Biology subjects

Bhat, S.

Publications and source records attributed to Bhat, S..

At least 19 recordsLinked to original sources

bioRxiv2025

Chromosomal genome assemblies of Antarctic Notothenia coriiceps and temperate relative Paranotothenia angustata

Antarctic notothenioid fish genomes contain evidence of an ancient evolutionary history including their remarkable adaptive radiation beginning [~]10 MYA amid extreme selective pressures of Southern Ocean glaciation. Several Antarctic lineages subsequently escaped and recolonized temperate waters, thus their genomes chronicle the re-adaptation to warmer climates. Here, we report two chromosome-scale genomes assembled from HiFi long reads and Hi-C data for the Antarctic cryonotothenioid Notothenia coriiceps and its secondarily temperate close relative Paranotothenia angustata. The N. coriiceps assembly comprises 11 pseudo-chromosomes, spans 1.12 Gbp with a N90 of 81.48 Mbp and 97.8% gene complete with 24,272 annotated genes. The P. angustata assembly comprises 13 pseudo-chromosomes, spans 1.00 Gbp with a N90 of 62.3 Mbp and 97.9% gene complete with 23,828 annotated genes. We report two new N. coriiceps mitochondrial genome structures, map the antifreeze glycoprotein loci, and corroborate the unusual notothenioid ribosomal RNA gene arrangement. The high-quality assemblies of this species pair provide valuable resources for investigating both the historical evolution of polar fish and their adaptive potential to climate change.

genomics↗

bioRxiv2025

Hypoxia-induced metastatic heterogeneity in pancreatic cancer

In most solid tumors, hypoxia constitutes a defining microenvironmental feature that reprograms malignant cells into a highly metastatic state by driving cellular plasticity and exacerbating chromosomal instability (CIN). However, the mechanisms by which cancer cells concurrently co-opt these elements of hypoxic adaptation to promote metastasis remains poorly understood. Here, we report that hypoxia promotes metastasis by suppressing the JmjC-containing histone lysine demethylase Kdm8. CRISPR/Cas9-mediated targeting of Kdm8 in a Kras;Trp53-driven mouse model of pancreatic ductal adenocarcinoma (PDA) robustly rewires the malignant cell transcriptomic programs, leading to a profound loss of the epithelial morphology and widespread metastatic disease. In PDA patients, a high KDM8-induced gene signature is associated with reduced metastatic burden and better survival in advanced disease. Notably, Kdm8 suppression in normoxia recapitulates key aspects of the global epigenetic and transcriptomic rewiring, mitotic spindle defects, and CIN induced by hypoxia. Moreover, disruption of Kdm8s demethylase activity phenocopies Kdm8 loss, whereas expression of hypermorphic Kdm8 variants resistant to hypoxic suppression markedly reduces metastasis beyond the levels achieved by the wildtype protein. Through the suppression of Kdm8 demethylase function, hypoxia unleashes a potent metastatic program by simultaneously advancing cellular plasticity and CIN.

cell biology↗

bioRxiv2025

Therapeutic Potential of Cyclodextrins Targeting Dengue Virus and SARS-CoV-2 Infection and Pathogenesis

Cyclodextrins (CDs) are cyclic oligosaccharides with promising therapeutic applications, including antiviral activity. During viral infections, pathogenesis arises not only from viral replication but also from viral proteins that act as "toxins", disrupting cellular barriers and inducing endothelial dysfunction, a hallmark of severe diseases such as dengue and COVID-19. Dengue virus (DENV) NS1 and SARS-CoV-2 Spike proteins induce endothelial hyperpermeability, contributing to severe complications. Here we explored the potential of a panel of 18 CDs in mitigating endothelial dysfunction caused by these viral proteins and evaluated the CDs antiviral activity in vitro and in vivo. The effect of CDs on endothelial hyperpermeability was assessed using a trans-endothelial electrical resistance assay with human pulmonary microvascular endothelial cells exposed to DENV NS1 and SARS-CoV-2 Spike proteins. Antiviral efficacy of CDs was evaluated in Vero cells infected with DENV2 and Calu-3 cells infected with SARS-CoV-2, and in vivo protection was assessed in a lethal DENV2 mouse model. CDs effectively inhibited DENV NS1-induced endothelial hyperpermeability in vitro, demonstrating their potential to counteract NS1-mediated barrier disruption. In the murine model, CD1 treatment provided partial protection against DENV-induced morbidity and mortality. Further, CDs significantly reduced SARS-CoV-2 infection in vitro and inhibited Spike-induced endothelial dysfunction. These findings indicate that CDs can prevent endothelial hyperpermeability induced by DENV NS1 and SARS-CoV-2 Spike proteins and exhibit antiviral activity against SARS-CoV-2, positioning them as promising candidates for mitigating endothelial complications associated with viral infections. Further research is needed to explore the clinical relevance of CDs and their mechanisms of action.

