Molecular Evolution of Small Peptide Hormones and Their Receptors: the Case of Relaxin and Insulin-like Peptide Signaling Systems in Deuterostomes

Parts of the research presented in this thesis have appeared in two peer reviewed publications: 1. Good, S., S. Yegorov, J. Martijn, J. Franck and J. Bogerd (2012) New Insights into Ligand-Receptor Pairing and Coevolution of Relaxin Family Peptides and their Receptors in Teleosts. International Journal of Evolutionary Biology. http://dx.doi.org/10.1155/2012/310278 . 2. Yegorov, S and Good, S.V. (2012) Using Ancestral Genome Reconstructions to study the Evolution of Ligand- Receptor Systems: the Case of Relaxin Hormones and Their Receptors. Plos One. http://dx.doi.org/10.1371/journal.pone.0032923Relaxin family peptides are a diverse family of signalling molecules that play important roles in the regulation of reproductive and neuroendocrine processes in vertebrates. The signalling of relaxin peptides is mediated by G protein-coupled receptors of two distinct classes, small peptide receptors and leucine-rich repeat-containing receptors. The origins and evolutionary history of both relaxin family peptides and their receptors have been a matter of debate for several reasons, among which the small size of peptide molecules (~ 60 aa, often providing insufficient information for phylogenetic reconstructions) and low coverage of vertebrate taxa by functional studies have been most prominent. In this study, I combined traditional bioinformatic approaches with ancestral genome reconstructions to reassess some of the debated aspects of the evolution of relaxin peptides and their receptors. To cover a broad range of taxa, I performed thorough data mining of the focal genes in 29 publicly available genome databases of both vertebrate and invertebrate deuterostomes. Ancestral genome reconstruction-based analyses provided clear evidence for the strong influence of whole genome duplications (WGDs) on the diversification of the relaxin signaling system from a tripartite system, consisting of one hormone and two receptor-encoding genes in the vertebrate ancestor, to the present day system. The results presented here indicate that relaxin family peptide systems are more diverse than previously thought, in particular with respect to the number of genes present in different vertebrate lineages. Based on the duplication model presented here, I propose that the ancestral tripartite signaling system had a dual function which was partitioned after the first round of WGD such that two sets of ligand-receptor pairs subfunctionalized into predominantly neuroendocrine- or reproductive-focused functions. My further analyses indicated that the suite of four ligand-receptor pairs common to the majority of modern mammals and teleosts, and already present in their gnathostome ancestor, have mostly evolved under similar selection pressures, suggesting a similar function of the genes across vertebrates. However, there are some distinct patterns of selection and evidence of differential codon-specific selection in mammals versus teleosts. Lastly, the reconstruction of the ancestral states of relaxin family peptides demonstrates how the ancestral structure shared by all four peptides has changed over time and in different lineages to acquire the specific structural characteristics of the peptides that we are familiar with today. Overall, by creating an evolutionary framework for future analyses, this study should facilitate further investigation into the properties of relaxin family peptides and their receptors.1-the Province of Manitoba 2-University of Winnipeg 3-Natural Sciences and Engineering Research Council of CanadaMaster of Science in Bioscience, Technology and Public Polic

Effects of Vitamin D Supplementation and Seasonality on Circulating Cytokines in Adolescents: Analysis of Data From a Feasibility Trial in Mongolia

Vitamin D deficiency is prevalent in human populations and has been linked to immune dysfunction. Here we explored the effects of cholecalciferol supplementation on circulating cytokines in severely vitamin D deficient [blood 25(OH)D << 30 nmol/L] adolescents aged 12-15 from Mongolia. The study included 28 children receiving 800 IU daily cholecalciferol for 6 months spanning winter and spring, and 30 children receiving placebo during the same period. The levels of 25(OH)D were assessed at baseline, 3 and 6 months. Twenty-one cytokines were measured in serum at baseline and at 6 months. Changes in 25(OH)D and cytokines were assessed using paired parametric tests. The median blood 25(OH)D concentration at baseline was 13.7 nmol/L (IQR = 10.0-21.7). Supplementation tripled blood 25(OH)D levels (p < 0.001) and was associated with elevated interleukin (IL)-6 (p = 0.043). The placebo group had reduced macrophage inflammatory protein (MIP)-1 alpha (p = 0.007) and IL-8 (p = 0.034) at 6 months. Although limited by a small sample size, these findings suggest that cholecalciferol supplementation and seasonality may impact systemic immunity in adolescents, identifying chemokines as potentially important biomarkers of vitamin D status in this Northeast Asian population. Larger clinical trials are warranted to validate these results

