Biology subjects

Verma, S.

Publications and source records attributed to Verma, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

KAT3 mutations impair neural crest migration through EMT regulators snai1b and snai2 in Rubinstein Taybi Syndrome

Rubinstein Taybi syndrome, a rare congenital disease is caused by mutation in KAT3 genes, EP300 and CREBBP. A subset of tissues affected in RSTS have their origin in neural crest cells, prompting our exploration into the role of KAT3 in neural crest development. Our zebrafish RSTS models generated by knocking down or mutating ep300a and cbpa genes, reveal defects in neural crest migration and its derived tissues when KAT3 genes are perturbed. We also demonstrate that the effects on neural crest can be reversed by HDAC inhibition in in morphant embryos. KAT3 knockdown causes downregulation of EMT regulators, snai1b and snai2. Snai2 is known to repress cdh6b in neural crest cells facilitating their delamination from neural tube and migration. We generated RSTS patient-derived iPSC line and differentiated them into neural crest cells in vitro. We show that role of KAT3 proteins in neural crest migration is conserved in human iPSC derived neural crest cells. Our findings make a case for classifying RSTS as a neurocristopathy. HighlightsO_LIPerturbation of KAT3 gene expression in zebrafish recapitulates the Rubinstein Taybi patient defects C_LIO_LIThe zebrafish model of Rubinstein Taybi model reveals defects in neural crest cell migration C_LIO_LIKAT3 proteins regulate snai2, snai1b and cdh6, genes important for neural crest migration C_LIO_LIThe neural crest migration defects in the zebrafish model can be partially rescued by modulating the global acetylation levels C_LIO_LIStudy of RSTS patient-derived neural crest cells reveals that the role of KAT3 in neural crest migration is conserved across vertebrates C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/593474v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1761fceorg.highwire.dtl.DTLVardef@220e21org.highwire.dtl.DTLVardef@1cdd311org.highwire.dtl.DTLVardef@1dc0e53_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

Epg5 links proteotoxic stress due to defective autophagic clearance and epileptogenesis in Drosophila and Vici Syndrome patients

Epilepsy is a common neurological condition that arises from dysfunctional neuronal circuit control due to either acquired or innate disorders. Autophagy is an essential neuronal housekeeping mechanism, which causes severe proteotoxic stress when impaired. Autophagy impairment has been associated to epileptogenesis through a variety of molecular mechanisms. Vici Syndrome (VS) is the paradigmatic congenital autophagy disorder in humans due to recessive variants in the ectopic P-granules autophagy tethering factor 5 (EPG5) gene that is crucial for autophagosome-lysosome fusion and ultimately for effective autophagic clearance. VS is characterized by a wide range of neurodevelopmental, neurodegenerative, and neurological features, including epilepsy. Here, we used Drosophila melanogaster to study the importance of epg5 in development, ageing, and seizures. Our data indicate that proteotoxic stress due to impaired autophagic clearance and seizure-like behaviors correlate and are commonly regulated, suggesting that seizures occur as a direct consequence of proteotoxic stress and age-dependent neurodegenerative progression in epg5 Drosophila mutants, in the absence of evident neurodevelopmental abnormalities. We provide complementary evidence from EPG5-mutated patients demonstrating an epilepsy phenotype consistent with Drosophila predictions and propose autophagy stimulating diets as a feasible approach to control EPG5-related pharmacoresistant seizures.

neuroscience↗

bioRxiv2024

Genome-wide association studies in a diverse strawberry collection unveil loci controlling agronomic and fruit quality traits