microbiology↗

bioRxiv2025

The G57 genotype of the BJ/94-like H9N2 lineage exhibits increased replication and virulence in chickens compared to the G1 Middle East Group B lineage

Avian influenza H9N2 viruses cause significant economic losses to the poultry industry and pose a public health risk due to their potential to reassort with other avian influenza viruses, generating strains with zoonotic and pandemic potential. Two major H9N2 lineages dominate globally: the G1 lineage (genotype G1-B), prevalent in the Middle East, Africa and the Indian subcontinent, and the BJ/94 lineage (predominantly genotype G57), dominant in China, Vietnam, South Korea, Indonesia, and the Far East. We investigated replication, transmission, and pathogenicity of representatives of these two lineages, linking genotype to phenotype. The G57 strain A/Ck/Vietnam/H7F-14-BN4-315/2014 (Vietnam/315) was more lethal to chicken embryos than the G1-B strain A/chicken/Pakistan/UDL-01/2008 (Pakistan/UDL-01). Vietnam/315 exhibited higher replication in both directly infected and contact chickens, with increased virus shedding from the oropharynx and cloaca. In contrast, Pakistan/UDL-01 virus was primarily shed from the oropharynx, highlighting differences in replication, tissue tropism and transmission. Gene analysis showed the M gene of Vietnam/315 enhanced replication in primary chicken kidney cells, whereas the PB2, HA, NA, and M genes promoted increased replication in Madin-Darby Canine Kidney cells. Both viruses showed preferential binding to avian-like receptors over human-like receptors. However, Vietnam/315, however, exhibited higher neuraminidase activity and a more acid-stable HA (pH fusion 5.2) than Pakistan/UDL-01 virus. These findings suggest G57 genotype viruses possess greater replication and transmission fitness than G1-B viruses in vivo, ex vivo and in vitro. Reassortment events involving G1-B strains acquiring G57 genes may enhance replication and virulence, potentially increasing the risk of animal and human infection. ImportanceH9N2 avian influenza viruses are widespread in poultry, resulting in significant economic losses and occasional human infections. Different genetic variants dominate in various regions, but their ability to cause disease and spread in poultry remains unclear. These viruses can exchange genes with other avian influenza strains, altering their infectivity and transmission. This study compared two major H9N2 genotypes: G1-B (common in the Indian subcontinent and the Middle East) and G57 (dominant in China and Vietnam). The G57 virus showed higher replication in laboratory tests and infected chickens, shedding more virus through the orofecal route. It also exhibited a stronger attachment to bird cells and had a more acid-stable HA, suggesting an increased potential for infection and spread. Our findings indicate that G57 genotype viruses are more infectious and adaptable in poultry than G1 viruses. Gene exchange with other avian influenza strains may generate more virulent viruses with increased transmission potential. This study supports the risk assessment of emerging strains and enhances disease mitigation strategies.

microbiology↗

bioRxiv2025

Rapid Detection of Carbapenemase-Producing Organisms Using a Luminescent Biosensor

