Biology subjects

Sekar, A.

Publications and source records attributed to Sekar, A..

15 recordsLinked to original sources

bioRxiv2026

Aptamer-Targeted PLGA Nanoparticles for Biofilm-Directed Intra-articular Antibiotic Delivery in Prosthetic Joint Infection

Introduction: Prosthetic joint infection (PJI) is a severe complication of orthopedic surgery, driven by biofilm-forming bacteria such as Staphylococcus aureus. Conventional antibiotics are limited in penetrating biofilms or remaining localized at the infection site. We engineered a targeted nanocarrier system by conjugating DNA aptamers specific to S. aureus biofilm components onto poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) encapsulating vancomycin. PLGA NPs were synthesized using flow-focusing microfluidics, followed by surface functionalization via EDC/NHS chemistry. Aptamer conjugation was validated using gel electrophoresis and DLS. Targeting efficiency and antimicrobial activity were evaluated through fluorescence microscopy, bacterial viability assays and in vitro biofilm models. Biodistribution and joint retention were assessed using fluorescent aptamer-conjugated NPs following intra-articular injection in rat knee joints. Results: The optimized vancomycin-loaded NPs exhibited uniform morphology, high vancomycin loading capacity (7.9 {+/-} 2.2%), and a sustained biphasic release profile with an early high-release phase followed by slower extended release. PK modeling indicated prolonged intra-articular drug retention with concentrations maintained above the minimum inhibitory concentration (MIC) for an extended period. Aptamer conjugation preserved nanoparticle stability and enhanced binding and retention within S. aureus biofilms in vitro, supporting improved biofilm localization. In-vivo fluorescence imaging demonstrated high local retention of aptamer-conjugated NPs, supporting localized joint retention with limited off-target tissue signal. Conclusions: S. aureus-specific aptamer conjugated nanoparticles (SA Apt-P(V)) represent a targeted intra-articular delivery platform with potential for localized PJI therapy. The combined in-vitro and in-vivo findings highlight their potential to improve local therapeutic concentration, minimize systemic exposure, and enhance infection control within prosthetic joints.

bioengineering↗

bioRxiv2026

Tumor cell-intrinsic mtDNA instability drives cGAS-dependent temporal remodeling of the melanoma immune microenvironment

Mitochondrial DNA (mtDNA) instability is an emerging feature of cancer, yet its biological effects on the tumor microenvironment (TME) and tumor progression remain incompletely defined. Using melanocyte-specific conditional silencing of mitochondrial transcription factor A (TFAM) in an autochthonous BrafV600E/Pten-/- melanoma model, we demonstrate that tumor cell-intrinsic mtDNA instability accelerates melanoma growth and metastatic dissemination in a cGAS-dependent manner. Transcriptomic profiling of early and late tumor stages revealed that mtDNA instability drives temporal remodeling of the tumor microenvironment (TME), characterized by an early type I interferon (IFN)-rich inflammatory state that licenses immunoregulatory programs as tumors progress. Tumor-associated macrophages emerged as the principal immune cell type reshaped by tumor cell-intrinsic mtDNA stress signaling, with early enrichment of inflammatory states and a shift toward efferocytosis-associated, tissue-adaptive states. The early mtDNA instability-associated macrophage signatures mapped to human melanoma states associated with favorable survival and therapy response, whereas the late signatures mapped to states associated with poor prognosis and therapy resistance across independent cohorts. Anti-PD-1 treatment delayed progression of mtDNA-instable tumors, consistent with a therapeutic sensitivity window associated with the early IFN-rich immune state. These findings establish tumor cell-intrinsic mtDNA instability as a driver of temporal immune-stromal remodeling that promotes melanoma progression and shapes therapeutic response.

immunology↗

bioRxiv2025

Inducible re-epithelialization of cancer cells increases autophagy and DNA damage: implications for breast cancer dormancy

