Biology subjects

Srinivasan, S.

Publications and source records attributed to Srinivasan, S..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

Small RNA Sequencing Reveals a Distinct MicroRNA Signature between Glucocorticoid Responder and Glucocorticoid Non-responder Primary Human Trabecular Meshwork Cells after Dexamethasone Treatment

The present study aimed to understand the role of miRNAs in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using small RNA sequencing. For this, total RNA was extracted from cultured HTM cells with known GC responsiveness using Human organ-cultured anterior segment (HOCAS) (GC-responder GC-R; n=4) and GC-non-responder (GC-NR; n=4) after treatment with either 100nM dexamethasone (DEX) or ethanol (ETH) for 7 days. Differentially expressed miRNAs (DEMIRs) were compared among 5 groups and validated by RT-PCR. There were 13 and 21 DEMIRs identified in Group #1 (ETH vs DEX-treated GC-R) and Group #2 (ETH vs DEX-treated GC-NR) respectively. Seven miRNAs were found as common miRNAs dysregulated in both GC-R and GC-NR (Group #3). There were 6 and 14 unique DEMIRs were identified in GC-R (Gropu#4) and GC-NR (Group#5) HTM cells respectively. Ingenuity Pathway Analysis identified enriched pathways and biological processes associated with differential GC responsiveness in HTM cells. Integrative analysis of miRNA-mRNA of the same set of HTM cells revealed several molecular regulators for GC non-responsiveness. This is the first study revealed a unique miRNA signature between GC-R and GC-NR HTM cells which raises the possibility of developing new molecular targets for the management of steroid-OHT/glaucoma.

cell biology↗

bioRxiv2023

Enhanced muscle regeneration in mdx mice, Duchenne muscular dystrophy animal model, proven by CD44 & MYH3 expression, on oral feeding of N-163 strain of Aureobasidium Pullulans produced B-Glucan

IntroductionDuchenne muscular dystrophy (DMD) is a rare genetic disease, causing muscle degeneration due to lack of dystrophin with inadequate muscle regeneration culminating in muscle dysfunction. The N-163 strain of Aureobasidium Pullulans produced Beta-1,3-1,6-glucan (Neu REFIX) reported to be safe with anti-inflammatory and anti-fibrotic efficacy earlier, herein we evaluated its effects on muscle regeneration in mdx mice. MethodsForty-five mice in three groups (n=15 each): Group 1 (normal), Group 2 (mdx control), and Group 3 (mdx fed Neu REFIX) were evaluated for 45 days. IGF-1, Dystrophin, CD44 and MYH3 in diaphragm, plasma and skeletal muscle were evaluated by ELISA and immunohistochemistry. ResultsMean IGF-1 expression was 20.32% and 16.27% higher in plasma (p = 0.03) and diaphragm respectively in Neu-REFIX group. Mean dystrophin was higher in Neu-REFIX group by 70.3% and 4.7% in diaphragm and plasma respectively than control. H-score intensity of CD44+ was >2.0 with an MYH3-positivity 20% higher in Neu-REFIX than control. ConclusionOral administration of Neu REFIX was safe. Significantly enhanced plasma IGF-1 beside increased Dystrophin, MYH3 and CD44, proving a restoration of muscle regeneration and differentiation, especially in diaphragm, makes us recommend it as a disease modifying adjuvant in both early and advanced stages of DMD.

genetics↗

bioRxiv2023

Recalibration of protein interactions in Martini 3

Multidomain proteins with flexible linkers and disordered regions play important roles in many cellular processes, but characterizing their conformational ensembles is diffcult. We have previously shown that the coarse-grained model, Martini 3, produces too compact ensembles in solution, that may in part be remedied by strengthening protein-water interactions. Here, we show that decreasing the strength of protein-protein interactions leads to improved agreement with experimental data on a wide set of systems. We show that the symmetry between rescaling protein-water and protein-protein interactions breaks down when studying interactions with or within membranes; rescaling protein-protein interactions better preserves the binding specificity of proteins with lipid membranes, whereas rescaling protein-water interactions preserves oligomerization of transmembrane helices. We conclude that decreasing the strength of protein-protein interactions improves the accuracy of Martini 3 for IDPs and multidomain proteins, both in solution and in the presence of a lipid membrane.