Carbapenemase-producing organisms (CPOs) pose an urgent global health threat due to their ability to inactivate carbapenems, a group of last resort antibiotics. Infections caused by these pathogens are associated with poor patient outcomes, high mortality rates, and added burden to infection prevention and control programs, making early detection vital to ensure optimal antimicrobial therapy and appropriate implementation of infection control practices. In this study, we report the application of a luminescent whole-cell biosensor for the rapid detection of CPOs. This biosensor provides positive test results within 2.5 hours, inclusive of setup time, and has been validated with a panel of laboratory and clinical isolates producing a diverse range of carbapenemases (KPC, NDM, IMP, VIM and OXA-48-like). The assay successfully identified all CPO isolates tested with a sensitivity of 100%, including strains producing weak OXA-48-like carbapenemases which are sometimes missed by currently used detection methods. The assay also demonstrated a specificity of 100 %, with all non-CPO clinical isolates testing negative under the optimized assay conditions. Due to the rapid time-to-positivity, minimal setup requirements, and high sensitivity, this test could serve as an attractive alternative to CPO detection methods currently employed by clinical microbiology labs, and could also facilitate CPO screening in other settings (e.g., environmental, agricultural).

microbiology↗

bioRxiv2024

Notch signaling stabilizes lengths of motile cilia in multiciliated cells in the lung

Airway multiciliated cells (MCs) maintain respiratory health by clearing mucus and trapped particles through the beating of motile cilia. While it is known that ciliary lengths decrease along the proximal-distal (P-D) axis of the tracheobronchial tree, how this is regulated is unclear. Here, we demonstrate that canonical Notch signaling in MCs plays a critical role in stabilizing ciliary length. Inhibition of Notch signaling in MCs results in ciliary shortening in the trachea, lengthening in the distal airway, and to region-specific alterations in gene expression. We probe how environmental challenges impact MC homeostasis using germ-free and Mycobacterium tuberculosis (M. tb) infection models. While germ-free conditions do not perturb ciliary lengths, M. tb infection leads to lengthening of distal airway cilia, correlating with a downregulation of Notch signaling. These findings reveal that ciliary length and the P-D gradient in the airways are actively regulated, with Notch signaling serving as a stabilizing mechanism.

cell biology↗

bioRxiv2024

Mechanism-based approach in designing patient-specific combination therapies for nonsense mutation diseases

Premature termination codon (PTC) diseases, arising as a consequence of nonsense mutations in a patients DNA, account for approximately 12% of all human disease mutations. Currently there are no FDA approved treatments for increasing PTC readthrough in nonsense mutation diseases, although one translational readthrough inducing drug, ataluren, has had conditional approval for treatment of Duchenne muscular dystrophy in Europe and elsewhere for 10 years. Ataluren displays consistent low toxicity in clinical trials for treatment of several different PTC diseases, but its therapeutic effects on such diseases are inconsistent. The identity of the stop codon and its sequence context are major determinants of PTC readthrough efficiency in both the absence and presence of nonsense suppressors. Previously we have shown that ataluren stimulates readthrough exclusively by competitively inhibiting release factor complex (RFC, eRF1.eRF3.GTP)-dependent catalysis of translation termination. Here, using an in vitro reconstituted system (PURE-LITE) and both ensemble and single molecule assays, we demonstrate that PTC identity and the immediately adjacent mRNA sequence contexts modulate the catalytic activity of RFC in terminating peptide elongation. Such modulation largely determines the effectiveness of ataluren in stimulating readthrough, whether added alone or in combination with either the aminoglycoside G418 or an anticodon edited aa-tRNA, each of which stimulate readthrough by mechanisms orthogonal to that of ataluren. Our results provide an attractive rationale for the variability of ataluren effectiveness in stimulating readthrough in clinical trials. Patients harboring a PTC mutation with a sequence context promoting strong interaction with RFC are predicted to be resistant to ataluren, whereas ataluren treatment should be more effective for patient sequences conferring weaker interaction with RFC.

biochemistry↗

bioRxiv2024

Kingdom-specific lipid unsaturation shapes up sequence evolution in membrane arm subunits of eukaryotic respiratory complexes