Strawberries are cherished for their organoleptic properties and nutritional value. However, breeding new cultivars involves the simultaneous selection of many agronomical and fruit quality traits. The strawberry germplasm collection here studied exhibited extensive phenotypic variation in 26 agronomic and fruit quality traits across three consecutive seasons. Phenotypic correlations and Principal Component Analysis (PCA) revealed relationships among traits and accessions, emphasizing the impact of plant breeding. Genetic diversity analysis on 124 accessions using 44,408 markers denoted a population structure divided into six subpopulations that still retain considerable diversity. Genome-wide association studies (GWAS) for the 26 traits unveiled 121 significant marker-trait associations distributed across 95 quantitative trait loci (QTL). Multiple associations were detected for fruit firmness, a key breeding target, including a prominent locus on chromosome 6A. The candidate gene FaPG1, controlling fruit softening, was identified within this QTL region. Differential expression of FaPG1 confirmed its role as the main contributor to natural variation in fruit firmness. A Kompetitive Allele-Specific PCR (KASP) assay based on SNP AX-184242253, associated with the 6A QTL, predicts a substantial increase in fruit firmness, validating its utility for Marker-Assisted Selection (MAS). In essence, this comprehensive study provides insights into the phenotypic and genetic landscape of the strawberry collection and lays a robust foundation for propelling the development of superior strawberry cultivars through precision breeding. HighlightSeveral QTL controlling agronomic and fruit quality traits detected by genome wide association studies. Natural variation on FaPG1 expression is associated with a mayor and stable QTL for fruit firmness.

plant biology↗

bioRxiv2024

A Machine Learning-based approach for Simultaneous Electrochemical Detection of Interfering Analytes

Electrochemical biosensors can be used to detect analytes of importance precisely. These sensors generate rapid and accurate electrical signals that reveal the presence and concentration of the targeted analyte. Detecting multiple analytes simultaneously with an electrochemical biosensor is advantageous. It provides cost and time efficiency, multiplexing capability, and flexibility, making it valuable in diverse applications such as medical diagnostics, environmental monitoring, and industrial processes. However, simultaneous detection of analytes may suffer from the problem of interference. The interference effect causes the signal of an analyte at a particular concentration to deviate from the expected one. We observe a similar effect in the simultaneous detection of Folic Acid and Uric Acid using a nanomaterial-based electrochemical sensor. To address this effect, we propose a machine learning (ML) approach. ML algorithms handle complex interactions by autonomously identifying patterns, dependencies, and nonlinear relationships within data, enabling it to make predictions and decisions in intricate and dynamic scenarios. Our approach can be generalised to any two analytes showing interference and would scale well to interference between multiple analytes. We test several regression algorithms and compare their performance to the standard calibration plot method. As compared to the standard method, our approach shows a 4.49 {micro}M decrease in concentration prediction error.

bioengineering↗

bioRxiv2024

Zinc Alpha-2-Glycoprotein (ZAG/AZGP1) secreted by triple-negative breast cancer promotes tumor microenvironment fibrosis.

Obesity is a predisposition factor for breast cancer, suggesting a localized, reciprocal interaction between breast cancer cells and the surrounding mammary white adipose tissue. To investigate how breast cancer cells alter the composition and function of adipose tissue, we screened the secretomes of ten human breast cancer cell lines for the ability to modulate the differentiation of adipocyte stem and progenitor cells (ASPC). The screen identified a key adipogenic modulator, Zinc Alpha-2-Glycoprotein (ZAG/AZGP1), secreted by triple-negative breast cancer (TNBC) cells. TNBC-secreted ZAG inhibits adipogenesis and instead induces the expression of fibrotic genes. Accordingly, depletion of ZAG in TNBC cells attenuates fibrosis in white adipose tissue and inhibits tumor growth. Further, high expression of ZAG in TNBC patients, but not other clinical subtypes of breast cancer, is linked to poor prognosis. Our findings suggest a role of TNBC-secreted ZAG in promoting the transdifferentiation of ASPCs into cancer-associated fibroblasts to support tumorigenesis.

cancer biology↗

bioRxiv2024

The complement regulator CD55 modulates TLR9 signaling and supports survival in marginal zone B cells

Marginal zone (MZ) B cells bridge innate and adaptive immunity by sensing bloodborne antigens and producing rapid antibody and cytokine responses. CD55 is a membrane-bound complement regulator that interferes with complement activation, an important component of innate immunity. CD55 also regulates adaptive immunity--CD55 downregulation is critical for germinal center reactions. MZ B cells also express low CD55, but its role in MZ B cell function is unknown. Using germline knockout mice, we found that similar numbers of MZ B cells are initially established in 3-week-old CD55-deficient mice compared to wild-type (WT) mice. However, MZ B cells fail to accumulate as mice age and undergo increased apoptosis. Following ex vivo stimulation of MZ B cells through Toll-like receptor 9, we observed a proinflammatory phenotype with increased IL-6 expression. These findings demonstrate a critical role for CD55 in supporting MZ B cell survival while also regulating cellular function.