biophysics↗

bioRxiv2023

The CaliAli tool for long-term tracking of neuronal population dynamics in calcium imaging

Neuron-tracking algorithms exhibit suboptimal performance in calcium imaging when the same neurons are not consistently detected, as unmatched features hinder intersession alignment. CaliAli addresses this issue by employing an alignment-before-extraction strategy that incorporates vasculature information to improve the detectability of weak signals and maximize the number of trackable neurons. By excelling in neural remapping and high spatial overlap scenarios, CaliAli paves the way toward further understanding long-term neural network dynamics.

neuroscience↗

bioRxiv2023

Molecular signatures associated with successful implantation of the human blastocyst

Embryo implantation in humans is remarkably inefficient for reasons that remain largely unexplained, and high rates of implantation failure remain one of the greatest obstacles in treating infertility. The volume of gene expression data available from human embryos has rapidly accumulated in recent years. However, prioritization of these data to identify the subset of genes that determine successful implantation remains a challenge, in part, because comprehensive analyses cannot be performed on the same embryos that are transferred. Here, we leverage clinical morphologic grading--known for decades to correlate with implantation potential--and transcriptome analyses of matched embryonic and abembryonic samples to identify genes and cell-cell interactions enriched and depleted in human blastocysts of good and poor morphology, genome-wide. Unexpectedly, we discovered that the greatest molecular difference was in the state of the extraembryonic primitive endoderm (PrE), with relative deficiencies in PrE development in embryos of poor morphology at the time of embryo transfer. Together, our results support a model in which implantation success is most strongly reflected by factors and signals from the embryonic compartment and suggest that deficiencies in PrE development, in particular, are common among embryos with reduced implantation potential. Our study provides a valuable resource for those investigating the markers and mechanisms of human embryo implantation.

developmental biology↗

bioRxiv2023

Conformational dynamics of lipid transfer domains provide a general framework to decode their functional mechanism.

Lipid Transfer Proteins (LTPs) are key players in cellular homeostasis and regulation, as they coordinate the exchange of lipids between different cellular organelles. Despite their importance, our mechanistic understanding of how LTPs function at the molecular level is still in its infancy, mostly due to the large number of existing LTPs and to the low degree of conservation at the sequence and structural level. In this work, we use molecular simulations to characterize dynamical and mechanistic aspects of a representative dataset of Lipid Transport Domains (LTDs) of 12 LTPs that belong to 8 distinct families. We find that LTDs display common dynamical, rather than structural, features despite no sequence homology nor structural conservation. These dynamical features correlate with their mechanistic mode of action, allowing to interpret and design experimental strategies to further dissect their mechanism. Our findings indicate the existence of a conserved, fold-independent mechanism of lipid transfer across LTPs of various families and offer a general framework for understanding their functional mechanism.

biophysics↗

bioRxiv2023

Integrative transcriptomic and metabolic analyses of the mammalian hibernating brain identifies a key role for succinate dehydrogenase in ischemic tolerance

Ischemic stroke results in a loss of tissue homeostasis and integrity, the underlying pathobiology of which stems primarily from the depletion of cellular energy stores and perturbation of available metabolites1. Hibernation in thirteen-lined ground squirrels (TLGS), Ictidomys tridecemlineatus, provides a natural model of ischemic tolerance as these mammals undergo prolonged periods of critically low cerebral blood flow without evidence of central nervous system (CNS) damage2. Studying the complex interplay of genes and metabolites that unfolds during hibernation may provide novel insights into key regulators of cellular homeostasis during brain ischemia. Herein, we interrogated the molecular profiles of TLGS brains at different time points within the hibernation cycle via RNA sequencing coupled with untargeted metabolomics. We demonstrate that hibernation in TLGS leads to major changes in the expression of genes involved in oxidative phosphorylation and this is correlated with an accumulation of the tricarboxylic acid (TCA) cycle intermediates citrate, cis-aconitate, and -ketoglutarate-KG. Integration of the gene expression and metabolomics datasets led to the identification of succinate dehydrogenase (SDH) as the critical enzyme during hibernation, uncovering a break in the TCA cycle at that level. Accordingly, the SDH inhibitor dimethyl malonate (DMM) was able to rescue the effects of hypoxia on human neuronal cells in vitro and in mice subjected to permanent ischemic stroke in vivo. Our findings indicate that studying the regulation of the controlled metabolic depression that occurs in hibernating mammals may lead to novel therapeutic approaches capable of increasing ischemic tolerance in the CNS.