Sequence evolution of protein complexes (PCs) is constrained by protein-protein interactions (PPIs). PPI-interfaces are predominantly conserved and hotspots for disease-related mutations. How lipid-protein interactions (LPIs) constrain sequence evolution of membrane- PCs? We explore Respiratory Complexes (RCs) as a case study as these allow to compare sequence evolution in subunits exposed to both lipid-rich inner-mitochondrial membrane (IMM) and aqueous matrix. We find that lipid-exposed surfaces of the IMM-subunits but not of the matrix subunits are populated with non-PPI disease-causing mutations signifying LPIs in stabilizing RCs. Further, IMM-subunits including their exposed surfaces show high intra- kingdom sequence conservation but remarkably diverge beyond. Molecular Dynamics simulation suggests contrasting LPIs of structurally superimposable but sequence-wise diverged IMM-exposed helices of Complex I (CI) subunit Ndufa1 from human and Arabidopsis depending on kingdom-specific unsaturation of cardiolipin fatty acyl chains. in cellulo assays consolidate inter-kingdom incompatibility of Ndufa1-helices due to the lipid- exposed amino acids. Plant-specific unsaturated fatty acids in human cells also trigger CI- instability. Taken together, we posit that altered LPIs calibrate sequence evolution at the IMM-arms of eukaryotic RCs.

cell biology↗

bioRxiv2024

KCNG4 genetic variant linked to migraine prevents expression of KCNB1

AO_SCPLOWBSTRACTC_SCPLOWMigraines are a common type of headaches affecting around 15% of the population. The signalling pathways leading to migraines have not been fully understood, but neuronal voltage-gated ion channels have been linked to this pathology, among them KCNG4. KCNG4 (Kv6.4) is a silent member of the superfamily of voltage-gated potassium (Kv) channels, which expresses in heterotetramers with members of the KCNB (Kv2) family. The genetic variant Kv6.4-L360P has previously been linked to migraine, but their mode of action remains unknown. Here, we characterized the molecular characteristics of Kv6.4-L360P when co-expressed with Kv2.1. We found that Kv6.4-L360P almost completely abolishes Kv2 currents, and we propose that this mechanism in the trigeminal system, linked to the initiation of migraine, leads to the pathology.

biophysics↗

bioRxiv2023

An explainable machine learning data analytics method using TIGIT-linked genes for identifying biomarker signatures to clinical outcomes.

In the last decade, immunotherapies targeting immune checkpoint inhibitors have been extremely effective in eliminating subsets of some cancers in some patients. Multi-modal immune and non-immune factors that contribute to clinical outcomes have been utilized for predicting response to therapies and developing diagnostics. However, these data analytic methods involve a combination of complex mathematical data analytics, and even-more complex biological mechanistic pathways. In order to develop a method for data analytics of transcriptomics data sets, we have utilized an explainable machine learning (ML) model to investigate the genes involved in the signaling pathway of T-cell-immunoreceptor with immunoglobulin and ITIM domain (TIGIT). TIGIT is a receptor on T, NK, and T-regulatory cells, that has been classified as a checkpoint inhibitor due to its ability to inhibit innate and adaptive immune responses. We extracted gene whole genome sequencing data of 1029 early breast cancer patient tumors, and adjacent normal tissues from the TCGA and UCSC Xena Data Hub public databases. We followed a workflow which involved the following steps: i) data acquisition, processing, and visualization followed by ii) developed of a predictive prognostic model using input (gene expression data) and output (survival time) parameters iii) model interpretation was performed by calculating SHAP (Shapely-Additive-exPlanations); iv) the application of the model involved a Cox-regression model, trained with L-2 regularization and optimization using 5 fold cross validation. The model identified gene signatures associated with TIGIT that predicted survival outcome with a test set with a score of 0.601. In summary, we have utilized this case study of TIGIT-mediated signaling pathways to develop a roadmap for biologists to harness ML methods effectively.

cancer biology↗

bioRxiv2023

Hierarchical regulation of functionally antagonistic neuropeptides expressed in a single neuron pair