immunology↗

bioRxiv2024

Methyl substituted β-lactam framework based antibiotics and β-lactamase inhibitors: Proof of concept by computations

Increasing the life-time of the acyl-enzyme complex formed between an inhibitor or drug molecule and the {beta}-lactamase through chemical modifications of existing drug molecules is an important strategy towards developing inhibitors. In this direction, our group proposed a methyl-substituted {beta}-lactam framework for the design of inhibitors for {beta}-lactamases (J. Phys. Chem. B. 2018, 122, 4299). This unconventional design was guided by the transition state structure of the deacylation reaction of the acyl-enzyme complex. Here, we present a proof of principle study of this concept through detailed molecular simulations and free energy calculations. In particular, we improve the antimicrobial activity of the first-generation cephalosporin antibiotic, cephalothin, through C6-methylation. The proposed molecule, (6R,7R)-3-(acetyloxymethyl)-6-methyl-8-oxo-7-[(2-thiophen-2-ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate) slows down the deacylation of the acyl-enzyme complex 109-fold with no apparent effect on its binding to class-C {beta}-lactamase and formation of the acyl-enzyme intermediate. The design strategy presented in this work can be further extended to all {beta}-lactam antibiotics, like monobactams, carbapenems, cephalosporins, and penicillins.

biochemistry↗

bioRxiv2023

A mitochondrial quality control mechanism reverses the phagosome maturation arrest caused by Mycobacterium tuberculosis

Phagosome maturation arrest (PMA) imposed by Mycobacterium tuberculosis (Mtb) is a classic tool that helps Mtb evade macrophage anti-bacterial responses. The exclusion of RAB7, a small GTPase, from Mtb-phagosomes causes PMA. Here, we report an unexpected mechanism that triggers crosstalk between the mitochondrial quality control (MQC) and the phagosome maturation pathways that reverses the PMA. CRISPR-mediated p62/SQSTM1 depletion (p62KD) does not appear to impact mitochondrial quality. The p62KD cells are restrictive to Mtb growth, triggered by an increasingly oxidative environment and increased lysosomal targeting. The lysosomal targeting of Mtb is facilitated by enhanced TOM20+ mitochondria-derived vesicles (MDVs) biogenesis, a key MQC mechanism. In p62KD cells, TOM20+-MDVs biogenesis is MIRO1/MIRO2-dependent and gets delivered to lysosomes for degradation in a RAB7-dependent manner. Upon infection in p62KD cells, TOM20+-MDVs get extensively targeted to Mtb-phagosomes, inadvertently facilitating RAB7 recruitment, PMA reversal and lysosomal targeting of Mtb; the phenotype also replicated in p62/SQSTM1 knockout cells. Triggering MQC collapse in p62KDcells further diminishes Mtb survival, signifying cooperation between redox- and lysosome-mediated mechanisms. The MQC-anti-bacterial pathway crosstalk could be exploited for host-directed anti-tuberculosis therapies.

microbiology↗

bioRxiv2023

SARS-CoV-2 infection leads to sustained testicular injury and functional impairments in K18 hACE2 mice

Compromised male reproductive health is one of the symptoms of long COVID with a decrease in male fertility markers including testosterone levels and sperm count for months in recovering patients. However, the long-term impact of SARS-CoV-2 infection on testicular injury and underlying mechanisms remains unknown. We previously demonstrated a disrupted tissue architecture with no evidence of virus replication in the testis during the acute stage of the disease in K18-hACE2 mice. Here, we systematically delineate the consequences of SARS-CoV-2 infection on the testis injury and function both during the acute stage of the disease and up to 4 weeks after infection in survivor K18-hACE2 mice. The gross morphological defects included sloughing of healthy spermatids and spermatocytes into the lumen, lack of lumen, and increase in apoptotic cells that sustained for at least 2 weeks after infection. Testis injury correlated with systemic and testicular inflammation, and infiltration of immune cells in the interstitial space and seminiferous tubules. Transcriptomic analysis identified dysregulation of key pathways of testicular immune homeostasis, spermatogenesis, and cell death at the symptomatic and short-term recovery stages. Further, a significant reduction in testosterone levels was associated with transient reduction in sperm count and mouse fertility. Most of the testicular impairments except testosterone levels were resolved within 4 weeks, which is almost one spermatogenesis cycle in mice. These findings provide much-needed mechanistic insights beyond our current understanding of testicular pathogenesis, suggesting that recovering COVID-19 patients should be closely monitored to rescue the pathophysiological effects on male reproductive health.

