Biology subjects

Debnath, A.

Publications and source records attributed to Debnath, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Beyond Direct Binding: Serotonin Rewires Model Brain Plasma Membrane-Mediated Communication Pathways in Y4R

Serotonin, an endogenous neurotransmitter, modulates pancreatic polypeptide (PP) recognition by the neuropeptide Y4 receptor (Y4R), a mechanism relevant to altered serotonergic signaling associated with metabolic disorder and anxiety. Although recent experiment demonstrates serotonin modulated PP dissociation kinetics of Y4R through membrane allostery, the molecular mechanism of serotonin induced lipidomic reorganization of the brain plasma membrane (BPM) relevant for Y4R activation is unknown. To this end, a total of 825.9 ns long all atom molecular dynamics simulations at 310 K elucidate the concentration-dependent coupling between serotonin partitioning, selective lipid reorganization, and PP binding and Y4R activation relevant intra-receptor communication in a heterogeneous, asymmetric model BPM. In the absence of serotonin, Y4R preferentially associates with the anionic lipids. Serotonin preferentially localizes at the lipid water interface over the PP binding and activation-associated regions of Y4R. At 6 mM, serotonin preferentially contacts phosphocholine species depleted from Y4R, whereas at 50 mM, phosphatidylinositol 4,5 bisphosphate dominates both microenvironments. These changes align with the selective remodeling of lipid networks, enhanced lipid chain ordering, and modest bilayer compaction. Weighted implementation of suboptimal paths analysis reveals concentration-dependent reorganization of communication pathways between experimentally implicated PP binding and activation associated Y4R residues. Collectively, these findings connects serotonin dependent selective lipid redistribution to altered intramolecular communication between Y4R residues associated with PP binding and G-protein coupling, and provide a framework for future experiments investigating membrane-mediated neuromodulation and neuronal signaling. These findings provide a basis for exploring lipid-targeted pharmacology as a complementary strategy to modulate GPCR allostery by altering membrane composition rather than directly targeting the receptor.

biophysics↗

bioRxiv2026

Stepwise Proteinase K Digestion Improves Genomic DNA Yield and Purity from Goat Semen: A Comparative Pilot Study of Five Extraction Protocols

Background: Genomic DNA of high quality is a prerequisite for molecular genetic analyses. However, DNA extraction from goat semen is frequently challenged by highly compacted sperm chromatin and the presence of enzymatic inhibitors within the seminal plasma. Objectives: This pilot study aimed to identify a reliable and cost-effective method for isolating high-quality genomic DNA from caprine semen. Methods: Five extraction protocols were evaluated: an unmodified blood protocol (Protocol B), two modifications of a sperm protocol (Modified Protocols 1 and 2), and a stool protocol (Protocol BS) using a commercial spin-column kit (Kit A), alongside a second commercial silica membrane kit (Kit B). Three technical replicates per protocol were processed from a single frozen semen straw of a Black Bengal buck (with one replicate evaluated for Protocol B). Extracted DNA was evaluated based on concentration, purity (absorbance ratios A260/A280 and A260/A230), cost per sample, and PCR amplification success of the mitochondrial hypervariable region 1 (HVR1). Results: Modified Protocol 2 utilizing 100 uL of semen and two sequential 1.5-hour proteinase K incubations at 55 degrees C yielded the highest normalized DNA concentration (mean +/- SD: 55.9 +/- 30.4 ng/uL semen), optimal purity ratios (A260/A280 = 1.78; A260/A230 = 2.06), and the lowest cost per nanogram of DNA (INR 0.073 +/- 0.037). All extracts from this protocol were successfully amplified via PCR. While the Kruskal-Wallis test for normalized yield did not reach statistical significance (p = 0.075), rendering yield differences exploratory, Modified Protocol 2 demonstrated the strongest overall performance. Conclusions: Modified Protocol 2 offers a promising, cost-efficient workflow for caprine sperm DNA isolation. Given that this pilot study sampled a single animal, these findings must be validated using biological replicates from multiple bucks prior to widespread implementation.

molecular biology↗

bioRxiv2026

Nanoscopic Origin of Enhanced Tyrosine-Mediated Water Ordering over Canonical Threonine Motifs in a Bacterial Ice-Nucleating Protein