neuroscience↗

bioRxiv2023

Click chemistry selectively activates an auristatin protodrug with either intratumoral or systemic tumor-targeting agents

The Click Activated Protodrugs Against Cancer (CAPAC) platform enables activation of powerful cancer drugs at tumor sites, maximizing efficacy and minimizing systemic toxicity. CAPAC utilizes a potent click chemistry reaction between tetrazine and trans-cyclooctene, called tetrazine ligation. The reaction between the activator, linked to a tumor targeting agent, and the protodrug leads to targeted activation of the drug. In this study, activation is accomplished either by intratumoral injection of a tetrazine-modified hyaluronic acid biopolymer (SQL70) or by systemic infusion of a tetrazine-modified HER2-targeting antigen-binding fragment (SQT01). The drug used is monomethyl auristatin E (MMAE), a cytotoxic agent hindered in its clinical use by severe toxicity. MMAE modification with a trans-cyclooctene moiety to form the protodrug SQP22 reduced its cytotoxicity in vitro and in vivo. Treatment of SQP22 paired with SQL70 biopolymer demonstrated anti-tumor effects in Karpas 299 and RENCA murine tumor models, establishing the requirement of click chemistry for protodrug activation. Furthermore, SQP22 paired with SQT01 induced anti-tumor effects in the HER2-positive NCI-N87 murine tumor model, showing that activation could be accomplished by systemic dosing of a tumor-targeting antibody conjugate. Observed toxicities were limited to modest, transient myelosuppression and moderate body weight loss in these tumor models. This study further delineates the capabilities of the CAPAC platform by demonstrating anti-tumor activity of SQP22 with two differentiated targeting approaches and underscores the power of click chemistry to precisely control the activation of cancer drugs at tumor sites. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/534625v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1563a70org.highwire.dtl.DTLVardef@16ac78org.highwire.dtl.DTLVardef@95214eorg.highwire.dtl.DTLVardef@bdbb78_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisClick chemistry efficiently activates protodrugs at tumors. A novel protodrug delivering monomethyl auristatin E, SQP22, and preclinical experiments with a biopolymer and antibody fragment as targeting agents are reported.

pharmacology and toxicology↗

bioRxiv2023

SQ3370, the first clinical click chemistry-activated cancer therapeutic, shows safety in humans and translatability across species

BackgroundSQ3370 is the first demonstration of the Click Activated Protodrugs Against Cancer (CAPAC) platform that uses click chemistry to activate drugs directly at tumor sites, maximizing therapeutic exposure. SQ3370 consists of a tumor-localizing biopolymer (SQL70) and a chemically-attenuated doxorubicin (Dox) protodrug SQP33; the protodrug is activated upon clicking with the biopolymer at tumor sites. Here, we present data from preclinical studies and a Phase 1 dose-escalation clinical trial in adult patients with advanced solid tumors (NCT04106492) demonstrating SQ3370s activation at tumor sites, safety, systemic pharmacokinetics (PK), and immunological activity. MethodsTreatment cycles consisting of an intratumoral or subcutaneous injection of SQL70 biopolymer followed by 5 daily intravenous doses of SQP33 protodrug were evaluated in tumor-bearing mice, healthy dogs, and adult patients with solid tumors. ResultsSQL70 effectively activated SQP33 at tumor sites, resulting in high Dox concentrations that were well tolerated and unachievable by conventional treatment. SQ3370 was safely administered at 8.9x the veterinary Dox dose in dogs and 12x the conventional Dox dose in patients, with no dose-limiting toxicity reported to date. SQ3370s safety, toxicology, and PK profiles were highly translatable across species. SQ3370 increased cytotoxic CD3+ and CD8+ T-cells in patient tumors indicating T-cell-dependent immune activation in the tumor microenvironment. ConclusionsSQ3370, the initial demonstration of click chemistry in humans, enhances the safety of Dox at unprecedented doses and has the potential to increase therapeutic index. Consistent safety, toxicology, PK, and immune activation results observed with SQ3370 across species highlight the translatability of the click chemistry approach in drug development. Trial registrationNCT04106492; 7 September 2019

pharmacology and toxicology↗

bioRxiv2023

Identification of a Micropeptide Linked to Cancer Stem Cell Regulation and Chemoresistance