Epithelial lineage differentiation is pivotal to mammary gland development and it can pause metastasis of breast cancer (BC) by inducing tumor dormancy. To simulate this, we expressed epithelial genes in mesenchymal BC cells. Inducible expression of the epithelial OVOL genes in metastatic BC cells suppressed proliferation and migration. We found that C1ORF116, an OVOLs target, is susceptible to genetic and epigenetic aberrations in BC. It is regulated by steroids and functions as a putative autophagy receptor that inhibits antioxidants like thioredoxin. Accordingly, boosting epithelialization lowered glutathione, elevated reactive oxygen species and increased both DNA oxidation and double strand breaks. Epithelialization also associated with redistribution of NRF2 and an altered interplay among p38, ATM, and the other kinases regulating the DNA damage response. Hence, hormonal regulation of OVOLs and chronic stress might permit epithelial differentiation and retard exit from dormancy, while altering redox homeostasis and permitting DNA damage accumulation, which may awaken dormant tumors.

cancer biology↗

bioRxiv2025

TMPRSS2-ERG confers resistance to antiandrogens: mechanism and therapeutic implications

Approximately 50% of prostate cancer (PCa) patients harbor fusions involving the TMPRSS2 and ERG genes. Despite this, tailored therapies targeting the fused gene, tERG, remain undeveloped. Our study analyzed biopsy samples from two clinical trials assessing the efficacies of androgen receptor (AR) signaling inhibitors (ARSIs). The results revealed that tERG promotes resistance to ARSIs and is associated with elevated levels of the glucocorticoid receptor (GR). Subsequent assays showed that GR directly interacts with tERG, alleviates allosteric autoinhibition and prevents chemotherapy-induced tERG degradation. In PCa models, either inhibiting GR or lowering cortisol levels suppressed tumor growth in tERG-positive models, but not in fusion-negative models. In addition, patient-derived fusion-positive xenografts displayed enhanced sensitivity to combined GR and AR inhibitors. Collectively, these findings highlight TMPRSS2-ERG as a new biomarker and propose that simultaneous inhibition of GR and AR may specifically benefit tERG-positine patients. However, GR stimulatory corticosteroid therapies may not be advisable for this patient subgroup.

cancer biology↗

bioRxiv2024

A Dual Role for the PP2A Phosphatase in Hippo Signalling Regulation

Hippo signalling is an evolutionarily conserved pathway that regulates tissue growth. The FERM domain protein Expanded plays a crucial role in integrating polarity cues to activate the Hippo pathway. Previous work has shown that the apicobasal polarity protein Crumbs can limit Hippo activity by promoting the phosphorylation and degradation of Expanded. Here, we provide evidence that PP2AWrd can counteract the effects of Crumbs, by dephosphorylating and stabilising Expanded. Indeed, we demonstrate that the PP2AWrd holoenzyme can increase Hippo signalling activity, in contrast to the previously established Hippo pathway inhibitory role of the PP2ACka-containing STRIPAK complex. We also uncover a role for PP2AWrd and PP2ATws in the regulation of Expanded proteostasis. Remarkably, the upstream Hippo regulator, Kibra interacts with PP2AWrd and prevents Expanded degradation. However, Kibra is unable to antagonise Crumbs-mediated Expanded regulation, in agreement with the previously established role of Crumbs in inhibiting Kibra function. Overall, our work characterises a novel Hippo-activating role for PP2A in the stabilisation of Expanded and provides new insights into how PP2A tightly controls Hippo activity in response to polarity stimuli.

developmental biology↗

bioRxiv2024

Gene expansions contributing to human brain evolution

Duplicated genes expanded in the human lineage likely contributed to brain evolution, yet challenges exist in their discovery due to sequence-assembly errors. We used a complete telomere-to-telomere genome sequence to identify 213 human-specific gene families. From these, 362 paralogs were found in all modern human genomes tested and brain transcriptomes, making them top candidates contributing to human-universal brain features. Choosing a subset of paralogs, long-read DNA sequencing of hundreds of modern humans revealed previously hidden signatures of selection, including for T-cell marker CD8B. To understand roles in brain development, we generated zebrafish CRISPR "knockout" models of nine orthologs and introduced mRNA-encoding paralogs, effectively "humanizing" larvae. Our findings implicate two genes in possibly contributing to hallmark features of the human brain: GPR89B in dosage-mediated brain expansion and FRMPD2B in altered synapse signaling. Our holistic approach provides insights and a comprehensive resource for studying gene expansion drivers of human brain evolution.

