WormCat 2.0 defines characteristics and conservation of poorly annotated genes in Caenorhabditis elegans

Genome-wide measurement of mRNA or protein levels provides broad data sets for biological discovery. However, subsequent computational methods are essential for uncovering the functional implications of the data as well as intuitively visualizing the findings. Current computational tools are biased toward well-described pathways, limiting their utility for novel discovery. Recently, we developed an annotation and category enrichment tool for Caenorhabditis elegans genomic data, WormCat, that provides an intuitive visualization output. Unlike GO, which excludes genes with no annotation information retains these genes as a special UNASSIGNED category. Here, we show that the UNASSIGNED gene category shows tissue-specific expression patterns and include genes with biological functions. Poorly annotated genes have previously been considered to lack homologs in closely related species. Instead, we find that around 3% of the UNASSIGNED genes have poorly characterized human orthologs. These human orthologs are themselves poorly characterized. A recently developed method that incorporates lineage relationships (abSENSE) indicates that failure of BLAST to detect homology explains the apparent lineage specificity for many UNASSIGNED genes, suggesting that a larger subset could be related to human genes. WormCat provides an annotation strategy that allows association of UNASSIGNED genes with specific phenotypes and known pathways. Our analysis indicates that the UNASSIGNED gene category contains candidates that merit further functional study which could yield insight into understudied areas of biology.

X-linked palindromic gene families 4930567H17Rik and Mageb5 are dispensable for male mouse fertility

Mammalian sex chromosomes are enriched for large, nearly-identical, palindromic sequences harboring genes expressed predominately in testicular germ cells. Discerning if individual palindrome-associated gene families are essential for male reproduction is difficult due to challenges in disrupting all copies within a gene family. Here we generate precise, independent, deletions to assess the reproductive roles of two X-linked palindromic gene families with spermatid-predominant expression, 4930567H17Rik or Mageb5. Via sequence comparisons, we find mouse 4930567H17Rik and Mageb5 have human orthologs, 4930567H17Rik is rapidly diverging in rodents and primates, and 4930567H17Rik is harbored in a palindrome in humans and mice, while Mageb5 is not. Mice lacking either 4930567H17Rik or Mageb5 gene families do not have detectable defects in male fertility, fecundity, spermatogenesis, or in gene regulation, but do show differences in sperm head morphology, suggesting a potential role in sperm function. We conclude that while all palindrome-associated gene families are not essential for male fertility, large palindromes influence the evolution of their associated gene families.

Loxl2 is a mediator of cardiac aging in Drosophila melanogaster; genetically examining the role of aging clock genes

Transcriptomic, proteomic, and methylation aging clocks demonstrate that aging has a predictable preset program, while Transcriptome Trajectory Turning Points indicate that the 20 to 40 age range in humans is the likely stage at which the progressive loss of homeostatic control, and in turn aging, begins to have detrimental effects. Turning points in this age range overlapping with human aging clock genes revealed five candidates that we hypothesized could play a role in aging or age-related physiological decline. To examine these gene’s effects on lifespan and health-span, we utilized whole body and heart specific gene knockdown of human orthologs in Drosophila melanogaster. Whole body Loxl2, fz3, and Glo1 RNAi positively affected lifespan as did heart-specific Loxl2 knockdown. Loxl2 inhibition concurrently reduced age-related cardiac arrythmia and collagen (Pericardin) fiber width. Loxl2 binds several transcription factors in humans and RT-qPCR confirmed that a conserved transcriptional target CDH1 (Drosophila CadN2), has expression levels which correlate with Loxl2 reduction in Drosophila. These results point to conserved pathways and multiple mechanisms by which inhibition of Loxl2 can be beneficial to heart health and organismal aging.

Changes in the urinary proteome in rats with regular swimming exercise

Purpose Urine can sensitively reflect early pathophysiological changes in the body. The purpose of this study was to explore the changes of urine proteome in rats with regular swimming exercise. Methods In this study, experimental rats were subjected to daily moderate-intensity swimming exercise for 7 weeks. Urine samples were collected at weeks 2, 5, and 7 and were analyzed by using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Results Unsupervised clustering analysis of all urinary proteins identified at week 2 showed that the swimming group was distinctively different from the control group. Compared to the control group, a total of 112, 61 and 44 differential proteins were identified in the swimming group at weeks 2, 5 and 7, respectively. Randomized grouping statistical analysis showed that more than 85% of the differential proteins identified in this study were caused by swimming exercise rather than random allocation. According to the Human Protein Atlas, the differential proteins that have human orthologs were strongly expressed in the liver, kidney and intestine. Functional annotation analysis revealed that these differential proteins were involved in glucose metabolism and immunity-related pathways. Conclusion Our results revealed that the urinary proteome could reflect significant changes after regular swimming exercise. These findings may provide an approach to monitor the effects of exercise of the body.