Short open reading frames encoding micropeptides (miPEPs) less than 100 amino acids in length have recently emerged as important regulators of diverse biological functions. However, the functional role of cancer-specific miPEPs in cancer progression and therapeutic response remains largely unexplored. Genome-wide association studies have identified an association of Iroquois (IRX) clusters with multiple cancer risk. In this study, we identified 17 miPEPs generated from IRX clusters in prostate, breast, endometrial, and ovarian cancers using SWATH-MS/MS-based proteomic analysis. We found that IRX4-derived miPEP, IRX4_PEP1, promotes prostate cancer (PCa) cell proliferation, migration, and invasion by interacting with heterogeneous nuclear ribonucleoprotein K (hnRNPK). Overexpression of IRX4_PEP1 leads to dysregulation of stem cell pathways by co-interaction with Catenin beta-1 (CTNB1) and upregulation of prominent PCa stem markers, resulting in docetaxel resistance in PCa. IRX4_PEP1 expression is significantly upregulated in prostate tumour tissues compared to normal and is positively correlated with disease aggressiveness. SIGNIFICANCEOur findings highlight the critical role of IRX4_PEP1 in regulating PCa stem cells and chemotherapy resistance and suggest it as a novel therapeutic target for the treatment of PCa. Moreover, IRX4_PEP1 expression can serve as a novel potential diagnostic and prognostic biomarker for PCa.

cancer biology↗

bioRxiv2023

The Dietary Fermentable Fiber Inulin Alters the Intestinal Microbiome and Improves Chronic Kidney Disease Mineral-Bone Disorder in a Rat Model of CKD

BackgroundDietary fiber is important for a healthy diet, but intake is low in CKD patients and the impact this has on the manifestations of CKD-Mineral Bone Disorder (MBD) is unknown. MethodsThe Cy/+ rat with progressive CKD was fed a casein-based diet of 0.7% phosphate with 10% inulin (fermentable fiber) or cellulose (non-fermentable fiber) from 22 weeks to either 30 or 32 weeks of age (~30 and ~15 % of normal kidney function). We assessed CKD-MBD, cecal microbiota, and serum gut-derived uremic toxins. Two-way ANOVA was used to evaluate the effect of age and inulin diet, and their interaction. ResultsIn CKD animals, dietary inulin led to changes in microbiota alpha and beta diversity at 30 and 32 weeks, with higher relative abundance of several taxa, including Bifidobacterium and Bacteroides, and lower Lactobacillus. Inulin reduced serum levels of gut-derived uremic toxins, phosphate, and parathyroid hormone, but not fibroblast growth factor-23. Dietary inulin decreased aorta and cardiac calcification and reduced left ventricular mass index and cardiac fibrosis. Bone turnover and cortical bone parameters were improved with inulin; however, bone mechanical properties were not altered. ConclusionsThe addition of the fermentable fiber inulin to the diet of CKD rats led to changes in the gut microbiota composition, lowered gut-derived uremic toxins, and improved most parameters of CKD-MBD. Future studies should assess this fiber as an additive therapy to other pharmacologic and diet interventions in CKD. Significance StatementDietary fiber has well established beneficial health effects. However, the impact of fermentable dietary fiber on the intestinal microbiome and CKD-MBD is poorly understood. We used an animal model of progressive CKD and demonstrated that the addition of 10% of the fermentable fiber inulin to the diet altered the intestinal microbiota and lowered circulating gut-derived uremic toxins, phosphorus, and parathyroid hormone. These changes were associated with improved cortical bone parameters, lower vascular calcification, and reduced cardiac hypertrophy, fibrosis and calcification. Taken together, dietary fermentable fiber may be a novel additive intervention to traditional therapies of CKD-MBD.

cell biology↗

bioRxiv2023

Poles apart - the structure and composition of the bird community in bamboo in the Eastern Himalaya