Neuronal communication involves small-molecule transmitters, gap junctions, and neuropeptides. While neurons often express multiple neuropeptides, our understanding of the coordination of their action and their mutual interactions remains limited. Here, we demonstrate that two neuropeptides, NLP-10 and FLP-1, released from the same interneuron pair, AVKL/R, exert antagonistic effects on locomotion speed in Caenorhabditis elegans. NLP-10 accelerates locomotion by activating the G protein-coupled receptor NPR- 35 on premotor interneurons that promote forward movement. Notably, we establish that NLP-10 is crucial for the aversive response to mechanical and noxious light stimuli. Conversely, AVK-derived FLP-1 slows down locomotion by suppressing the secretion of NLP-10 from AVK, through autocrine feedback via activation of its receptor DMSR-7 in AVK neurons. Our findings suggest that peptidergic autocrine motifs, exemplified by the interaction between NLP-10 and FLP-1, might represent a widespread mechanism in nervous systems across species. These mutual functional interactions among peptidergic co-transmitters could fine-tune brain activity. Highlights* A pair of neurons elicits opposing behaviors via two distinct neuropeptides * The neuropeptide NLP-10 accelerates locomotion by activating premotor interneurons * Release of the neuropeptide FLP-1 reduces NLP-10 release via autocrine feedback * NLP-10 is crucial for escaping aversive mechanical stimuli and noxious blue light eTOC blurbNeuropeptidergic communication is complex due to the hundreds of proteinaceous transmitters and receptors involved. Aoki and colleagues demonstrate a further layer of regulation, where one neuropeptide negatively influences release of an antagonistic neuropeptide by autocrine feedback, in a single neuron pair. This may represent a general principle in neuropeptidergic signaling.

neuroscience↗

bioRxiv2023

Investigation of H9N2 avian influenza immune escape mutant that lacks haemagglutination activity

H9N2 avian influenza viruses pose a global threat to animal and human health. While vaccination is essential for mitigating disease impact, these viruses evolve to evade vaccine immunity through changes in the haemagglutinin (HA) glycoprotein. In this study, we identified immune escape mutation in an H9N2 virus resulting from pressure exerted by homologous chicken antisera. The immune-escape variant acquired an amino acid substitution, replacing glycine (G) with glutamic acid (E) at position 149 in the HA protein. The G149E mutant virus lost the ability to agglutinate chicken erythrocytes, while still maintaining replication comparable to the wild-type virus in chicken embryos and cells. This led to the hypothesis that the G149E substitution, leading to a shift from a neutral to a negative charge polarity at HA position 149, might be crucial for the optimal interaction between the virus and receptors on erythrocytes. Investigation indicated that agglutination could be restored by substituting E to positively charged amino acids histidine (H), arginine (R) or lysine (K). These findings suggest that the H9N2 virus may be likely acquire the G149E mutation under immune pressure in nature. This mutation poses challenges to vaccination and surveillance efforts as it partially evades immune protection and is not easily detectable by conventional haemagglutination assays. This underscores the intricate interplay between antigenic variation and viral traits, emphasising the critical need for ongoing surveillance and research to effectively mitigate the risks associated with avian influenza H9N2 viruses. IMPORTANCEUnderstanding how avian influenza viruses evolve to persist in nature is crucial for enhancing disease mitigation tools such as vaccines, diagnostics, and risk assessment. In this study, we identified an H9N2 virus antibody escape mutant with G149E mutation in the haemagglutinin that had lost the ability to agglutinate chicken erythrocytes, while retaining infectivity and replication fitness. The lack of haemagglutination activity potentially negatively impacts routine surveillance and commonly used diagnostics such as haemagglutination assay or haemagglutination inhibition assay. Therefore, it is urgent to develop and adopt alternative methods for viral detection. Difficult to detect variants potentially that are not compatible with common surveillance techniques could circulate remain silent while reassort with other influenza viruses, which posing unpredictable risks to animal and human health. This research helps us better understand avian influenza, leading to improved disease control, diagnostics, and risk assessment to protect both animals and humans.

microbiology↗

bioRxiv2023

Hydrogel-Polyurethane Fiber Composites With Enhanced Microarchitectural Control For Heart Valve Replacement

