Transcriptome Assembly and Molecular Evolutionary Analysis of Sex-Biased Genes in the Guppy (Poecilia reticulata)

It is a phenomenon universally seen that males and females of a species show phenotypic differences as they evolve under often diverging sex-specific selection pressures. The evolution and maintenance of their sexual dimorphism is generally associated with gene expression divergence between the sexes. Genes that show enriched expression in one sex, also called sex-biased genes, often show rapid molecular evolution. Furthermore, sex-biased genes have also been found to be over-represented on X or Z chromosomes in several species with differentiated sex chromosomes or neo-sex chromosomes. While research on sex-biased genes in drosophilids, mammals and birds has developed in the last decade, there is relatively little known about sex-biased genes in teleost species with largely undifferentiated sex-chromosomes of recent origin. A case in point is that of the Trinidadian guppy, Poecilia reticulata, which is the focal species of my thesis. In this dissertation, I investigate sex-biased gene expression in guppy, a fresh-water fish with XY sex-determination and Y-linked inheritance of male-advantageous traits. Guppies display sexual dimorphism in size, ornaments, and behavior, traits that are shaped by both natural and sexual selection in the wild. My first task was to assemble a transcriptome reference using deep sequencing of cDNA. I compared several methods of assembly with RNA sequencing (RNA-seq) data and assembled and annotated a reference transcriptome combining a genome-independent and a genome-guided assembly. Subsequently, I analyzed sex-biased gene expression in brain, tail and gonads, tissues with overt sexual dimorphism in adult guppies. I found tissue-specific expression generally related to the phenotypic sexual dimorphism. For example, genes related to signal transduction, pigmentation processes and spermatogenesis were expressed more in males; while female-biased genes related to growth, cell-division, extra-cellular matrix organization, nutrient transport, and folliculogenesis. As male sex-determination and differentiation in guppies is believed to be associated with the male-specific pigment patterns, I analyzed the gene-expression and genomic locations of guppy orthologs of candidate genes functional in these processes in other vertebrates. The list of candidate genes could be specifically aligned to the female genome and no male-limited candidate could be identified. I found tissue-specificity in the magnitude and direction of sex-bias in the expression of several sex-related and pigmentation genes. I then studied the genomic distribution of all sex-biased genes. I observed the accumulation of ovary-biased genes on the putative sex linkage group, LG12. Genome-wide comparison of rates of evolution of sex-biased and unbiased genes, measured by the ratio of non-synonymous substitution rate (dN) to the synonymous substitution rate (dS), indicated faster evolution of testis-biased genes, and female-biased genes in all three studied tissues. Among these, the female-biased genes in brain showed elevated ratios of non-synonymous substitutions irrespective of the breadth and magnitude of expression. In this study, I describe a comprehensive annotated guppy reference transcriptome that is compiled after extensive evaluation of different existing methods for assembly using de novo strategies as well as reference-guided strategies. The reference transcriptome of the guppy provides a resource for investigating the molecular genetics of the guppy’s complex adaptive traits. The methods and pipelines are generally applicable for developing and utilizing transcriptomic resources in organisms with limited molecular resources. Genome-wide differential expression between male and female tissues, allowed us to identify genes with strong characteristic differential expression in the differentiated gonads as well as genes with small but significant expression differences in the somatic tissues. These sets of sex-biased genes may be relevant for the tissue-associated sexual dimorphism. Differential genomic distributions of ovary- and testis-biased genes provide evidence for sex-specific selection pressures acting on the slightly differentiated sex chromosomes of the guppy. Elevated rates of molecular evolution observed in testis-biased and all categories of female-biased genes suggest evolution under distinct selection pressures on the reproductive versus non-reproductive tissues. Overall, these results are useful for guppy researchers and for further understanding the evolution of sex differences in diverse species

Virtual screening, identification and experimental testing of novel inhibitors of PBEF1/Visfatin/NMPRTase for glioma therapy

Background: Pre-B-cell colony enhancing factor 1 gene (PBEF1) encodes nicotinamide phosphoribosyltransferase (NMPRTase), which catalyses the rate limiting step in the salvage pathway of NAD+ metabolism in mammalian cells. PBEF1 transcript and protein levels have been shown to be elevated in glioblastoma and a chemical inhibitor of NMPRTase has been shown to specifically inhibit cancer cells. Methods: Virtual screening using docking was used to screen a library of more than 13,000 chemical compounds. A shortlisted set of compounds were tested for their inhibition activity in vitro by an NMPRTase enzyme assay. Further, the ability of the compounds to inhibit glioma cell proliferation was carried out. Results: Virtual screening resulted in short listing of 34 possible ligands, of which six were tested experimentally, using the NMPRTase enzyme inhibition assay and further with the glioma cell viability assays. Of these, two compounds were found to be significantly efficacious in inhibiting the conversion of nicotinamide to NAD+, and out of which, one compound, 3-amino-2-benzyl-7-nitro-4-(2-quinolyl-)-1,2-dihydroisoquinolin-1-one, was found to inhibit the growth of a PBEF1 over expressing glioma derived cell line U87 as well. Conclusions: Thus, a novel inhibitor has been identified through a structure based drug discovery approach and is further supported by experimental evidence

Protective Role of Herbal Drugs in Diabetic Neuropathy: An Updated Review

Medicinal plants play a beneficial role in health care and are commonly used in preventing and testing diseases and specific ailments. The advantage associated with herbals plants are numerous and cannot be ignored as they have less adherence issues and are accepted widely by the population due to greater belief in Ayurveda since ancient times. Neuropathic pain has immersed as a serious threat to patient that occurs by damaging the blood vessels leading to morbidity and mortality. The present review paper aims in providing an account of various herbal plants that could be employed in treatment of neuropathic pain