Bamboos (Poaceae: Bambuseae) are a unique and diverse group of plants (> 1500 species) that are found in tropical and subtropical regions of the world. Various birds, mammals, and insects use bamboo either directly or indirectly, and some species are obligate bamboo specialists. For instance, several species of Neotropical birds are found in bamboo alone. However, the mechanisms underlying dependence on/associations with bamboo are almost entirely unknown. We studied bird communities in bamboo and rainforest across two seasons in the Eastern Himalaya, where we explored a possible mechanism for bird associations with bamboo - dietary resource specialization. We calculated use of bamboo versus rainforest by bird species and described the structure and composition of the bird and arthropod communities in both habitats and seasons. We provide the first systematic evidence of bamboo specialist bird species from the Eastern Himalaya. Although the species richness of birds and arthropods in both habitats and seasons did not vary starkly, we found that the composition of bird and arthropod communities in both habitats and both seasons to be distinct. Interestingly, we found arthropod communities in different substrates of bamboo to also be distinct. Bird specialization in bamboo in the Eastern Himalaya could be because of their dietary specialization to the unique arthropods found in bamboo. The results from this study emphasise the importance of bamboo in the Eastern Himalaya and provide baseline information that might aid in conservation.

ecology↗

bioRxiv2022

Enrichment of phenotype among biological forms of Anopheles stephensi Liston through establishment of isofemale lines

The success of vector management programs relies on knowledge of the biology and genetic make-up of mosquitoes so that they can be interlaced with modern tools for developing suitable intervention strategies. There are many reports available for rearing varied species of mosquito vectors. However, there are limited studies addressing the development of isofemale lines among mosquitoes to homogenize the population to obtain both high-quality genome assemblies and enrichment of phenotype. Anopheles stephensi, an urban malaria vector, is one of the major invasive vectors of malaria distributed throughout the Indian subcontinent, Middle East, and has recently been expanding its range in Africa. With the existence of three biological forms, distinctly identifiable based on the number of ridges on eggs with varying vectorial competence, An. stephensi is a perfect species for developing a method for the successful establishment of isofemale lines, which can be tested for retention of the expected vectorial competence for the various forms. We describe the key steps in the establishment and validation of isofemale lines, which include monitoring the transgenerational fitness traits, morphometrics of eggs, and adult wing size during every generation. After the initial inbreeding depression, as proof of the tedious selection process, no significant morphometric differences were observed in the wings and egg size between the parental and their respective isofemale lines. We observed a significant change in the vectorial competence between the respective isofemale and parental lines enriching expected differential susceptibility towards malaria parasites by the type and intermediate forms. Interestingly, IndCh and IndInt strains showed variations in resistance to different insecticides belonging to all the four major classes. These variant lines have been characterized for their levels of homozygosity both at the phenotype and genotype levels and can be used as a standard reference or as a biological resource for other studies related to urban malaria research. Author summaryIsofemale lines can be a valuable resource for characterizing and enhancing several genotypic and phenotypic traits. This is the first detailed report of the establishment of two isofemale lines of type and intermediate biological forms in Anopheles stephensi. The work encompasses the characterization of fitness traits among the two lines through a transgenerational study. Further, isofemale colonies were established and used to characterize insecticide susceptibility and vector competence. The study provides valuable insights into the differential susceptibility status of the parental and isofemale to different insecticides belonging to the same class. Corroborating with the earlier hypothesis we exemplify the high vector competence in type form than the intermediate from using homozygous isofemale lines. Using these isofemale lines it is now possible to study host-parasite interactions and identify factors that might be responsible for altered susceptibility and increased vector competence in An. stephensi biological forms that would also pave way for developing better vector management strategies.

genetics↗

bioRxiv2022

Common and distinct genetic architecture of age at diagnosis of diabetes in South Indian and European populations

