Resistance Evolution against Host-directed Antiviral Agents: Buffalopox Virus Switches to Use p38-ϒ under Long-term Selective Pressure of an Inhibitor Targeting p38-α.

Host-dependency factors have increasingly been targeted to minimize antiviral drug resistance. In this study, we have demonstrated that inhibition of p38 mitogen-activated protein kinase (a cellular protein) suppresses buffalopox virus (BPXV) protein synthesis by targeting p38-MNK1-eIF4E signaling pathway. In order to provide insights into the evolution of drug resistance, we selected resistant mutants by long-term sequential passages (P; n = 60) in the presence of p38 inhibitor (SB239063). The P60-SB239063 virus exhibited significant resistance to SB239063 as compared to the P60-Control virus. To provide mechanistic insights on the acquisition of resistance by BPXV-P60-SB239063, we generated p38-α and p38-ϒ (isoforms of p38) knockout Vero cells by CRISPR/Cas9-mediated genome editing. It was demonstrated that unlike the wild type (WT) virus which is dependent on p38-α isoform, the resistant virus (BPXV-P60-SB239063) switches over to use p38-ϒ so as to efficiently replicate in the target cells. This is a rare evidence wherein a virus was shown to bypass the dependency on a critical cellular factor under selective pressure of a drug.

KDM6B cooperates with Tau and regulates synaptic plasticity and cognition via inducing VGLUT1/2.

The excitatory neurotransmitter glutamate shapes learning and memory, but the underlying epigenetic mechanism of glutamate regulation in neuron remains poorly understood. Here, we showed that lysine demethylase KDM6B was expressed in excitatory neurons and declined in hippocampus with age. Conditional knockout of KDM6B in excitatory neurons reduced spine density, synaptic vesicle number and synaptic activity, and impaired learning and memory without obvious effect on brain morphology in mice. Mechanistically, KDM6B upregulated vesicular glutamate transporter 1 and 2 (VGLUT1/2) in neurons through demethylating H3K27me3 at their promoters. Tau interacted and recruited KDM6B to the promoters of Slc17a7 and Slc17a6, leading to a decrease in local H3K27me3 levels and induction of VGLUT1/2 expression in neurons, which could be prevented by loss of Tau. Ectopic expression of KDM6B, VGLUT1, or VGLUT2 restored spine density and synaptic activity in KDM6B-deficient cortical neurons. Collectively, these findings unravel a fundamental mechanism underlying epigenetic regulation of synaptic plasticity and cognition.

Clean water production from plastic and heavy metal contaminated waters using redox-sensitive iron nanoparticle-loaded biochar.

The unceasing release of tiny plastics (microplastics and nanoplastics) and their additives, like metal ions, into the aquatic systems from industries and other sources is a globally escalating problem. Their combined toxic effects and human health hazard are already proven; hence, their remediation is requisite. This study utilised the nano-zerovalent iron-loaded sugarcane bagasse-derived biochar (nZVI-SBC) for simultaneous removal of Nanoplastics (NPs) of different functionality and size along with metal ions (Ni2+, Cd2+, AsO43-, and CrO42-). Batch and column experiments were conducted, and the results showed an efficient removal of contaminants with maximum sorption of carboxylate-modified NPs of size 500 nm (qmax = 90.3 mg/g) among all three NPs types. Significant removal was observed in Cd2+ in case of cations and CrO42- in case of anions with qmax = 44.0 and 87.8 mg/g, respectively. Kinetics and the isotherm modelling better fitted the pseudo-second-order kinetic model and Sips isotherm model, respectively for both NPs and metal ions. The designed material worked well in pH range of 4-8, ionic strength 1-20 mM and in complex aqueous matrices, with >90% removal. FTIR, zeta potential and the imaging analysis of the reaction precipitates confirmed the electrostatic attraction, pore retention and complexation as the potential mechanisms for removing NPs, whereas, XPS studies confirmed the reduction co-precipitation and surface complexation as the possible mechanism for removing metal ions. High values of attachment efficiency factor calculated from colloidal filtration theory (CFT) validated the experimental results and justified the high sorption of carboxylate modified 500 nm NPs particles. The synthesized material successfully removed both NPs of varying size and functionality and metal ions simultaneously with significant efficacy in complex environmental samples proving the broad applicability of material in realistic environmental conditions and different types of water treatment processes.