Polymeric heart valves offer the potential to overcome the limited durability of tissue based bioprosthetic valves and the need for anticoagulant therapy of mechanical valve replacement options. However, developing a single-phase material with requisite biological properties and target mechanical properties remains a challenge. In this study, a composite heart valve material was developed where an electrospun mesh provides tunable mechanical properties and a hydrogel coating confers an antifouling surface for thromboresistance. Key biological responses were evaluated in comparison to glutaraldehyde-fixed pericardium. Platelet and bacterial attachment were reduced by 38% and 98%, respectively, as compared to pericardium that demonstrated the antifouling nature of the hydrogel coating. There was also a notable reduction (59%) in the calcification of the composite material as compared to pericardium. A custom 3D printed hydrogel coating setup was developed to make valve composites for device-level hemodynamic testing. Regurgitation fraction (9.6 {+/-} 1.8%) and effective orifice area (1.52 {+/-} 0.34 cm2) met ISO 5840-2:2021 requirements. Additionally, the mean pressure gradient was comparable to current clinical bioprosthetic heart valves demonstrating preliminary efficacy. Although the hemodynamic properties are promising, it is anticipated that the random microarchitecture will result in suboptimal strain fields and peak stresses that may accelerate leaflet fatigue and degeneration. Previous computational work has demonstrated that bioinspired fiber microarchitectures can improve strain homogeneity of valve materials toward improving durability. To this end, we developed advanced electrospinning methodologies to achieve polyurethane fiber microarchitectures that mimic or exceed the physiological ranges of alignment, tortuosity, and curvilinearity present in the native valve. Control of fiber alignment from a random fiber orientation at a normalized orientation index (NOI) 14.2 {+/-} 6.9% to highly aligned fibers at a NOI of 85.1 {+/-} 1.4%. was achieved through increasing mandrel rotational velocity. Fiber tortuosity and curvilinearity in the range of native valve features were introduced through a post-spinning annealing process and fiber collection on a conical mandrel geometry, respectively. Overall, these studies demonstrate the potential of hydrogel-polyurethane fiber composite as a heart valve material. Future studies will utilize the developed advanced electrospinning methodologies in combination with model-directed fabrication toward optimizing durability as a function of fiber microarchitecture.

bioengineering↗

bioRxiv2023

Cooperative activities of dICA69 N-BAR and dCIP4 F-BAR domain proteins regulate membrane tubule organization in vivo

Intracellular membrane tubules play a crucial role in diverse cellular processes, and their regulation is facilitated by Bin-Amphiphysin-Rvs (BAR) domain-containing proteins. This study investigates the roles of dICA69N-BAR and dCIP4F-BAR in vivo, focusing on their impact on in vivo tubule organization. Through cell culture and immunofluorescence staining, we observed co-localization of endogenous dICA69 with dCIP4-induced membrane tubules, indicating their potential recruitment for tubule formation and maintenance. Additionally, dCIP4-positive tubules exhibit enrichment of actin regulatory proteins such as Wasp, SCAR, Arp2, Arp3, and Syndapin. Overexpressing dICA69N-BAR in S2R+ cells reveals distinct punctate patterns in the perinuclear region. An earlier study indicated that F-BAR proteins spontaneously segregate from the N-BAR domain-containing proteins during membrane tubule formation. In contrast, our observation supports a model in which different BAR-domain family members can associate with the same tubule and cooperate to fine-tune the tubule width. Moreover, our analysis highlights how dCIP4F-BAR facilitates the redistribution of dICA69N-BAR punctae, leading to altered patterns within the cells. These cooperative activities of dICA69N-BAR and dCIP4F-BAR are vital for the precise organization of intracellular tubules. Understanding the underlying mechanisms governing this cooperation provides valuable insights into cellular dynamics and the organization of membrane tubules. The implications extend to various physiological and pathological conditions related to intracellular membrane dynamics.

cell biology↗

bioRxiv2023

De Novo Generation and Prioritization of Target-Binding Peptide Motifs from Sequence Alone

Designing binders to target undruggable proteins presents a formidable challenge in drug discovery, requiring innovative approaches to overcome the lack of putative binding sites. Recently, generative models have been trained to design binding proteins via three-dimensional structures of target proteins, but as a result, struggle to design binders to disordered or conformationally unstable targets. In this work, we provide a generalizable algorithmic framework to design short, target-binding linear peptides, requiring only the amino acid sequence of the target protein. To do this, we propose a process to generate naturalistic peptide candidates through Gaussian perturbation of the peptidic latent space of the ESM-2 protein language model, and subsequently screen these novel linear sequences for target-selective interaction activity via a CLIP-based contrastive learning architecture. By integrating these generative and discriminative steps, we create a Peptide Prioritization via CLIP (PepPrCLIP) pipeline and validate highly-ranked, target-specific peptides experimentally, both as inhibitory peptides and as fusions to E3 ubiquitin ligase domains, demonstrating functionally potent binding and degradation of conformationally diverse protein targets in vitro. Overall, our design strategy provides a modular toolkit for designing short binding linear peptides to any target protein without the reliance on stable and ordered tertiary structure, enabling generation of programmable modulators to undruggable and disordered proteins such as transcription factors and fusion oncoproteins.