South Asians are diagnosed with type 2 diabetes (T2D) more than a decade earlier in life than seen in European populations. We hypothesised that studying the genomics of age of onset in these populations may give insight into earlier age at onset of T2D among individuals of South Asian descent. We conducted a meta-analysis of GWAS of age at diagnosis of T2D in 34,001 individuals from four independent cohorts of European and Asian Indians. We identified two signals near the TCF7L2 and CDKAL1 associated with age at onset of T2D. The strongest genome-wide significant variants at chromosome 10q25.3 in TCF7L2 (lead SNP rs7903146; p = 2.4 x10-12; p-het =0.01; Beta = -0.436; SE = 0.02) and chromosome 6 p22.3 in CDKAL1 (rs9368219; p = 2.29 x10-8; p-het =0.007; Beta = -0.053; SE=0.01) were directionally consistent across ethnic groups and present at similar frequencies, however both loci harboured additional independent signals that were only present in the South Asian cohorts. A genome wide signal was also obtained at chromosome 10q26.12 in WDR11 (rs3011366; p = 2.4 x10-8; p-het =0.25; Beta = 1.44; SE=0.25) specifically in the South Asian Indian cohorts. Our study estimated 17% of heritability (h2) for age onset of T2D among South Indians; higher than the 5% heritability we observed in Europeans suggesting different genetic architectures between these populations. We further estimated the Asian Indian genome-wide polygenic risk score for T2D risk and identified that the variance explained by the Indian specific polygenic risk score (PRS) for age at onset of T2D is substantially higher (2%) in an independent cohort of Asian Indians compared to that seen in White Europeans (<0.1%). This reveals that although variants do exist that are shared between the two population there are also genetic disparities of age onset of T2D between the ethnicities. Author SummaryRecent large multi-ancestry genome-wide meta-analyses have identified over 277 genetic loci associated with type 2 diabetes (T2D). Most association studies for T2D have focused on populations of European ancestry, and the specific genetic architecture of T2D in the South Asian population has not been extensively investigated. Increasing evidence indicates that the prevalence of T2D is higher in South Asians with a strikingly earlier age at diagnosis when compared to Europeans. Genome-wide association studies (GWAS) on age at age at diagnosis of T2D are limited and can help elucidate underlying mechanisms. We conducted the largest meta-analysis to date of genome-wide association studies of age at diagnosis as a surrogate for age of onset of T2D in South Indians and Europeans. Our trans-ancestry meta-analysis identified genetic loci near TCF7L2 and CDKAL1 with substantial ethnic heterogeneity in both allele frequencies and effect sizes in early diagnosis with T2D. Two novel variants near TCF7L2 (rs570193324) and CDKAL1 (rs143316471) were associated with age of diagnosis of T2D only in South Indians and were present at much lower frequency in the European populations. Heritability estimates for age of onset were much stronger in Asian Indians compared to Europeans and a polygenic risk score was constructed using a South Indians, which explained about 2% trait variance compared to European ancestry (<0.1%). Our novel findings provide a better understanding of ethnic differences in the age at onset of T2D and indicate the potential importance of ethnic differences in the genetic architecture underpinning T2D.

genetics↗

bioRxiv2022

Absorbable Conductive Electrotherapeutic Scaffolds (ACES) for Enhanced Peripheral Nerve Regeneration and Stimulation

While peripheral nerve stimulation (PNS) has shown promise in applications ranging from peripheral nerve regeneration after injury to therapeutic organ stimulation, clinical implementation has been impeded by various technological limitations, including surgical placement, lead migration, and atraumatic removal. Here, we describe the design and validation of a new platform for nerve regeneration and interfacing: Absorbable, Conductive, Electrotherapeutic Scaffolds (ACES). ACES are comprised of an alginate/poly-acrylamide interpenetrating network hydrogel optimized for both open and minimally invasive percutaneous approaches. In a rodent model of sciatic nerve repair, ACES significantly improved motor and sensory recovery (p < 0.05), increased muscle mass (p < 0.05), and increased axonogenesis (p < 0.05). Triggered dissolution of ACES enabled atraumatic, percutaneous removal of leads at forces significantly lower than controls (p < 0.05). In a porcine model, ultrasound-guided percutaneous placement of leads with an injectable ACES near the femoral and cervical vagus nerves facilitated stimulus conduction at significantly greater lengths than saline controls (p < 0.05). Overall, ACES facilitated lead placement, stabilization, stimulation and atraumatic removal enabling therapeutic PNS as demonstrated in small and large animal models.

bioengineering↗

bioRxiv2022

DLC3/Cv-c function in testis development in humans and Drosophila: implication for variants of sex development

