testes, which closely resembled the global downregulation of piRNA-metabolic gene expression at the transcriptional and post-transcriptional levels. Together with proteomic and transcriptional analyses, our data suggest that Ubb expression is essential for the maintenance of testicular RNA-binding regulators and piRNA-metabolic proteins to complete spermatogenesis in mice.