Bacterial ice-nucleating proteins (INPs) are known as the most efficient biological heterogeneous ice nucleators. Ice-nucleation ability is conventionally attributed to the threonine-based conserved motifs of {beta} -helical INPs, whereas the role of other conserved motifs remains overlooked. To identify the motif with the highest hydration-ordering ability, a 16-loop model INP and its two mutants are simulated using all-atom molecular dynamics at 260 K, just below the ice nucleation temperature, using the CHARMM36m force field and TIP4P/Ice water model. Among four potential ice-nucleating sites present in four exposed surfaces of the {beta} -helical INP, the glycine-tyrosine-glycine (GYG) motif-associated interface water (IW) exhibits the probability distribution of the Steinhardt bond-orientational and tetrahedral order parameters closest to that of the Ih. The GYG arrange themselves into a ladder-like array of tyrosine hydroxyls, compatible with the Ih-like lattice geometry, suggesting that this motif provides the IW a favorable template for ice-like water organization. The Y-to-phenylalanine mutation produces the largest disruption in IW order compared to the threonine mutations. Although the difference in entropy of threonine-associated IW and Ih is the least among all sites, ice-like hydration ordering near GYG may exceed prior assumptions for a threonine-based ice-nucleating surface. The ordering is collectively determined by lattice compatibility, entropy reduction, hydrophobicity, interfacial geometry, and sequence conservation. The study unfolds a molecular framework of biological ice nucleation and provides a foundation for the rational engineering of more efficient artificial ice-nucleators for applications in climate-related technologies.

biophysics↗

bioRxiv2026

Hyperexcitable but seizure-free: hippocampal network dysfunction in a Wolfram syndrome model

Wolfram syndrome (WS) is a rare, life-limiting progressive neurodegenerative disorder caused by pathogenic variants in the WFS1 gene. It is a prototype endoplasmic reticulum (ER) stress disorder, and the mechanisms underlying neurological phenotypes in WS remain incompletely understood. Although WFS1 protein is abundant in the hippocampus, the role of this region in WS remains relatively understudied. Here we characterize hippocampal function across scales in mice carrying a patient orthologous Wfs1 variant (p.W542*) on both alleles. Male mutants lose excitatory synapses, and dissociated hippocampal neurons are markedly hyperexcitable yet generate no increase in network bursting and no shift in network criticality, indicating that their additional firing is not recruited into coordinated population activity. This finding translates in vivo wherein intracranial electroencephalogram (EEG) recordings show no epileptiform activity or seizures in either sex, with no hippocampal astrogliosis, alongside a male-specific reduction in hippocampal low-gamma power. Additionally, EEG demonstrates a sex-specific disruption of sleep, which parallels a sex-divergent synaptic phenotype. Mutant male mice show impaired hippocampus-dependent fear memory and reduced voluntary motor activity. Taken together, we propose that cellular hyperexcitability in the WS hippocampus does not translate into increased network bursting, potentially due to disproportionate loss of excitatory synapses. This dissociation may provide a functional substrate for the neurological features of WS.

neuroscience↗

bioRxiv2026

Palmitoylation calibrates mitochondrial transport of insulin across the endothelium in insulin resistance

Impaired insulin transport across the endothelium contributes to insulin resistance, a poorly understood condition implicated in diabetes and many other chronic diseases. Insulin has been reported to undergo non-receptor-mediated endocytosis resembling fluid-phase uptake through unclear mechanisms. Here we show in mice with diet-induced insulin resistance that endothelial-specific deletion of the depalmitoylase acyl-protein thioesterase 1 (APT1) improved glucose tolerance and insulin sensitivity without affecting chronic inflammation or capillary structure. Endothelial APT1 deficiency increased interstitial insulin levels in mice confirmed by in-situ microneedle-based sampling. In cultured human microvascular cells, APT1 inhibition enhanced cell transport of high-dose insulin independent of the insulin receptor and canonical endocytic machinery. Unexpectedly, live-cell imaging revealed insulin rapidly localizing to mitochondria prior to endolysosomal trafficking, even at physiological insulin concentrations. APT1 inhibition delayed mitochondrial discharge of insulin to lysosomes. Cyclosporin A, an immunosuppressant known to affect mitochondrial function, preserved mitochondrial insulin content and promoted insulin transport in cultured cells, and enhanced interstitial insulin delivery in mice. Proteomic analysis revealed two palmitoylated proteins, PACS1 and YTHDF2, required for APT1-mediated mitochondrial-endolysosomal trafficking of insulin. A mitochondrial insulin shuttle in endothelial cells may participate in the physiological adaptation to hyperinsulinemia and its regulation by palmitoylation suggests a novel approach to insulin resistance.