FOXP1 negatively regulates intrinsic excitability in D2 striatal projection neurons by promoting inwardly rectifying and leak potassium currents.

Heterozygous loss-of-function mutations in the transcription factor FOXP1 are strongly associated with autism. Dopamine receptor 2 expressing (D2) striatal projection neurons (SPNs) in heterozygous Foxp1 (Foxp1+/-) mice have higher intrinsic excitability. To understand the mechanisms underlying this alteration, we examined SPNs with cell-type specific homozygous Foxp1 deletion to study cell-autonomous regulation by Foxp1. As in Foxp1+/- mice, D2 SPNs had increased intrinsic excitability with homozygous Foxp1 deletion. This effect involved postnatal mechanisms. The hyperexcitability was mainly due to down-regulation of two classes of potassium currents: inwardly rectifying (KIR) and leak (KLeak). Single-cell RNA sequencing data from D2 SPNs with Foxp1 deletion indicated the down-regulation of transcripts of candidate ion channels that may underlie these currents: Kcnj2 and Kcnj4 for KIR and Kcnk2 for KLeak. This Foxp1-dependent regulation was neuron-type specific since these same currents and transcripts were either unchanged, or very little changed, in D1 SPNs with cell-specific Foxp1 deletion. Our data are consistent with a model where FOXP1 negatively regulates the excitability of D2 SPNs through KIR and KLeak by transcriptionally activating their corresponding transcripts. This, in turn, provides a novel example of how a transcription factor may regulate multiple genes to impact neuronal electrophysiological function that depends on the integration of multiple current types - and do this in a cell-specific fashion. Our findings provide initial clues to altered neuronal function and possible therapeutic strategies not only for FOXP1-associated autism but also for other autism forms associated with transcription factor dysfunction.

Role of p38 mitogen-activated protein kinase signalling in virus replication and potential for developing broad spectrum antiviral drugs.

Mitogen-activated protein kinases (MAPKs) play a key role in complex cellular processes such as proliferation, development, differentiation, transformation and apoptosis. Mammals express at least four distinctly regulated groups of MAPKs which include extracellular signal-related kinases (ERK)-1/2, p38 proteins, Jun amino-terminal kinases (JNK1/2/3) and ERK5. p38 MAPK is activated by a wide range of cellular stresses and modulates activity of several downstream kinases and transcription factors which are involved in regulating cytoskeleton remodeling, cell cycle modulation, inflammation, antiviral response and apoptosis. In viral infections, activation of cell signalling pathways is part of the cellular defense mechanism with the basic aim of inducing an antiviral state. However, viruses can exploit enhanced cell signalling activities to support various stages of their replication cycles. Kinase activity can be inhibited by small molecule chemical inhibitors, so one strategy to develop antiviral drugs is to target these cellular signalling pathways. In this review, we provide an overview on the current understanding of various cellular and viral events regulated by the p38 signalling pathway, with a special emphasis on targeting these events for antiviral drug development which might identify candidates with broad spectrum activity.

Effect of clay colloid - CuO nanoparticles interaction on retention of nanoparticles in different types of soils: role of clay fraction and environmental parameters.