genomics↗

bioRxiv2024

Investigating the translational value of Periprosthetic Joint Infection (PJI) models to determine the risk and severity of Staphylococcal biofilms.

With the advent of antibiotic-eluting polymeric materials for targeting recalcitrant infections, using preclinical models to study biofilm is crucial for improving the treatment efficacy in periprosthetic joint infections. The stratification of risk and severity of infections is needed to develop an effective clinical dosing framework with better outcomes. Here, using in-vivo and in-vitro implant-associated infection models, we demonstrate that methicillin-sensitive and resistant Staphylococcus aureus (MSSA and MRSA) have model-dependent distinct implant and peri-implant tissue colonization patterns. The maturity of biofilms and the location (implant vs tissue) were found to influence the antibiotic susceptibility evolution profiles of MSSA and MRSA and the models could capture the differing host-microbe interactions in vivo. Gene expression studies revealed the molecular heterogeneity of colonizing bacterial populations. The comparison and stratification of the risk and severity of infection across different preclinical models provided in this study can guide clinical dosing to effectively prevent or treat PJI.

microbiology↗

bioRxiv2024

The deubiquitylating enzyme Fat Facets modulates Fat signalling and regulates tissue growth

Tissue growth is regulated by many signals, including polarity cues. The Hippo signalling pathway restricts tissue growth and receives inputs from the planar cell polarity-controlling Fat signalling pathway. The atypical cadherin Fat restricts growth via several mechanisms that ultimately control the activity of the pro-growth transcriptional co-activator Yorkie. The Fat pathway modulates the activity of the Yorkie inhibitory kinase Warts, as well as the function of the FERM protein Expanded, which promotes Hippo signalling and also directly inhibits Yorkie. Although several Fat pathway activity modulators are known to be involved in ubiquitylation, the role of this post-translational modification in the pathway remains unclear. Moreover, no deubiquitylating enzymes have been described in this pathway. Here, using an in vivo RNAi screening approach, we identify the deubiquitylating enzyme Fat facets as a positive regulator of Fat signalling that is important for tissue growth control. Fat facets interacts genetically and physically with Fat signalling components and regulates transcription of Yorkie target genes. Thus, we uncover a role for reversible ubiquitylation in the control of Fat signalling and, by extension, in the regulation of tissue growth.

developmental biology↗

bioRxiv2021

Rbf/E2F1 control growth and endoreplication via steroid-independent Ecdysone Receptor signalling in Drosophila prostate-like secondary cells

Dysregulation of cell cycle components results in the development and progression of several cancer types. Unusually, loss of the tumour suppressor gene, Retinoblastoma (Rb), and consequent activation of transcription factor E2F1 have been linked to late-stage tumour progression in prostate cancer, rather than early-stage events. This change is associated with an androgen-independent form of cancer, castration-resistant prostate cancer (CRPC), which frequently still requires androgen receptor (AR) signalling. We have previously shown that binucleate secondary cells (SCs) of the Drosophila melanogaster male accessory gland (AG) share several functional and signalling similarities with human prostate epithelial cells. Upon mating, SC growth regulation switches from a steroid-dependent to a steroid-independent form of Ecdysone Receptor (EcR) control that induces genome endoreplication. Here, we demonstrate that the Drosophila Rb homologue, Rbf, and E2F1, as well as cell cycle regulators, Cyclin D (CycD) and Cyclin E (CycE), are key mediators of SC growth and endoreplication both in virgin and mated males. Importantly, we show that the CycD/Rbf/E2F1 axis requires the EcR, but not ecdysone, to trigger CycE-dependent endoreplication and associated growth in SCs after mating, mirroring changes in CRPC. We also demonstrate that excess Rbf activity reversibly suppresses binucleation in adult SCs. Overall, our work reveals mechanistic parallels between the physiological switch to hormone-independent EcR signalling in SCs, and the pathological switch seen in CRPC, and suggests that the latter may represent the dysregulation of a currently unidentified physiological process, which permits AR signalling when androgen levels are low.