physiology↗

bioRxiv2026

A cell type-resolved proteomic atlas of the human body

Proteins define what cells do, yet their systematic quantification across human cell types has remained out of reach. Using Deep Visual Proteomics on tissue from a healthy female donor, we built an atlas of 27 cell types across 14 tissues, quantifying two-thirds of all human protein-coding genes, with up to 8,500 per population. The proteome partitions bimodally into a universal core and highly specialized programs. Integration into the Human Protein Atlas Single Cell Resource enabled comparison of RNA and protein abundance at cell type resolution, revealing that concordance depends on pathway rather than cellular identity. This resolution uncovered cancer-testis antigens in oocytes invisible to bulk profiling. Our openly accessible resource provides a foundation for cell type-resolved proteomics in health and disease.

biochemistry↗

bioRxiv2026

Linguistic and Acoustic Biomarkers from Simulated Speech Reveal Early Cognitive Impairment Patterns in Alzheimers Disease

BackgroundAlzheimers disease (AD) causes progressive decline in language and cognition. Automated speech analysis has emerged as a promising screening tool, yet clinical data scarcity limits progress. To address this, we generated a large-scale simulated speech dataset to model linguistic and acoustic deterioration across cognitive stages, Control, Mild Cognitive Impairment (MCI), and AD. MethodsUsing Monte Carlo simulations, we emulated the Pitt DementiaBank "Cookie Theft" narratives. Acoustic features (speech rate, pause duration, jitter, shimmer) and linguistic features (type-token ratio, unique-word count, filler usage) were synthetically sampled from real-world DementiaBank distributions. We trained an XGBoost classifier to distinguish diagnostic groups, and applied SHAP (Shapley Additive exPlanations) to assess feature importance. ResultsThe model achieved high discriminative performance (AUC {approx} 0.94; accuracy {approx} 85%). Compared to controls, simulated MCI and AD groups showed progressive declines in fluency and lexical diversity, and increases in disfluencies and voice instability. SHAP analysis revealed that key predictors included reduced type-token ratio, higher pause and filler rates, and elevated jitter/shimmer. Classification was most accurate for Control vs. AD; MCI misclassifications highlighted intermediate profiles. InterpretationOur framework, FMN (Forget Me Not), captures clinically relevant speech changes using simulated data, offering an explainable and scalable approach for cognitive screening. While not a substitute for real datasets, FMN validates a pipeline that mirrors known AD markers and can guide future real-world deployments. External validation remains a key next step for translational impact.

neuroscience↗

bioRxiv2025

Potential probiotic application of a novel commensal E. coli with antagonistic activity against different enteric pathogens

Diarrheal diseases remain a leading cause of global morbidity and mortality, and treatment options are increasingly compromised by the global rise in multidrug-resistant (MDR) pathogens. Furthermore, the absence of effective vaccines against many causative agents renders large populations vulnerable. This critical gap necessitates the development of novel alternatives that can function both prophylactically to prevent infection and therapeutically to treat established diseases. Here, we investigated the commensal Escherichia coli strain HK220822 (HK5), which was isolated from a healthy human, as a potential live biotherapeutic. Comprehensive genomic and phenotypic characterization established a robust safety profile, confirming the absence of key virulence genes while demonstrating essential probiotic traits, including high tolerance to gastrointestinal stressors, such as acid and bile. The functional efficacy of Escherichia coli HK5 has been validated in murine models of infection. Prophylactic administration before pathogen exposure significantly inhibited intestinal colonization by Salmonella Typhimurium, Shigella flexneri 2a, and Vibrio cholerae (El Tor O1 strain). Moreover, HK5 demonstrated therapeutic potential by significantly reducing pathogen shedding when administered to previously infected animals. These findings establish commensal E. coli (HK5) as a potent candidate with dual preventative and therapeutic efficacy, offering a promising non-antibiotic strategy to combat diarrheal diseases.