microbiology↗

bioRxiv2023

Antigen-level resolution of commensal-specific B cell responses enabled by phage-display screening and B cell tetramers

Induction of adaptive immune responses to commensal microbes is critical for tissue homeostasis, and perturbation of these responses is associated with multiple chronic inflammatory disorders. However, the mechanisms underlying the induction and regulation of mucosal B cells targeting commensal microbes remain poorly understood, in part due to a lack of tools to identify commensal-specific B cells ex vivo. To address this, we identified immunogenic protein epitopes recognized by Segmented Filamentous Bacteria (SFB)-specific serum antibodies using a whole-genome phage display screen and identified immunogenic proteins engaging IgA, IgG1 and IgG2b responses. Using these antigens, we generated B cell tetramers to identify and track SFB-specific B cell responses in the gut associated lymphoid tissue during natural and de novo colonization. We revealed a compartmentalized response in SFB-specific B cell activation between Peyers patches and mesenteric lymph nodes, with a gradient of IgA, IgG1 and IgG2b isotypes along the small intestine, and selective production of IgG2b with the mesenteric lymph node chain. VDJ sequencing analyses and generation of SFB-specific monoclonal antibodies identified that somatic hypermutation drives affinity maturation to SFB derived antigens under homeostatic conditions. By combining phage display screening and B cell tetramer technologies, we now enable antigen-level based studies of immunity to intestinal microbes, which will advance our understanding of the ontogeny and function of commensal-specific B cell responses in tissue immunity, inflammation and repair.

immunology↗

bioRxiv2023

Co-evolution of spheroid fluidization and mechanical fracture in a morphogenic cascade

Cells migrate and invade tissues during development, immune responses, and cancer. Collective invasion is generally understood to be driven by invading cells unjamming and pushing through barriers such as the extracellular matrix and surrounding tissues. Whether these barriers actively contribute to invasion remains unclear. Using ovarian adenocarcinoma spheroids invading mesothelium derived from benign pleural effusions as an experimental model, combined with modelling, we examine invasion across molecular to multicellular scales. We identify intercellular integrin adhesions linking invasive leader cells to the tissue barrier, triggering apical constrictions within the barrier. This constriction shrinks cell-cell contacts, leading to barrier rupture. Thus, the tissue barrier plays a mechanically active role in invasion. Rather than cells pushing through, we find that coordinated subcellular contractility between the invading leader cell and the barrier drives barrier tensile rupture and invasion, independent of a jamming transition. Together, our findings challenge prevailing paradigms of collective cell invasion.

cell biology↗

bioRxiv2023

Pre-existing tissue mechanical hypertension at adherens junctions disrupts apoptotic extrusion in epithelia

Apical extrusion is a tissue-intrinsic process that allows epithelia to eliminate unfit or surplus cells. This is exemplified by the early extrusion of apoptotic cells, which is critical to maintain the epithelial barrier and prevent inflammation. Apoptotic extrusion is an active mechanical process, which involves mechanotransduction between apoptotic cells and their neighbours, as well as local changes in tissue mechanics. Here we report that the pre-existing mechanical tension at adherens junctions conditions the efficacy of apoptotic extrusion. Specifically, increasing baseline mechanical tension by overexpression of a phosphomimetic Myosin II regulatory light chain (MRLC) compromises apoptotic extrusion. This occurs when tension is increased in either the apoptotic cell or its surrounding epithelium. Further, we find that the pro-inflammatory cytokine, TNF, stimulates Myosin II and increases baseline AJ tension to disrupt apical extrusion, causing apoptotic cells to be retained in monolayers. Importantly, reversal of mechanical tension with an inhibitory MRLC mutant or tropomyosin inhibitors is sufficient to restore apoptotic extrusion in TNF-treated monolayers. Together, these findings demonstrate that baseline levels of tissue tension are important determinants of apoptotic extrusion, which can potentially be co-opted by pathogenetic factors to disrupt the homeostatic response of epithelia to apoptosis.