cell biology↗

bioRxiv2020

Evaluation of CRISPR gene-editing tools in zebrafish identifies spurious mutations in 'mock control embryos

BackgroundZebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create knockout models. In particular, the use of G0 mosaic mutants has potential to increase throughput of functional studies significantly but may suffer from transient effects of introducing Cas9 via microinjection. Further, a large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods leaving uncertainty in choosing an optimal approach for zebrafish studies. MethodsTo systematically assess accuracy of tool predictions of on- and off-target gene editing, we subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes. We also investigate potential confounders of G0-based CRISPR screens by screening control embryos for spurious mutations and altered gene expression. ResultsWe compared our experimental in vivo editing efficiencies in mosaic G0 embryos with those predicted by eight commonly used gRNA design tools and found large discrepancies between methods. Assessing off-target mutations (predicted in silico and in vitro) found that the majority of tested loci had low in vivo frequencies (<1%). To characterize if commonly used mock CRISPR controls (larvae injected with Cas9 enzyme or mRNA with no gRNA) exhibited spurious molecular features that might exacerbate studies of G0 mosaic CRISPR knockout fish, we generated an RNA-seq dataset of various control larvae at 5 days post fertilization. While we found no evidence of spontaneous somatic mutations of injected larvae, we did identify several hundred differentially-expressed genes with high variability between injection types. Network analyses of shared differentially-expressed genes in the mock injected larvae implicated a number of key regulators of common metabolic pathways, and gene-ontology analysis revealed connections with response to wounding and cytoskeleton organization, highlighting a potentially lasting effect from the microinjection process that requires further investigation. ConclusionOverall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.

genetics↗

bioRxiv2020

Role of ERK activation in H. pylori-induced disruption of cell-cell tight junctions

BackgroundTight junctions, a network of claudins and other proteins, play an important role in maintaining barrier function and para-cellular permeability. H. pylori, the major etiological agent of various gastroduodenal diseases, is known to cause tight junction disruption. However, the molecular events that triggered cell-cell tight junction disruption in H. pylori-infected cells, remain largely elusive. Materials and MethodsTrans-epithelial electrical resistance (TEER) and FITC-Dextran permeability measurement were performed to determine the barrier function in H. pylori 88-3887-infected polarized MKN28 cells. For visualization of tight junction protein localization, immunofluorescence and immunoblotting techniques were used. To examine the role of ERK activation in tight junction disruption, U0126, a MEK inhibitor, was employed. To further support the study, computational analyses of H. pylori-infected primary gastric cells were carried out to decipher the transcriptomic changes. ResultsThe epithelial barrier of polarized MKN28 cells when infected with H. pylori displayed disruption of cell-cell junctions as shown by TEER & FITC-dextran permeability tests. Claudin-4 was shown to delocalize from host cytoplasm to nucleus in H. pylori-infected cells. In contrast, delocalization of claudin-4 was minimized when ERK activation was inhibited. Interestingly, transcriptomic analyses revealed the upregulation of genes associated with cell-junction assembly and ERK pathway forming a dense interacting network of proteins. ConclusionTaken together, evidence from this study indicates that H. pylori regulates ERK pathway triggering cell-cell junction disruption, contributing to host pathogenesis. It indicates the vital role of ERK in regulating key events associated with the development of H. pylori-induced gastroduodenal diseases.