The extensive application of metal oxide nanoparticles (NPs) in various sectors has raised concern about their subsequent release and potentially harmful impacts on the soil system. The present study has addressed the interaction of CuO NPs with bentonite clay colloids (CC) under varying environmental parameters as a model to represent the soil pore water scenario. Based on CuO - CC interaction in model and natural soil solution extracts (SSE), the role of clay fraction and their stability on CuO retention in various types of soils have been evaluated. Results suggested that increasing ionic strength (IS) in the system caused aggregation of CuO NPs, and in the presence of CC, critical coagulation concentration decreased drastically from 27.8 and 17.3 mM to 10.7 and 0.33 mM for NaCl and CaCl2 respectively, due to heteroaggregation in the system. Interestingly, in the SSE, the dominating role of ionic valency, dissolved organic carbon (DOC), and CC was observed in colloidal stabilization over IS. No significant impact of temperature was observed on the stability of CuO NPs both in model and SSE. Further, stability studies in the SSE were correlated with NPs retention behavior in soils. Observations suggest that retention of CuO NPs in soils is a function of binding of the colloidal fraction to the soil, which in turn depends on the colloidal stability. The highest retention was observed in black and laterite soils, whereas lower binding of clay fraction in red soil caused the least retention. A decrease in Kd values after a certain application concentration provided maximum sustainable application concentration of CuO NPs, which may vary with soil properties. Results suggest that the binding of clay and organic matter with a sandy matrix of soil plays a prime role in deciding the overall fate of CuO NPs in the soils.

Overcoming presynaptic effects of VAMP2 mutations with 4-aminopyridine treatment.

Clinical and genetic features of five unrelated patients with de novo pathogenic variants in the synaptic vesicle-associated membrane protein 2 (VAMP2) reveal common features of global developmental delay, autistic tendencies, behavioral disturbances, and a higher propensity to develop epilepsy. For one patient, a cognitively impaired adolescent with a de novo stop-gain VAMP2 mutation, we tested a potential treatment strategy, enhancing neurotransmission by prolonging action potentials with the aminopyridine family of potassium channel blockers, 4-aminopyridine and 3,4-diaminopyridine, in vitro and in vivo. Synaptic vesicle recycling and neurotransmission were assayed in neurons expressing three VAMP2 variants by live-cell imaging and electrophysiology. In cellular models, two variants decrease both the rate of exocytosis and the number of synaptic vesicles released from the recycling pool, compared with wild-type. Aminopyridine treatment increases the rate and extent of exocytosis and total synaptic charge transfer and desynchronizes GABA release. The clinical response of the patient to 2 years of off-label aminopyridine treatment includes improved emotional and behavioral regulation by parental report, and objective improvement in standardized cognitive measures. Aminopyridine treatment may extend to patients with pathogenic variants in VAMP2 and other genes influencing presynaptic function or GABAergic tone, and tested in vitro before treatment.

Increased stress and altered expression of histone modifying enzymes in brain are associated with aberrant behaviour in vitamin B12 deficient female mice.

A sub-optimal nutritional environment from early life can be envisaged as a stressor that translates into mental health problems in adulthood. After considering (a) the widespread prevalence of vitamin B12 deficiency especially amongst women in developing countries and (b) the importance of vitamin B12 in normal brain function, in this study we have elucidated the behavioural correlates of chronic severe and moderate vitamin B12 deficiency in C57BL/6 mice. Female weanling mice were assigned to three dietary groups: (a) control AIN-76A diet with cellulose as dietary fibre (b) vitamin B12 restricted AIN-76A diet with pectin as dietary fibre (severe deficiency group) and (c) vitamin B12 restricted AIN-76A diet with cellulose as dietary fibre (moderate deficiency group). The mice received these diets throughout pregnancy, lactation and thereafter. Nest-building, maternal care, anxiety and depressive behaviours were evaluated. Oxidative stress, activities of antioxidant enzymes and expression of various histone modifying enzymes in brain were investigated to unravel the probable underlying mechanisms. Our data suggests that both severe and moderate vitamin B12 deficiency induced anxiety and impaired maternal care. However, only severe vitamin B12 deficiency induced depression. Oxidative stress and poor antioxidant defense underlie the deleterious effects of both severe and moderate vitamin B12 deficiency. Altered expression of histone modifying enzymes in the brain of severely deficient mice is suggestive of epigenetic reprogramming. This study suggests that chronic vitamin B12 deficiency leads to behavioural anomalies in female C57BL/6 mice and the severity of these outcomes can be correlated to the level of deficiency.