microbiology↗

bioRxiv2025

Exposure to third generation cephalosporin induces L-form transition in Shigella sonnei, potentially acting as a bacterial reservoir for persistent infection

Shigellosis remains a major global health burden, and the increasing prevalence of multidrug-resistant (MDR) Shigella strains is complicating effective antibiotic therapy. Bacteria may survive antibiotics by transitioning into cell wall-deficient L-forms, which are intrinsically resistant to {beta}-lactams and can revert to a virulent state, potentially causing relapsing infections. Here we characterized a clinical MDR isolate Shigella sonnei HK8, a.k.a. PD552A, whose genome contains key resistance (gyrA, PBP3) and virulence (icsA) genes. Exposure to ceftriaxone induced a transition into a viable L-form state that was hyper-adhesive to macrophages in vitro. However, this survival adaptation was linked to a profound loss of pathogenicity. Using murine and guinea pig models, the L-form variant was shown to be profoundly attenuated, failing to cause the keratoconjunctivitis, diarrheal disease, or significant histopathology characteristic of the wild-type strain. These findings reveal a critical virulence-survival trade-off, positioning the L-form as a "stealth" phenotype that enables bacterial persistence at the expense of acute virulence. This offers a potential mechanism for asymptomatic carriage and recurrent infections, highlighting a previously underappreciated mechanism by which antibiotic treatment may resolve acute symptoms while permitting the persistence of a cryptic bacterial reservoir capable of driving recurrent infection. ImportanceOur research provides critical insight into the challenge of antibiotic treatment failure in shigellosis. By integrating experimental validation of reversible L-form transitions with mathematical modelling, we uncover a crucial virulence-survival trade-off. We show that MDR S. sonnei survives ceftriaxone by adopting a "stealth" phenotype, quantified by a high Stealth Index - maintaining bacterial burden while evading host inflammatory detection. These findings imply that standard antibiotics may resolve acute symptoms while inadvertently selecting for a cryptic, persistent reservoir poised for relapse. This work challenges the conventional view of therapeutic success and highlights an urgent need to develop novel diagnostic and therapeutic strategies capable of identifying and eliminating these resilient, "stealth" L-form persisters to achieve true bacterial clearance and prevent chronic infections.

microbiology↗

bioRxiv2025

Neural mechanisms of training in Brain-Computer Interface: ABiophysical modeling approach

Brain-Computer Interface (BCI) is a system that translates neural activity into commands, allowing direct communication between the brain and external devices. Despite its clinical application, BCI systems are unable to robustly capture subjects intent due to a limited understanding of the neural mechanisms underlying BCI control. To address this issue, we introduce a biophysical linear neural mass model to investigate the associated neural mechanisms of motor imagery-based BCI experiments by modulating the scale-free spectral characteristics of afferent inputs. We tailor this motor imagery neural mass model (mi-NMM) to simulate both motor imagery task and resting state, and apply this approach to a cohort of 19 healthy subjects trained over four sessions where magnetoencephalography (MEG) and electroencephalography (EEG) signals were simultaneously recorded. Task-dependent modulation of scale-free cortical dynamics reveals training-induced excitatory-inhibitory reconfiguration in BCI training. The intra-regional neural connectivity strengths and time scales of the modeled excitatory and inhibitory neural mass populations capture changes in neural activity across conditions and sessions. Those changes appear in important areas of the sensorimotor cortex, relevant for motor imagery tasks. We observed these effects in both EEG and MEG modalities. These findings provide insights into the underlying neural mechanisms in a motor imagery task in BCI, paving the way to tailored BCI training protocols. Significance StatementBrain-computer interface (BCI) holds great promise in allowing direct communication between the brain and external devices, yet many users struggle to learn it reliably, largely because the neural mechanisms underlying BCI training remain poorly understood - thus lack of biologically grounded training protocols. This study introduces a biophysically grounded modeling framework that links large-scale brain dynamics to learning during motor imagery based BCI training. By explicitly modeling task-dependent cortical background input, we reveal how excitatory-inhibitory population dynamics reorganize with training, shifting from widespread cortical to targeted sensorimotor recruitment. Our results, replicated across EEG and MEG, identify excitatory population time scales as a robust neural marker of motor imagery learning. These findings provide mechanistic insight into BCI learning and offer principled targets for designing individualized BCI training protocols.