Short and long-read genome sequencing methodologies for somatic variant detection; genomic analysis of a patient with diffuse large B-cell lymphoma

Recent advances in throughput and accuracy mean that the Oxford Nanopore Technologies PromethiON platform is a now a viable solution for genome sequencing. Much of the validation of bioinformatic tools for this long-read data has focussed on calling germline variants (including structural variants). Somatic variants are outnumbered many-fold by germline variants and their detection is further complicated by the effects of tumour purity/subclonality. Here, we evaluate the extent to which Nanopore sequencing enables detection and analysis of somatic variation. We do this through sequencing tumour and germline genomes for a patient with diffuse B-cell lymphoma and comparing results with 150 bp short-read sequencing of the same samples. Calling germline single nucleotide variants (SNVs) from specific chromosomes of the long-read data achieved good specificity and sensitivity. However, results of somatic SNV calling highlight the need for the development of specialised joint calling algorithms. We find the comparative genome-wide performance of different tools varies significantly between structural variant types, and suggest long reads are especially advantageous for calling large somatic deletions and duplications. Finally, we highlight the utility of long reads for phasing clinically relevant variants, confirming that a somatic 1.6 Mb deletion and a p.(Arg249Met) mutation involving TP53 are oriented in trans

Distinct regulation of hippocampal neuroplasticity and ciliary genes by corticosteroid receptors

Glucocorticoid hormones (GCs) are of critical importance for maintaining brain health, but their involvement in mental disorders is poorly understood. Here the authors show how GCs act through hippocampal mineralocorticoid and glucocorticoid receptors to impact the gene regulatory programs underpinning neuronal plasticity, ciliogenesis and behavioral adaptation

Early detection of doxorubicin-induced cardiotoxicity in rats by its cardiac metabolic signature assessed with hyperpolarized MRI.

Doxorubicin (DOX) is a widely used chemotherapeutic agent that can cause serious cardiotoxic side effects culminating in congestive heart failure (HF). There are currently no clinical imaging techniques or biomarkers available to detect DOX-cardiotoxicity before functional decline. Mitochondrial dysfunction is thought to be a key factor driving functional decline, though real-time metabolic fluxes have never been assessed in DOX-cardiotoxicity. Hyperpolarized magnetic resonance imaging (MRI) can assess real-time metabolic fluxes in vivo. Here we show that cardiac functional decline in a clinically relevant rat-model of DOX-HF is preceded by a change in oxidative mitochondrial carbohydrate metabolism, measured by hyperpolarized MRI. The decreased metabolic fluxes were predominantly due to mitochondrial loss and additional mitochondrial dysfunction, and not, as widely assumed hitherto, to oxidative stress. Since hyperpolarized MRI has been successfully translated into clinical trials this opens up the potential to test cancer patients receiving DOX for early signs of cardiotoxicity

Exploring potato seed research: a bibliometric approach towards sustainable food security

IntroductionPotato is considered to be complete food that will not only ensure food security but also alleviate poverty. Seed production of potato requires specific temperatures and conditions. In response to the growing emphasis on sustainable production, there has been an increasing focus on research on tuber seed production.MethodsIn our study, we have employed bibliometric analysis to investigate the trends in potato seed research and assess its correlation with sustainable development. Tabular analysis and network analysis are employed in the study to understand the prominent authors and institutions and research trends across time. For this purpose, Biblioshiny and Vosviewer software were used. The steps of bibliometric analysis were used, which included data retrieval from Dimensions software. Owing to its limitations, a major analysis was conducted without affecting the results.Result and discussionIt was found from the analysis that it was SDG 2 that was mostly linked with the theme of potato seed production. The results depicted an increasing trend of publications and citations. Co-authorship analysis of authors showed high linkage among groups of authors that formed clusters while other authors remained disconnected. Among countries United States, China and the United Kingdom had a higher impact on publications and citations. Our analysis showed that there is still scope for collaboration among countries as there is no evidence of multidisciplinary interlinkages. By understanding the current research landscape, identifying influential works and authors, and uncovering collaboration patterns, we can pave the way for future advancements in potato seed production. Ultimately, this research contributes to achieving sustainable agriculture and ensuring food security for future generations

Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations.

SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology

The genome of the Trinidadian guppy, Poecilia reticulata, and variation in the Guanapo population

For over a century, the live bearing guppy, Poecilia reticulata, has been used to study sexual selection as well as local adaptation. Natural guppy populations differ in many traits that are of intuitively adaptive significance such as ornamentation, age at maturity, brood size and body shape. Water depth, light supply, food resources and predation regime shape these traits, and barrier waterfalls often separate contrasting environments in the same river. We have assembled and annotated the genome of an inbred single female from a high-preda- tion site in the Guanapo drainage. The final assembly comprises 731.6 Mb with a scaffold N50 of 5.3 MB. Scaffolds were mapped to linkage groups, placing 95% of the genome assembly on the 22 autosomes and the X-chromosome. To investigate genetic variation in the population used for the genome assembly, we sequenced 10 wild caught male individu- als. The identified 5 million SNPs correspond to an average nucleotide diversity (π) of 0.0025. The genome assembly and SNP map provide a rich resource for investigating adap- tation to different predation regimes. In addition, comparisons with the genomes of other Poeciliid species, which differ greatly in mechanisms of sex determination and maternal resource allocation, as well as comparisons to other teleost genera can begin to reveal how live bearing evolved in teleost fish