Progress towards completing the mutant mouse null resource

The generation of a comprehensive catalog of null alleles covering all protein-coding genes is the goal of the International Mouse Phenotyping Consortium. Over the past 20 years, significant progress has been made towards achieving this goal through the combined efforts of many large-scale programs that built an embryonic stem cell resource to generate knockout mice and more recently employed CRISPR/Cas9-based mutagenesis to delete critical regions predicted to result in frameshift mutations, thus, ablating gene function. The IMPC initiative builds on prior and ongoing work by individual research groups creating gene knockouts in the mouse. Here, we analyze the collective efforts focusing on the combined null allele resource resulting from strains developed by the research community and large-scale production programs. Based upon this pooled analysis, we examine the remaining fraction of protein-coding genes focusing on clearly defined mouse–human orthologs as the highest priority for completing the mutant mouse null resource. In summary, we find that there are less than 3400 mouse–human orthologs remaining in the genome without a targeted null allele that can be further prioritized to achieve our overall goal of the complete functional annotation of the protein-coding portion of a mammalian genome.

Drosophila: A model to study the pathogenesis of Parkinson’s disease

: Human central nervous system (CNS) is the complex part of the human body, which regulates multiple cellular and molecular events taking place simultaneously. Parkinsons disease (PD) is the second most common neurodegenerative disease after Alzheimer’s disease (AD). The pathological hallmarks of PD are loss of dopaminergic neurons in the substantianigra (SN) pars compacta (SNpc) and accumulation of misfolded α-synuclein, in intra-cytoplasmic inclusions called Lewy bodies (LBs). So far, there is no cure for PD, due to the complexities of molecular mechanisms and events taking place during the pathogenesis of PD. Drosophila melanogaster is an appropriate model organism to unravel the pathogenicity not only behind PD but also other NDs. In this context as numerous biological functions are preserved between Drosophila and humans. Apart from sharing 75% of human disease-causing genes homolog in Drosophila, behavioral responses like memory-based tests, negative geotaxis, courtship and mating are also well studied. The genetic, as well as environmental factors, can be studied in Drosophila to understand the gene-environment interactions behind the disease condition. Through genetic manipulation, mutant flies can be generated harboring human orthologs, which can prove to be an excellent model to understand the effect of the mutant protein on the pathogenicity of NDs.

#23: Investigation of Phosphomannomutase as an Antimalarial Drug Target

Background Malaria continues to pose an enormous economic and global health threat, killing over 200,000 people annually, primarily children under the age of 5. With the constant barrier of antimalarial resistance and the rise of delayed clearance by artemisinin, it is especially important to identify drug/target pairs that rapidly kill parasites. We study targetable metabolic pathways in the malaria parasite, Plasmodium falciparum, to guide such future drug development against this disease. In recent years, we have discovered that a large family of hydrolases, the Haloacid Dehalogenase (HAD) Superfamily of proteins, are implicated in regulating a variety of P. falciparum metabolic pathways, which can lead to dramatic changes in central carbon metabolism and drug resistance. We now turn our attention to a related HAD protein, the putative phosphomannomutase in these parasites, HAD5, responsible for the interconversion of mannose-6-phosphate and mannose-1-phosphate. This is an essential process for all stages of the parasite, and thus has potential as a broad antimalarial target. We examined the role of HAD5 in these parasites, and its potential to be chemically inhibited. Methods Recombinant protein was generated and purified for enzymatic assays to determine HAD5 activity and test inhibitor potency against HAD5 compared to recombinant human orthologs, PMM1 and PMM2. In parallel, CRISPR/Cas9 was used to generate inducible knockdown parasite strains to demonstrate this gene’s essentiality and its role in parasite biology. Parasite growth was measured by flow cytometry and light microscopy. Immunofluorescence analysis (IFA) was used to track the parasite development on a molecular scale. Results Inhibition of HAD5 was achieved in biochemical assays, with an IC50 of 68µM in our most potent compound, representing roughly 10-fold increased potency against the parasite protein compared to human orthologs. In culture, knockdown of HAD5 leads to interrupted egress from and reinvasion into red blood cells, culminating in parasite death. In IFA-visualized parasites, reinvasion-facilitating proteins were no longer anchored to parasite surfaces, accounting for the inhibition of the parasite life cycle. Conclusion In the search for new antimalarial targets, identifying proteins that are essential across multiple parasite life-stages while being distinct from human orthologs is necessary to block parasite transmission, cure symptomatic infection, and minimize off-target effects. HAD5 is an essential protein in malaria parasites that is expressed throughout the parasite’s life cycle, and can be specifically targeted by inhibitors, giving it promise as a future drug target.