Relaxin gene family in teleosts: phylogeny, syntenic mapping, selective constraint, and expression analysis

<p>Abstract</p> <p>Background</p> <p>In recent years, the relaxin family of signaling molecules has been shown to play diverse roles in mammalian physiology, but little is known about its diversity or physiology in teleosts, an infraclass of the bony fishes comprising ~ 50% of all extant vertebrates. In this paper, 32 relaxin family sequences were obtained by searching genomic and cDNA databases from eight teleost species; phylogenetic, molecular evolutionary, and syntenic data analyses were conducted to understand the relationship and differential patterns of evolution of relaxin family genes in teleosts compared with mammals. Additionally, real-time quantitative PCR was used to confirm and assess the tissues of expression of five relaxin family genes in <it>Danio rerio </it>and <it>in situ </it>hybridization used to assess the site-specific expression of the insulin 3-like gene in <it>D. rerio </it>testis.</p> <p>Results</p> <p>Up to six relaxin family genes were identified in each teleost species. Comparative syntenic mapping revealed that fish possess two paralogous copies of human <it>RLN3</it>, which we call <it>rln3a </it>and <it>rln3b</it>, an orthologue of human <it>RLN2</it>, <it>rln</it>, two paralogous copies of human <it>INSL5</it>, <it>insl5a and insl5b</it>, and an orthologue of human <it>INSL3</it>, <it>insl3</it>. Molecular evolutionary analyses indicated that: <it>rln3a, rln3b </it>and <it>rln </it>are under strong evolutionary constraint, that <it>insl3 </it>has been subject to moderate rates of sequence evolution with two amino acids in <it>insl3/INSL3 </it>showing evidence of positively selection, and that <it>insl5b </it>exhibits a higher rate of sequence evolution than its paralogue <it>insl5a </it>suggesting that it may have been neo-functionalized after the teleost whole genome duplication. Quantitative PCR analyses in <it>D. rerio </it>indicated that <it>rln3a </it>and r<it>ln3b </it>are expressed in brain, <it>insl3 </it>is highly expressed in gonads, and that there was low expression of both <it>insl5 </it>genes in adult zebrafish. Finally, <it>in situ </it>hybridization of <it>insl3 </it>in <it>D. rerio </it>testes showed highly specific hybridization to interstitial Leydig cells.</p> <p>Conclusions</p> <p>Contrary to previous studies, we find convincing evidence that teleosts contain orthologues of four relaxin family peptides. Overall our analyses suggest that in teleosts: 1) <it>rln3 </it>exhibits a similar evolution and expression pattern to mammalian <it>RLN3</it>, 2) <it>insl3 </it>has been subject to positive selection like its mammalian counterpart and shows similar tissue-specific expression in Leydig cells, 3) <it>insl5 </it>genes are highly represented and have a relatively high rate of sequence evolution in teleost genomes, but they exhibited only low levels of expression in adult zebrafish, 4) <it>rln </it>is evolving under very different selective constraints from mammalian <it>RLN</it>. The results presented here should facilitate the development of hypothesis-driven experimental work on the specific roles of relaxin family genes in teleosts.</p

SPUTNIK-V REACTOGENICITY AND IMMUNOGENICITY IN THE BLOOD AND MUCOSA: A PROSPECTIVE COHORT STUDY

Sputnik-V (Gam-COVID-Vac) is a heterologous, recombinant adenoviral (rAdv) vector-based, COVID- 19 vaccine now used in > 70 countries. Yet there is a shortage of data on this vaccine’s performance in diverse populations. Here, we performed a prospective cohort study to assess the reactogenicity and immunologic outcomes of Sputnik-V vaccination in Kazakhstan. COVID-19-free participants (n = 82 at baseline) were followed at day 21 after Sputnik-V dose 1 (rAd5) and dose 2 (rAd26). Self-reported local and systemic adverse events were captured using questionnaires. Blood and nasopharyngeal swabs were collected to perform SARS-CoV-2 diagnostic and immunologic assays. We observed that most of the reported adverse events were mild-to-moderate injection site or systemic reactions, no severe or potentially life-threatening conditions were reported, and dose 1 appeared to be more reactogenic than dose 2. The seroconversion rate was 97% post-dose 1, remaining the same post-dose 2. The proportion of participants with detectable virus neutralization was 83% post-dose 1, increasing to 98% post-dose 2, with the largest relative increase observed in participants without prior COVID- 19 exposure. Dose 1 boosted nasal S-IgG and S-IgA, while the boosting effect of dose 2 on mucosal S-IgG, but not S-IgA, was only observed in subjects without prior COVID-19. Systemically, vaccination reduced serum levels of growth regulated oncogene (GRO), which correlated with an elevation in blood platelet count. Overall, Sputnik-V dose 1 elicited both blood and mucosal SARS-CoV-2 immunity, while the immune boosting effect of dose 2 was minimal. Thus, adjustments to the current vaccine dosing regimen are necessary to optimize immunization efficacy and cost-effectiveness. While Sputnik-V reactogenicity is similar to that of other COVID-19 vaccines, the induced alterations to the GRO/platelet axis warrant investigation of the vaccine’s effects on systemic immunology