neuroscience↗

bioRxiv2025

PEDOT:PSS Microparticles for Extrudable and Bioencapsulating Conducting Granular Hydrogel Bioelectronics

Conducting hydrogels are promising materials for forming physiomimetic bioelectronic interfaces to monitor and stimulate biological activity. However, most developed materials are non-microporous and possess fixed shapes, both of which can limit the integration of cells and tissues with devices. In non-conducting biomaterials, materials fabrication strategies imparting microporosity and dynamic mechanical properties have been shown to support cell infiltration and support biointerfaces of various geometries. Specifically, granular hydrogels have enabled encapsulating, conformal, and injectable interfaces through these features. However, granular hydrogels remain largely unexplored as conducting biomaterials. We present methods for fabricating spherical, poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) hydrogel microparticles. When densely packed, these microparticles form a conducting granular hydrogel with microporosity as well as shear-thinning and self-healing dynamic mechanical properties. The PEDOT:PSS granular hydrogel can be extruded and maintain structure post-3D printing. Modulating microparticle PSS content achieves high granular hydrogel conductivity (137 S/m), and microparticles exhibit excellent cytocompatibility (>98% viability). Finally, we demonstrate utility as bioencapsulating electrodes for electrophysiological monitoring. These results highlight the functionality of our PEDOT:PSS conducting granular hydrogel, suggesting its potential as 3D printed bioencapsulating electrodes, 3D tissue engineering scaffolds for monitoring encapsulated cells, and injectable therapies for enhanced cell recruitment and tissue regeneration combined with electronic stimulation.

bioengineering↗

bioRxiv2025

Dynamic Disorder in Chlorophyll Aggregation and Light-Harvesting Complex II in the Plant Thylakoid Membrane using Coarse-Grained Simulations

The dynamics of the aggregated light-harvesting complex (LHCII) associated with its antennae pigments can be crucial for a transition between light harvesting and dissipative states pivotal for non-photochemical quenching (NPQ). To this end, aggregation of chlorophyll-a (CLA) without the LHCII and pigment binding LHCII monomers in the plant thylakoid membranes have been investigated using coarse-grained molecular dynamics simulations at 293 K. Both CLA without the LHCII and pigment-binding LHCII monomers dynamically form and break dimers and higher-order aggregates in thylakoids within the simulation time. The contact lifetime and waiting time distributions of CLA dimers exhibit multiple time scales including most populated fast time scales and less populated slow time scales. The survival probability of CLA dimer in the absence of the LHCII follows a non-exponential decay with multiple residence time scales, leading to a time-dependent rate, unlike conventional rate theory. Such non-exponential decay of survival manifests the emergence of dynamic disorder in CLA without the LHCII resulting from the coupling between time scales of dimer formation and higher-order aggregates. The conformational fluctuations of the LHCII known for inter-CLA coupling variation occur on multiple time scales comparable to the LHCII dimer residence time scales leading to less probable but comparable and more probable slower inter-CLA fluctuations. This indicates the dynamic coupling in the LHCII conformations and their aggregates with the antennae pigments can result in dynamic disorder which will be highly relevant for the light-harvesting efficiency and regulation of NPQ. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/638782v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@5fd895org.highwire.dtl.DTLVardef@8456cforg.highwire.dtl.DTLVardef@5f5c69org.highwire.dtl.DTLVardef@abf6d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

The Role of Negatively Charged Groups in Antimicrobial Cationic Peptide Mimics: Insights into Membrane Interactions