Changes in the urinary proteome in rats with regular swimming exercise

Purpose: Urine can sensitively reflect early pathophysiological changes in the body. The purpose of this study was to determine whether the urine proteome could reflect changes in regular swimming exercise. Methods: In this study, experimental rats were subjected to daily moderate-intensity swimming exercise for 7 weeks. Urine samples were collected at weeks 2, 5, and 7 and were analyzed by using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Results: Unsupervised clustering analysis of all urinary proteins identified at week 2 showed that the swimming group was distinctively different from the control group. Compared to the control group, a total of 112, 61 and 44 differential proteins were identified in the swimming group at weeks 2, 5 and 7, respectively. Randomized grouping statistical analysis showed that more than 85% of the differential proteins identified in this study were caused by swimming exercise rather than random allocation. According to the Human Protein Atlas, the differential proteins that have human orthologs were strongly expressed in the liver, kidney and intestine. Functional annotation analysis revealed that these differential proteins were involved in glucose metabolism and immunity-related pathways. Conclusion: Our results revealed that the urinary proteome could reflect significant changes following regular swimming exercise. These findings may suggest an approach to monitoring whether the amount of exercise is appropriate.

Sperm cryopreservation impacts the early development of equine embryos by downregulating specific transcription factors

Equine embryos were obtained by insemination with either fresh or frozen-thawed spermatozoa at 8, 10 and 12 h post spontaneous ovulation, maintaining the pairs mare-stallion for the type of semen used. Next generation sequencing (NGS) was performed in all embryos and bioinformatic and enrichment analysis performed on the 21,058 identified transcripts. A total of 165 transcripts were downregulated in embryos obtained with cryopreserved spermatozoa respect embryos resulting from an insemination with fresh spermatozoa (p=0.021, q=0.1). The enrichment analysis using human orthologs using g:profiler on the downregulated transcripts marked an enrichment in transcription factors (TFs) in mRNAs downregulated in embryos obtained after insemination with cryopreserved spermatozoa. The 12 mRNAs (discriminant variables) most significantly downregulated in these embryos included among others, the chromatin-remodeling ATPase INO80, Lipase maturation factor 1 LMF1, the mitochondrial mRNA pseudouridine synthase RPUSD3, LIM and cysteine-rich domains protein 1, LMCD1. Sperm cryopreservation also caused a significant impact on the embryos at 8 to 10 days of development, but especially in the transition from 10 to 12 days. Overall, our findings provide strong evidence that insemination with cryopreserved spermatozoa poses a major impact in embryo development that may compromise its growth and viability, probably due to modifications in sperm proteins induced by cryopreservation. Identification of specific factors in the spermatozoa causing these changes may improve cryopreservation.

Functional conservation in genes and pathways linking ageing and immunity

At first glance, longevity and immunity appear to be different traits that have not much in common except the fact that the immune system promotes survival upon pathogenic infection. Substantial evidence however points to a molecularly intertwined relationship between the immune system and ageing. Although this link is well-known throughout the animal kingdom, its genetic basis is complex and still poorly understood. To address this question, we here provide a compilation of all genes concomitantly known to be involved in immunity and ageing in humans and three well-studied model organisms, the nematode worm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the house mouse Mus musculus. By analysing human orthologs among these species, we identified 7 evolutionarily conserved signalling cascades, the insulin/TOR network, three MAPK (ERK, p38, JNK), JAK/STAT, TGF-β, and Nf-κB pathways that act pleiotropically on ageing and immunity. We review current evidence for these pathways linking immunity and lifespan, and their role in the detrimental dysregulation of the immune system with age, known as immunosenescence. We argue that the phenotypic effects of these pathways are often context-dependent and vary, for example, between tissues, sexes, and types of pathogenic infection. Future research therefore needs to explore a higher temporal, spatial and environmental resolution to fully comprehend the connection between ageing and immunity.