microbiology↗

bioRxiv2020

Drosophila Sex Peptide Controls the Assembly of Lipid Microcarriers in Seminal Fluid

Seminal fluid plays an essential role in promoting male reproductive success and modulating female physiology and behaviour. In the fruit fly, Drosophila melanogaster, Sex Peptide (SP) is the best-characterised protein mediator of these effects. It is secreted from the paired male accessory glands (AGs), which, like the mammalian prostate and seminal vesicles, generate most of the seminal fluid contents. After mating, SP binds to spermatozoa and is retained in the female sperm storage organs. It is gradually released by proteolytic cleavage and induces several long-term post-mating responses including ovulation, elevated feeding and reduced receptivity to remating, primarily signalling through the SP receptor (SPR). Here, we demonstrate a previously unsuspected SPR-independent function for SP. We show that, in the AG lumen, SP and secreted proteins with membrane-binding anchors are carried on abundant, large neutral lipid-containing microcarriers, also found in other SP-expressing Drosophila species. These microcarriers are transferred to females during mating, where they rapidly disassemble. Remarkably, SP is a key assembly factor for microcarriers and is also required for the female disassembly process to occur normally. Males expressing non-functional SP mutant proteins that affect SPs binding to and release from sperm in females also do not produce normal microcarriers, suggesting that this male-specific defect contributes to the resulting widespread defects in ejaculate function. Our data therefore reveal a novel role for SP in formation of seminal macromolecular assemblies, which may explain the presence of SP in Drosophila species, which lack the signalling functions seen in D. melanogaster. Significance StatementSeminal fluid plays a critical role in reprogramming female physiology and behaviour to promote male reproductive success. We show in the fruit fly that specific seminal proteins, including the archetypal female-reprogramming molecule Sex Peptide, are stored in male seminal secretions in association with large neutral lipid-containing microcarriers, which rapidly disperse in females. Related structures are also observed in other Sex Peptide-expressing Drosophila species. Males lacking Sex Peptide have structurally defective microcarriers, leading to abnormal cargo loading and transfer to females. Our data reveal that this key signalling molecule in Drosophila seminal fluid is also a microcarrier assembly factor that controls transfer of other seminal factors, and that this may be a more evolutionarily ancient role of this protein.

cell biology↗

bioRxiv2019

Complement component 4 genes contribute sex-specific vulnerability in diverse illnesses

Many common illnesses differentially affect men and women for unknown reasons. The autoimmune diseases lupus and Sjogrens syndrome affect nine times more women than men1,2, whereas schizophrenia affects men more frequently and severely3-5. All three illnesses have their strongest common-genetic associations in the Major Histocompatibility Complex (MHC) locus, an association that in lupus and Sjogrens syndrome has long been thought to arise from HLA alleles6-13. Here we show that the complement component 4 (C4) genes in the MHC locus, recently found to increase risk for schizophrenia14, generate 7-fold variation in risk for lupus (95% CI: 5.88-8.61; p < 10-117 in total) and 16-fold variation in risk for Sjogrens syndrome (95% CI: 8.59-30.89; p < 10-23 in total), with C4A protecting more strongly than C4B in both illnesses. The same alleles that increase risk for schizophrenia, greatly reduced risk for lupus and Sjogrens syndrome. In all three illnesses, C4 alleles acted more strongly in men than in women: common combinations of C4A and C4B generated 14-fold variation in risk for lupus and 31-fold variation in risk for Sjogrens syndrome in men (vs. 6-fold and 15-fold among women respectively) and affected schizophrenia risk about twice as strongly in men as in women. At a protein level, both C4 and its effector (C3) were present at greater levels in men than women in cerebrospinal fluid (p < 10-5 for both C4 and C3) and plasma among adults ages 20-5015-17, corresponding to the ages of differential disease vulnerability. Sex differences in complement protein levels may help explain the larger effects of C4 alleles in men, womens greater risk of SLE and Sjogrens, and mens greater vulnerability in schizophrenia. These results nominate the complement system as a source of sexual dimorphism in vulnerability to diverse illnesses.