bioengineering↗

bioRxiv2023

Guanine-containing ssDNA and RNA induce dimeric and tetrameric SAMHD1 in cryo-EM and binding studies

The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNA, and telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with [~]20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity.

biochemistry↗

bioRxiv2023

Generation of Schwann cell derived melanocytes from hPSCs identifies pro-metastatic factors in melanoma

Summary/AbstractThe neural crest (NC) is highly multipotent and generates diverse lineages in the developing embryo. However, spatiotemporally distinct NC populations display differences in fate potential, such as increased gliogenic and parasympathetic potential from later migrating, nerve-associated Schwann cell precursors (SCPs). Interestingly, while melanogenic potential is shared by both early migrating NC and SCPs, differences in melanocyte identity resulting from differentiation through these temporally distinct progenitors have not been determined. Here, we leverage a human pluripotent stem cell (hPSC) model of NC temporal patterning to comprehensively characterize human NC heterogeneity, fate bias, and lineage development. We captured the transition of NC differentiation between temporally and transcriptionally distinct melanogenic progenitors and identified modules of candidate transcription factor and signaling activity associated with this transition. For the first time, we established a protocol for the directed differentiation of melanocytes from hPSCs through a SCP intermediate, termed trajectory 2 (T2) melanocytes. Leveraging an existing protocol for differentiating early NC-derived melanocytes, termed trajectory 1 (T1), we performed the first comprehensive comparison of transcriptional and functional differences between these distinct melanocyte populations, revealing differences in pigmentation and unique expression of transcription factors, ligands, receptors and surface markers. We found a significant link between the T2 melanocyte transcriptional signature and decreased survival in melanoma patients in the cancer genome atlas (TCGA). We performed an in vivo CRISPRi screen of T1 and T2 melanocyte signature genes in a human melanoma cell line and discovered several T2-specific markers that promote lung metastasis in mice. We further demonstrated that one of these factors, SNRPB, regulates the splicing of transcripts involved in metastasis relevant functions such as migration, cell adhesion and proliferation. Overall, this study identifies distinct developmental trajectories as a source of diversity in melanocytes and implicates the unique molecular signature of SCP-derived melanocytes in metastatic melanoma.

developmental biology↗

bioRxiv2023

IL-13 promotes sensory-sympathetic neurons crosstalk in asthma

Nociceptor neurons play a crucial role in maintaining the bodys homeostasis by detecting and responding to potential dangers in the environment. However, this function can be detrimental during allergic reactions, since vagal nociceptors can contribute to immune cell infiltration, bronchial hypersensitivity, and mucus imbalance, in addition to causing pain and coughing. Despite this, the specific mechanisms by which nociceptors acquire pro-inflammatory characteristics during allergic reactions are not yet fully understood. In this study, we aimed to investigate the molecular profile of airway nociceptor neurons during allergic airway inflammation and identify the signals driving such reprogramming. Using retrograde tracing and lineage reporting, we identified a unique class of inflammatory vagal nociceptor neurons that exclusively innervate the airways. In the ovalbumin mouse model of airway inflammation, these neurons undergo significant reprogramming characterized by the upregulation of the NPY receptor Npy1r. A screening of cytokines and neurotrophins revealed that IL-1{beta}, IL-13 and BDNF drive part of this reprogramming. IL-13 triggered Npy1r overexpression in nociceptors via the JAK/STAT6 pathway. In parallel, sympathetic neurons and macrophages release NPY in the bronchoalveolar fluid of asthmatic mice, which limits the excitability of nociceptor neurons. Single-cell RNA sequencing of lung immune cells has revealed that a cell-specific knockout of Npy1r in nociceptor neurons in asthmatic mice leads to an increase in airway inflammation mediated by T cells. Opposite findings were observed in asthmatic mice in which nociceptor neurons were chemically ablated. In summary, allergic airway inflammation reprograms airway nociceptor neurons to acquire a pro-inflammatory phenotype, while a compensatory mechanism involving NPY1R limits nociceptor neurons activity.

neuroscience↗