In this study, we explore cationic amphiphilic methacrylate copolymers incorporating both positively charged AEMA and negatively charged PAMA functional groups, focusing on their interactions with bacterial membranes. Aggregation studies reveal that electrostatic interactions drive the formation of stable polymer aggregates, with block copolymers forming micelle-like structures and random copolymers exhibiting a more uniform distribution. These ternary polymers preferentially interact with deep lipid packing defects in bacterial membranes, stabilizing and expanding these defects, while shallow defects remain largely unaffected due to the unfavorable interaction of anionic groups with lipid headgroups. The role of interfacial water is also critical, as hydration layers surrounding anionic groups shield them from electrostatic repulsion, enabling deeper penetration into the membrane. Comparative analyses highlight the advantages of anionic-containing polymers over previously studied polar-containing systems, which predominantly engage shallow defects and exhibit limited structural adaptability near membranes. These findings underscore the role of anionic residues in enabling adaptable AMP conformations, enhanced membrane engagement, and effective disruption mechanisms, providing valuable insights for the design of biomimetic antimicrobial polymers incorporating different functional groups.

biophysics↗

bioRxiv2024

Light Harvesting Complex II Resists Non-Bilayer Lipid-Induced Polymorphism in Plant Thylakoid Membranes via Lipid Redistribution

The plant thylakoid membrane hosting the light-harvesting complex (LHCII) is the site of oxygenic photosynthesis. Contrary to the earlier consensus of a protein-driven single lamellar phase of the thylakoid despite containing 40% non-bilayer-forming lipids, recent experiments confirm the polymorphic state of the functional thylakoid. What, then, is the origin of this polymorphism, and what factors control it? The current Letter addresses the question using a total of 617.8 s long coarse-grained simulations of thylakoids with and without LHCII and varying concentrations of non-bilayer lipids using Martini-2.2 and 3.0 at 323 K. The LHCII redistributes the non-bilayer lipids into its annular region, increases the bending modulus and the stalk formation free energy, reduces the non-zero mean curvature propensity, and resists the polymorphism these lipids promote. The thermodynamic trade-off between non-bilayer lipids and LHCII dictates the degree of nanoscopic curvature leading to the polymorphism crucial for non-photochemical quenching under excess heat conditions. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/623718v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@7eab4forg.highwire.dtl.DTLVardef@20dd1forg.highwire.dtl.DTLVardef@19558c2org.highwire.dtl.DTLVardef@1f64f52_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Neuronal Maturation-dependent Nano-Neuro Interaction and Modulation

Nanotechnology-enabled neuromodulation, a rapidly growing technique, is a promising minimally-invasive tool in neuroscience and engineering for both fundamental studies as well as clinical applications. However, the nano-neuro interactions at different stages of maturation of a neural network and its implications on the nano-neuromodulation remain unclear. Here, we report heterogeneous to homogenous transformation of neuromodulation in a progressively maturing neural network. Utilizing plasmonic fluors as ultrabright fluorescent nanolabels, we reveal that negative surface charge of the nanoparticles renders selective nano-neuro interaction with a strong correlation between the maturation stage of the individual neurons in the neural network and the density of the nanoparticles bound on the neurons. In stark contrast to homogeneous neuromodulation in a mature neural network reported so far, the maturation-dependent density of the nanoparticles bound to neurons in a developing neural network resulted in a heterogeneous optical neuromodulation (i.e., simultaneous excitation and inhibition of neural network activity). This study advances our understanding of nano-neuro interactions and nano-neuromodulation with potential applications in minimally-invasive technologies for treating neuronal disorders in parts of mammalian brain where neurogenesis persists throughout aging.

bioengineering↗

bioRxiv2021

Discovery of highly potent pancoronavirus fusion inhibitors that also effectively inhibit COVID-19 variants from the UK and South Africa