genetics↗

bioRxiv2019

Mating Induces Switch From Hormone-Dependent to -Independent Steroid Receptor-Mediated Growth in Drosophila Prostate-Like Cells

Male reproductive glands like the mammalian prostate and the paired Drosophila melanogaster accessory glands secrete seminal fluid components that enhance fecundity. In humans, the prostate grows throughout adult life, stimulated by environmentally regulated endocrine and local androgens. We previously showed that in each fly accessory gland, secondary cells (SCs) and their nuclei also grow in adults, a process enhanced by mating and controlled by bone morphogenetic protein (BMP) signalling. Here we demonstrate that BMP-mediated SC growth is dependent on the receptor for the developmental steroid, ecdysone, whose concentration reflects socio-sexual experience in adults. BMP signalling regulates ecdysone receptor (EcR) levels post-transcriptionally, partly via EcRs N-terminus. Nuclear growth in virgin males is ecdysone-dependent. However, mating activates genome endoreplication to drive additional BMP-mediated nuclear growth via a cell type-specific form of hormone-independent EcR signalling. In virgin males with low ecdysone levels, this mechanism ensures resources are conserved. However, by switching to hormone-independence after mating, this control is overridden to hyper-activate growth of secretory secondary cells. Our data suggest parallels between this physiological, behaviour-induced switch and altered pathological signalling associated with prostate cancer progression.

cell biology↗

bioRxiv2018

Human local adaptation of the TRPM8 cold receptor along a latitudinal cline

Ambient temperature is a critical environmental factor for all living organisms. It was likely an important selective force as modern humans recently colonized temperate and cold Eurasian environments. Nevertheless, as of yet we have limited evidence of local adaptation to ambient temperature in populations from those environments. To shed light on this question, we exploit the fact that humans are a cosmopolitan species that inhabits territories under a wide range of temperatures. Focusing on cold perception - which is central to thermoregulation and survival in cold environments-- we show evidence of recent local adaptation on TRPM8. This gene encodes for a cation channel that is, to date, the only temperature receptor known to mediate an endogenous response to moderate cold. The upstream variant rs10166942 shows extreme population differentiation, with frequencies that range from 5% in Nigeria to 88% in Finland (placing this SNP in the 0.02% tail of the FST empirical distribution). When all populations are jointly analysed, allele frequencies correlate with latitude and temperature beyond what can be explained by shared ancestry and population substructure. Using a Bayesian approach, we infer that the allele originated and evolved neutrally in Africa, while positive selection raised its frequency to different degrees in Eurasian populations, resulting in allele frequencies that follow a latitudinal cline. We infer strong positive selection, in agreement with ancient DNA showing high frequency of the allele in Europe 3,000 to 8,000 years ago. rs10166942 is important phenotypically because its ancestral allele is protective of migraine. This debilitating disorder varies in prevalence across human populations, with highest prevalence in individuals of European descent -precisely the population with the highest frequency of rs10166942 derived allele. We thus hypothesize that local adaptation on previously neutral standing variation may have contributed to the genetic differences that exist in the prevalence of migraine among human populations today.\n\nAuthor SummarySome human populations were likely under strong pressure to adapt biologically to cold climates during their colonization of non-African territories in the last 50,000 years. Such putative adaptations required genetic variation in genes that could mediate adaptive responses to cold. TRPM8 is potentially one such gene, being the only known receptor for the sensation of moderate cold temperature. We show that a likely regulatory genetic variant nearby TRPM8 has several signatures of positive selection rising its frequency in Eurasian populations during the last 25,000 years. While the genetic variant was and is rare in Africa, it is now common outside of Africa, with frequencies that strongly correlate with latitude and are highest in northern European populations. Interestingly, this same genetic variant has previously been strongly associated with migraine. This suggests that adaptation to cold has potentially contributed to the variation in migraine prevalence that exists among human groups today.

genetics↗