Leucine-rich repeat-containing 8B protein is associated with the endoplasmic reticulum Ca2+ leak in HEK293 cells.

Leucine-rich repeat-containing 8 (LRRC8) proteins have been proposed to evolutionarily originate from the combination of the channel protein pannexin, and a leucine-rich repeat (LRR) domain. Five paralogs of LRRC8, namely LRRC8A, LRRC8B, LRRC8C, LRRC8D and LRRC8E have been reported. LRRC8A has been shown to be instrumental in cell swelling. Here, we identify LRRC8B as a key player in the cellular Ca2+ signaling network. Overexpression of human LRRC8B in HEK293 cells reduced the Ca2+ level in the endoplasmic reticulum (ER). LRRC8B-overexpressing cells exhibited a lesser release of Ca2+ from the ER in response to ATP, carbachol and intracellular administration of inositol (1,4,5)-trisphosphate (IP3). LRRC8B-knockdown cells showed a slower depletion of the ER Ca2+ stores when sarco-endoplasmic reticulum Ca2+-ATPase was blocked with thapsigargin (TG), while overexpression of LRRC8B had the opposite effect. LRRC8B-overexpressing cells exhibited a higher level of store-operated Ca2+ entry following store-depletion by TG. Collectively, LRRC8B participates in intracellular Ca2+ homeostasis by acting as a leak channel in the ER. This study gives a fundamental understanding of the role of a novel protein in the elemental cellular process of ER Ca2+ leak and expands the known roles for LRRC8 proteins.This article has an associated First Person interview with the first author of the paper.

Disrupting the hippocampal Piwi pathway enhances contextual fear memory in mice.

The Piwi pathway is a conserved gene regulatory mechanism comprised of Piwi-like proteins and Piwi-interacting RNAs, which modulates gene expression via RNA interference and through interaction with epigenetic mechanisms. The mammalian Piwi pathway has been defined by its role in transposon control during spermatogenesis; however, despite an increasing number of studies demonstrating its expression in the nervous system, relatively little is known about its function in neurons or potential contribution to behavioural regulation. We have discovered that all three Piwi-like genes are expressed in the adult mouse brain, and that viral-mediated knockdown of the Piwi-like genes Piwil1 and Piwil2 in the dorsal hippocampus leads to enhanced contextual fear memory without affecting generalised anxiety. These results implicate the Piwi pathway in behavioural regulation in the adult mammalian brain, likely through modulation of plasticity-related gene expression.

Sex difference in mouse hypothalamic transcriptome profile in stress-induced depression model.

The influence of sex on prevalence and presentation of human depression necessitates studying its role in depression. Additionally, stress can also affect the incidence and progression of depressive phenotypes. Hypothalamus is a well-reported sexually dimorphic brain region. Hence, we performed a gene-array to uncover the chronic stress-induced transcriptional changes in the hypothalamus of male, intact female (with ovaries) and ovariectomized female mice. Chronic variable mild stress model was used to induce depression-like behavior in mice, assessed by despair behavior in forced swim test. We observed 90, 205 and 106 uniquely expressed genes among the 114, 226 and 121 stress-regulated genes (fold change >1.2 and p < 0.05) in male, intact female, and ovariectomized female group mice respectively. Pathway analysis of the altered genes identified 'posttranslational processing of neuroendocrine peptides' as the most significantly enriched pathway and also differentially regulated among the groups. Arginine vasopressin and Cholecystokinin were upregulated in males whereas in ovariectomized females Arginine vasopressin alone was upregulated, and Oxytocin was downregulated in intact females. A greater number of nodes and connections found in intact females indicate their intricate molecular response to chronic stress condition. These results suggest that stress-regulated transcriptional changes in the hypothalamus are sex-specific and ovarian hormone-dependent, which warrants urgent sex-based medical attention in depressive disorder.