cell biology↗

bioRxiv2023

A subclass of evening cells promotes the switch from arousal to sleep at dusk

Animals exhibit rhythmic patterns of behavior that are shaped by an internal circadian clock and the external environment. While light intensity varies across the day, there are particularly robust differences at twilight (dawn/dusk). These periods are also associated with major changes in behavioral states, such as the transition from arousal to sleep. However, the neural mechanisms by which time and environmental conditions promote these behavioral transitions are poorly defined. Here, we show that the E1 subclass of Drosophila evening clock neurons promotes the transition from arousal to sleep at dusk. We first demonstrate that the cell-autonomous clocks of E2 neurons alone are required to drive and adjust the phase of evening anticipation, the canonical behavior associated with "evening" clock neurons. We next show that conditionally silencing E1 neurons causes a significant delay in sleep onset after dusk. However, rather than simply promoting sleep, activating E1 neurons produces time- and light- dependent effects on behavior. Activation of E1 neurons has no effect early in the day, but then triggers arousal before dusk and induces sleep after dusk. Strikingly, these phenotypes critically depend on the presence of light during the day. Despite their influence on behavior around dusk, in vivo voltage imaging of E1 neurons reveals that their spiking rate does not vary between dawn and dusk. Moreover, E1-specific clock ablation has no effect on arousal or sleep. Thus, we suggest that, rather than specifying "evening" time, E1 neurons act, in concert with other rhythmic neurons, to promote behavioral transitions at dusk.

neuroscience↗

bioRxiv2023

Caveola mechanotransduction reinforces the cortical cytoskeleton to promote epithelial resistance

As physical barriers, epithelia must preserve their integrity when challenged by mechanical stresses. Cell-cell junctions linked to the cortical cytoskeleton play key roles in this process, often with mechanotransduction mechanisms that reinforce tissues. Caveolae are mechanosensitive organelles that buffer tension via disassembly. Loss of caveolae, through caveolin-1 or cavin1 depletion, causes activation of PtdIns(4, 5)P2 signalling, recruitment of FMNL2 formin, and enhanced cortical actin assembly. How this equates to physiological responses in epithelial cells containing endogenous caveolae is unknown. Here we examined the effect of mechanically-inducing acute disassembly of caveolae in epithelia. We show that perturbation of caveolae, through direct mechanical stress, reinforces the actin cortex at adherens junctions. Increasing interactions with membrane lipids by introducing multiple phosphatidylserine-binding undecad cavin1 (UC1) repeat domains into cavin1 rendered caveolae more stable to mechanical stimuli. This molecular stabilization blocked cortical reinforcement in response to mechanical stress. Cortical reinforcement elicited by the mechanically-induced disassembly of caveolae increased epithelial resilience against tensile stresses. These findings identify the actin cortex as a target of caveola mechanotransduction that contributes to epithelial integrity.

cell biology↗

bioRxiv2023

Desmosome-anchored intermediate filaments facilitate tension-sensitive RhoA signaling for epithelial homeostasis

Epithelia are subject to diverse forms of mechanical stress during development and post-embryonic life. They possess multiple mechanisms to preserve tissue integrity against tensile forces, which characteristically involve specialized cell-cell adhesion junctions coupled to the cytoskeleton. Desmosomes connect to intermediate filaments (IF) via desmoplakin (DP) 1,2, while the E-cadherin complex links to the actomyosin cytoskeleton in adherens junctions (AJ) 3. These distinct adhesion-cytoskeleton systems support different strategies to preserve epithelial integrity, especially against tensile stress. IFs coupled to desmosomes can passively respond to tension by strain-stiffening 4-10, whereas for AJs a variety of mechanotransduction mechanisms associated with the E-cadherin apparatus itself 11,12, or proximate to the junctions 13, can modulate the activity of its associated actomyosin cytoskeleton by cell signaling. We now report a pathway where these systems collaborate for active tension-sensing and epithelial homeostasis. We found that DP was necessary for epithelia to activate RhoA at AJ on tensile stimulation, an effect that required its capacity to couple IF to desmosomes. DP exerted this effect by facilitating the association of Myosin VI with E-cadherin, the mechanosensor for the tension-sensitive RhoA pathway at AJ 12. This connection between the DP-IF system and AJ-based tension-sensing promoted epithelial resilience when contractile tension was increased. It further facilitated epithelial homeostasis by allowing apoptotic cells to be eliminated by apical extrusion. Thus, active responses to tensile stress in epithelial monolayers reflect an integrated response of the IF- and actomyosin-based cell-cell adhesion systems.