PSORIASIS IS ASSOCIATED WITH ELEVATED GUT IL-1Α AND INTESTINAL MICROBIOME ALTERATIONS

Psoriasis is a chronic inflammatory condition that predominantly affects the skin and is associated with extracutaneous disorders, such as inflammatory bowel disease and arthritis. Changes in gut immunology and microbiota are important drivers of proinflammatory disorders and could play a role in the pathogenesis of psoriasis. Therefore, we explored whether psoriasis in a Central Asian cohort is associated with alterations in select immunological markers and/or microbiota of the gut

Using Paleogenomics to Study the Evolution of Gene Families: Origin and Duplication History of the Relaxin Family Hormones and Their Receptors

Recent progress in the analysis of whole genome sequencing data has resulted in the emergence of paleogenomics, a field devoted to the reconstruction of ancestral genomes. Ancestral karyotype reconstructions have been used primarily to illustrate the dynamic nature of genome evolution. In this paper, we demonstrate how they can also be used to study individual gene families by examining the evolutionary history of relaxin hormones (RLN/INSL) and relaxin family peptide receptors (RXFP). Relaxin family hormones are members of the insulin superfamily, and are implicated in the regulation of a variety of primarily reproductive and neuroendocrine processes. Their receptors are G-protein coupled receptors (GPCR's) and include members of two distinct evolutionary groups, an unusual characteristic. Although several studies have tried to elucidate the origins of the relaxin peptide family, the evolutionary origin of their receptors and the mechanisms driving the diversification of the RLN/INSL-RXFP signaling systems in non-placental vertebrates has remained elusive. Here we show that the numerous vertebrate RLN/INSL and RXFP genes are products of an ancestral receptor-ligand system that originally consisted of three genes, two of which apparently trace their origins to invertebrates. Subsequently, diversification of the system was driven primarily by whole genome duplications (WGD, 2R and 3R) followed by almost complete retention of the ligand duplicates in most vertebrates but massive loss of receptor genes in tetrapods. Interestingly, the majority of 3R duplicates retained in teleosts are potentially involved in neuroendocrine regulation. Furthermore, we infer that the ancestral AncRxfp3/4 receptor may have been syntenically linked to the AncRln-like ligand in the pre-2R genome, and show that syntenic linkages among ligands and receptors have changed dynamically in different lineages. This study ultimately shows the broad utility, with some caveats, of incorporating paleogenomics data into understanding the evolution of gene families

Exploring the Effects of Endemic East African Co-infections on HIV Susceptibility in the Female Genital Tract