Identifying genes affecting gonad development is essential to understand the mechanisms causing Variants/Differences in Sex Development. Recently, a DLC3 mutation was associated with male gonadal dysgenesis in 46,XY DSD patients. We show that Cv-c, the Drosophila ortholog of DLC3, is also required to maintain testis integrity during fly development. We found that Cv-c and human DLC3 can perform the same function in fly embryos, as flies with wild type but not mutated DLC3 rescue gonadal dysgenesis, suggesting a functional conservation. Expression of different Cv-c protein variants demonstrated that the StART domain mediates the Cv-c function in the male gonad, independently from the GAP domain activity. This work demonstrates a role for DLC3/Cv-c in male gonadogenesis and highlights a novel StART-mediated function required for gonadal mesoderm-germ cell interaction during testis development. Significance StatementAssociating rare human genetic variants to specific conditions is complex. An amino acid change in the StART domain of the RhoGAP DLC3 protein has been found in female DSD patients with a 46,XY male karyotype. We present a second DSD patient with a mutation in the same region and show that the Cv-c/DLC3 homolog is also required in Drosophila testis. In cv-c mutant embryos the testis mesodermal cells display various defects: the testes are unable to retain the germ cells due to their abnormal ensheathment by mesodermal cells and the mesodermal sheet of cells surrounding the testis is discontinuous resulting in the liberation of the germ cells outside the gonad. Defects can be rescued by gonadal expression of Cv-c or DLC3 but not by the patients DLC3 protein variant. Testis development requires the StART lipid binding domain but not the GAP domain, revealing a novel function of this RhoGAP family.

developmental biology↗

bioRxiv2022

Identification of a TNF-TNFR-like system in malaria vectors (Anopheles stephensi) likely to influence Plasmodium resistance

Identification of Plasmodium-resistance genes in malaria vectors remains an elusive goal despite the recent availability of high-quality genomes of several mosquito vectors. An. stephensi, with its three distinctly-identifiable forms at the egg stage, correlating with varying vector competence, offers an ideal species to discover functional mosquito genes implicated in Plasmodium resistance. Recently, the genomes of several strains of An. stephensi of the type-form, known to display high vectorial capacity, have been reported. Here, we report a chromosomal-level assembly of an intermediate-form of An. stephensi strain (IndInt), shown to have reduced vectorial capacity relative to a strain of type-form (IndCh). The contig level assembly with a L50 of 4 was scaffolded into chromosomes by using the genome of IndCh as the reference. The final assembly shows a heterozygous paracentric inversion, 3Li, involving 8 Mbp, which is syntenic to the extensively-studied 2La inversion implicated in Plasmodium resistance in An. gambiae involving 21 Mbp. Deep annotation of genes within the 3Li region in IndInt assembly using the state-of-the-art protein-fold prediction and other annotation tools reveals the presence of a TNF-like gene, which is the homolog of the eiger gene in Drosophila. Subsequent chromosome-wide searches revealed homologs of wengen (wgn) and grindelwald (grnd) genes in IndInt, which are known to be the receptors for eiger in Drosophila. We have identified all the genes in IndInt required for eiger-mediated signaling by analogy to TNF-TNFR system, suggesting the presence of a functionally active eiger signaling pathway present in IndInt. Comparative genomics of high-quality genome assemblies of the three type-forms with that of IndInt, reveals structurally disruptive mutations in eiger gene in all three strains of the type-form, alluding to compromised innate immunity in the type-form as the cause of high vectorial capacity in these strains. This is the first report of the presence of an intact evolutionarily-conserved TNF-TNFR signaling system in malaria vectors, with a potential role in Plasmodium resistance.

genomics↗

bioRxiv2022

Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of β-hemoglobinopathies

Reactivation of fetal hemoglobin (HbF) is the commonly adapted strategy to ameliorate {beta}-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits the HbF tetramer production affecting the therapeutic benefits. Here, we tested various deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified a 11 kb sequence, encompassing Putative Repressor Region (PRR) to {beta}-globin Exon-1 ({beta}E1), as the core deletion that ablates HbA and exhibit superior production of HbF compared to HPFH or other well-established targets. The PRR-{beta}E1 edited hematopoietic stem and progenitor cells (HSPCs) retained engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice generating HbF+ cells in vivo. Importantly, the editing induces therapeutically relevant levels of HbF to reverse the phenotypes of both sickle cell disease and {beta}-thalassemia major. These results indicate that the PRR-{beta}E1 gene editing in patient HSPCs can potentially lead to superior therapeutic outcomes for {beta}-hemoglobinopathies gene therapy.

biochemistry↗