We report here the discovery of several highly potent small molecules that showed low nM potency against SARS-CoV (IC50: as low as 13 nM), SARS-CoV-2 (IC50: as low as 23 nM), and MERS-CoV (IC50: as low as 76 nM) in pseudovirus based assays with excellent selectivity indices (SI: as high as > 5000) demonstrating their pancoronavirus inhibition. Some compounds also show 100% inhibition of CPE (IC100) at 1.25 {micro}M against an authentic SARS-CoV-2 (US_WA-1/2020). Furthermore, the most active inhibitors also potently inhibited variants of concerns (VOCs), such as the UK (B.1.1.7), South Africa (B.1.351), and Delta variant (B.1.617.2), originated in India. We confirmed that one of the potent inhibitors binds to the prefusion spike protein trimer of SARS-CoV-2 by SPR. Besides, we showed that they inhibit virus-mediated cell-cell fusion. The ADME data of one of the most active inhibitors, NBCoV1, show drug-like properties. In vivo PK of NBCoV1 in rats demonstrated excellent half-life (t1/2) of 11.3 h, mean resident time (MRT) of 14.2 h, and oral bioavailability. We expect the lead inhibitors to pave the way for further development to preclinical and clinical candidates.

pharmacology and toxicology↗

bioRxiv2020

Antineoplastic kinase inhibitors: a new class of potent anti-amoebic compounds

Entamoeba histolytica is a protozoan parasite which infects approximately 50 million people worldwide, resulting in an estimated 70,000 deaths every year. Since the 1960s E. histolytica infection has been successfully treated with metronidazole. However, drawbacks to metronidazole therapy exist, including adverse effects, a long treatment course, and the need for an additional drug to prevent cyst-mediated transmission. E. histolytica possesses a kinome with approximately 300 - 400 members, some of which have been previously studied as potential targets for the development of amoebicidal drug candidates. However, while these efforts have uncovered novel potent inhibitors of E. histolytica kinases, none have resulted in approved drugs. In this study we took the alternative approach of testing a set of twelve previously FDA-approved antineoplastic kinase inhibitors against E. histolytica trophozoites in vitro. This resulted in the identification of dasatinib, bosutinib, and ibrutinib as amoebicidal agents at low-micromolar concentrations. Next, we utilized a recently developed computational tool to identify twelve additional drugs with human protein target profiles similar to the three initial hits. Testing of these additional twelve drugs led to the identification of ponatinib, neratinib, and olmutinib were identified as highly potent, with EC50 values in the sub-micromolar range. All of these six drugs were found to kill E. histolytica trophozoites as rapidly as metronidazole. Furthermore, ibrutinib was found to kill the transmissible cyst stage of the model organism E. invadens. Ibrutinib thus possesses both amoebicidal and cysticidal properties, in contrast to all drugs used in the current therapeutic strategy. These findings together reveal antineoplastic kinase inhibitors as a highly promising class of potent drugs against this widespread and devastating disease. Author SummaryEvery year, nearly a hundred thousand people worldwide die from infection by the intestinal parasite Entamoeba histolytica, despite the widespread availability of metronidazole as a treatment. Here we report that six anticancer drugs of the kinase inhibitor class possess potent anti-amoebic properties, with one of them killing both actively dividing parasite and its transmissible cysts. These anticancer kinase inhibitors, including the dual-purpose drug with both amoebicidal and cysticidal activities may be used to treat amoebiasis, especially in cancer patients or in life-threatening brain- and liver-infecting forms of the disease.

microbiology↗

bioRxiv2020

Characterizing the portability of RecT-mediated oligonucleotide recombination

Bacterial genome editing methods are used to engineer strains for biotechnology and fundamental research. Homologous recombination (HR) is the most versatile method of genome editing, but traditional techniques using endogenous RecA-mediated pathways are inefficient and laborious. Phage encoded RecT proteins can improve HR over 1000-fold, but these proteins have limited portability between species. Using Escherichia coli, Lactococcus lactis, Mycobacterium smegmatis, Lactobacillus rhamnosus, and Caulobacter crescentus we investigated the hostlimited functionality of RecTs. We find that these proteins specifically recognize the 7 C-terminal amino acids of the bacterial single-stranded DNA-binding protein (SSB), and are portable between species only if compatibility with this host domain is maintained. Furthermore, in some species, we find that co-expressing otherwise incompatible RecTs with a paired bacterial SSB is sufficient to establish functionality. Finally, we demonstrate that high-efficiency HR surpasses the mutational capacity of more widely used error-prone methods for genome diversification, and can be used to identify exceptional phenotypes inaccessible through sequential nucleotide conversions.

synthetic biology↗