cell biology↗

bioRxiv2023

Apical extrusion prevents apoptosis from activating an acute inflammatory program in epithelia.

Apoptosis is traditionally considered to be an immunologically silent form of cell death. Multiple mechanisms exist to ensure that apoptosis does not stimulate the immune system to cause inflammation or autoimmunity. Against this expectation, we now report epithelia are programmed to provoke, rather than suppress, inflammation in response to apoptosis. We found that an acute inflammatory response led by neutrophils occurs in zebrafish and cell culture when apoptotic epithelial cells cannot be expelled from the monolayer by apical extrusion. This reflects an intrinsic circuit where ATP released from apoptotic cells stimulates epithelial cells in the immediate vicinity to produce IL-8. As the epithelial barrier is compromised when apical extrusion fails, this juxta-apoptotic proinflammatory pathway may represent an early-response mechanism at sites of potential microbial ingress. Conversely, apical extrusion prevents inappropriate epithelial inflammation by physically eliminating apoptotic cells before they can activate this proinflammatory circuit.

cell biology↗

bioRxiv2022

Telomere-to-Telomere and Haplotype-Phased Genome Assemblies of the Heterozygous Octoploid 'Florida Brilliance' Strawberry (Fragaria x ananassa)

The available haplotype-resolved allo-octoploid strawberry (Fragaria x ananassa Duch.) (2n = 8x = 56) genomes were assembled with the trio-binning pipeline, supplied with parental short-reads. We report here a high-quality, haplotype-phased genome assembly of a short-day cultivar, Florida Brilliance (FaFB2) without the use of parental sequences. Using Pacific Biosciences (PacBio) long reads and high-throughput chromatic capture (Hi-C) data, we completed telomere-to-telomere phased genome assemblies of both haplotypes. The N50 continuity of the two haploid assemblies were 23.7 Mb and 26.6 Mb before scaffolding and gap-filling. All 56 pseudochromosomes from the phased-1 and phased-2 assemblies contained putative telomere sequences at the 5 and/or 3 ends. A high level of collinearity between the haplotypes was confirmed by high-density genetic linkage mapping with 10,269 SNPs, and a high level of collinearity with the Royal Royce FaRR1 reference genome was observed. Genome completeness was further confirmed by consensus quality. The LTR assembly Index score for entire genome assembly was 19.72. Moreover, the BUSCO analysis detected over 99% of conserved genes in the combined phased-1 and phased-2 assembly. Both haploid assemblies were annotated using Iso-Seq data from six different Florida Brilliance tissues and RNA-Seq data representing various F. x ananassa tissues from the NCBI sequence read archive, resulting in a total of 104,099 genes. This telomere-to-telomere reference genome of Florida Brilliance will advance our knowledge of strawberry genome evolution and gene functions, and facilitate the development of new breeding tools and approaches.

genomics↗

bioRxiv2022

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury.

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ failure including testicular injury and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury can likely result either from direct virus infection of resident cells or by exposure to systemic inflammatory mediators or virus antigens. We here characterized SARS-CoV-2 infection in different human testicular 2D and 3D models including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not establish a productive infection in any testicular cell types. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma depicted a significant decrease in cell viability and death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 envelope protein, but not Spike or nucleocapsid proteins led to cytopathic effects on testicular cells that was dependent on the TLR2 receptor. A similar trend was observed in the K18h-ACE2 mouse model which revealed gross pathology in the absence of virus replication in the testis. Collectively, data strongly indicates that the testicular injury is not due to direct infection of SARS-CoV-2 but more likely an indirect effect of exposure to systemic inflammation or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

microbiology↗