RATIONALE: Human immunodeficiency virus (HIV) remains a leading cause of global morbidity with the highest burden in Sub-Saharan Africa (SSA). For reasons that are incompletely understood, the likelihood of HIV transmission is several fold higher in SSA than in higher income countries, and most of these infections are acquired by women. Residents of SSA are also exposed to a variety of endemic infections that could elevate HIV susceptibility through effects on mucosal and systemic immunology. In the East African Lake Victoria region high HIV transmission geographically overlaps with endemic malaria and Schistosoma mansoni infections. Therefore in this thesis I aimed to explore the impact of these infections on HIV susceptibility in the female genital tract. MAIN FINDINGS: The prevalence of malaria in adult women from Entebbe, Uganda was much lower than expected, but this low prevalence was masked by high rates of over-diagnosis in public health facilities. The prevalence of S. mansoni infection approached 50% and was associated with systemic immune alterations and several socio-behavioral HIV risk factors, emphasizing the importance of controlling these confounders in mucosal studies. A longitudinal clinical trial of S. mansoni treatment effects [ClinicalTrials.gov ID: NCT02878564] demonstrated a substantial anthelminthic treatment-induced reduction of HIV entry into both genital and blood CD4 T cells for two months after standard therapy, despite transient mucosal and systemic immune activation. Furthermore, schistosomiasis treatment was associated with the induction of Type I Interferon pathways, providing a possible mechanism for the reduced HIV entry seen in the trial. CONCLUSIONS: Collectively, the findings presented in this thesis i) highlight the importance of understanding the epidemiology of endemic infections and associated socio-behavioral factors that could confound studies of mucosal HIV susceptibility, and ii) suggest that S. mansoni treatment may lead to new HIV prevention strategies in SSA. ОБОСНОВАНИЕ: Вирус иммунодефицита человека (ВИЧ) является ведущей причиной глобальной заболеваемости с самым высоким уровнем инфекций в Африке к Югу от Сахары (АЮС). По причинам, которые не полностью изучены, вероятность передачи ВИЧ в несколько раз выше в АЮС, чем в странах с более высоким уровнем дохода, и большинство ВИЧ инфекций наблюдается у женщин. Жители АЮС также подвергаются воздействию множества эндемичных инфекций, которые могут влиять на восприимчивость к ВИЧ на иммунитет слизистой и на общесистемном уровне. В Восточно-Африканском регионе, в районе озера Виктория, высокая передача ВИЧ географически перекрывается с эндемичной малярией и шистосомой Мэнсона (Schistosoma mansoni). Цель этой диссертации- изучить влияние этих инфекций на восприимчивость к ВИЧ в женском половом пути. ОСНОВНЫЕ РЕЗУЛЬТАТЫ: Распространенность малярии у взрослых женщин из Энтеббе, Уганда, была намного ниже по сравнению с ожидаемым уровнем, но эта низкая распространенность была замаскирована высокими показателями чрезмерного диагноза малярии в медицинских учреждениях. Распространенность инфекции S. mansoni достигала 50% и была связана с иммунными изменениями в крови и несколькими социально-поведенческими факторами связанными с риском ВИЧ инфекции, что указало на необходимость принятия во внимание эти факторов в дальнейших клинических исследованиях. Проспективное клиническое исследование эффектов лечения S. mansoni [ClinicalTrials.gov ID: NCT02878564] продемонстрировало существенное снижение уровня ВИЧ инфекции CD4 Т-клеток изолированных из эндошейки матки и крови в течение двух месяцев после стандартной терапии шистосомоза, несмотря на временную иммунную активацию. Кроме того, лечение шистосомоза было связано с индукцией антивирусного интерферона типа I, что объясняет возможный механизм снижения уровня ВИЧ-инфекции. ВЫВОДЫ: В совокупности результаты, представленные в этом тезисе, i) подчеркивают важность понимания эпидемиологии эндемических инфекций и связанных с ними социально-поведенческих факторов в исследованиях восприимчивости к ВИЧ ii) указывают на то, что лечение S. mansoni может быть включено в стратегическую профилактику ВИЧ в АЮС.Ph.D

Yegorov et al GCE 2014.pdf

This is a draft of a manuscript published in the General and Comparative Endocrinology Journal

The evolution and genetic linkage of <i>RLN/INSL</i> (ligand) and <i>RXFP3/4</i> (receptor) loci in the pre-3R teleost ancestor and three species of teleost fish.

<p>Notice that among the three fish species analysed, medaka's genome and <i>rln/insl-rxfp</i> gene sets are the most preserved and resemble those of the teleost ancestor. Tetraodon experienced lineage-specific loss of two genes, <i>rln3b and rxfp3-1</i>, which may indicate their co-evolution as a ligand-receptor pair. The <i>rxfp4</i> gene in zebrafish seems to have been replaced with an extra (zebrafish-specific) copy of an <i>rxfp3-3</i> gene. The syntenic linkage between <i>rln</i> and <i>insl3</i> (ligand) and <i>rxfp3-1</i> and <i>rxfp3-2</i>(b) (receptor) genes has been conserved in all three teleosts since the post-2R ancestor (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032923#pone-0032923-g002" target="_blank">Figure 2</a>). Overall this scheme demonstrates that the rln/insl-rxfp system in teleosts has taken a slightly different, and seemingly more complicated, evolutionary pathway compared to other vertebrates. Chromosome numbers in extant species are shown as numbers and in the teleost ancestor as letters.</p