Influence of magnetite and its weathering originated maghemite and hematite minerals on sedimentation and transport of nanoplastics in the aqueous and subsurface environments.

Persistent nanoplastics (NPs) and their interaction with ubiquitous iron oxide minerals (IOMs) require a detailed understanding to dictate NPs fate and transport in aqueous and subsurface environments. Current study emphasizes on understanding nanoplastics (NPs) interaction with magnetite, and its weathering-originated mineral colloids, i.e., maghemite and hematite under varying environmental conditions (pH, humic acid, ionic strength and water matrix). Results showed that the higher surface hydroxyl group, smaller particle size, and positive surface charge of magnetite led to maximum NPs sorption (805.8 mg/g) in comparison to maghemite (602 mg/g) and hematite (384.3 mg/g). Charge distribution and sedimentation kinetic studies in bimodal systems showed enhanced coagulation in magnetite-NPs system. FTIR and XPS analysis of NPs-IOMs reaction precipitate revealed the vital role of surface functionality in their interaction. Column experiments revealed higher NPs retention in IOMs-coated quartz sand than bare quartz sand. Further, in river water (RW), magnetite-coated sand has shown maximum NPs retention (>80 %) than maghemite (62 %) and hematite (52 %), suggesting limited NPs mobility in the presence of magnetite in subsurface conditions. These findings elucidated the dependence of NPs fate on IOMs in freshwater systems and illustrated IOMs impact on NPs mobility in the subsurface porous environment.

FOXP1 regulates the development of excitatory synaptic inputs onto striatal neurons and induces phenotypic reversal with reinstatement.

Long-range glutamatergic inputs originating from the cortex and thalamus are indispensable for striatal development, providing the foundation for motor and cognitive functions. Despite their significance, transcriptional regulation governing these inputs remains largely unknown. We investigated the role of a transcription factor encoded by a high-risk autism-associated gene, FOXP1, in sculpting glutamatergic inputs onto spiny projection neurons (SPNs) within the striatum. We find a neuron subtype-specific role of FOXP1 in strengthening and maturing glutamatergic inputs onto dopamine receptor 2-expressing SPNs (D2 SPNs). We also find that FOXP1 promotes synaptically driven excitability in these neurons. Using single-nuclei RNA sequencing, we identify candidate genes that mediate these cell-autonomous processes through postnatal FOXP1 function at the post-synapse. Last, we demonstrate that postnatal FOXP1 reinstatement rescues electrophysiological deficits, cell type-specific gene expression changes, and behavioral phenotypes. Together, this study enhances our understanding of striatal circuit development and provides proof of concept for a therapeutic approach for FOXP1 syndrome and other neurodevelopmental disorders.

Compensation between FOXP transcription factors maintains proper striatal function.

Spiny projection neurons (SPNs) of the striatum are critical in integrating neurochemical information to coordinate motor and reward-based behavior. Mutations in the regulatory transcription factors expressed in SPNs can result in neurodevelopmental disorders (NDDs). Paralogous transcription factors Foxp1 and Foxp2, which are both expressed in the dopamine receptor 1 (D1) expressing SPNs, are known to have variants implicated in NDDs. Utilizing mice with a D1-SPN-specific loss of Foxp1, Foxp2, or both and a combination of behavior, electrophysiology, and cell-type-specific genomic analysis, loss of both genes results in impaired motor and social behavior as well as increased firing of the D1-SPNs. Differential gene expression analysis implicates genes involved in autism risk, electrophysiological properties, and neuronal development and function. Viral-mediated re-expression of Foxp1 into the double knockouts is sufficient to restore electrophysiological and behavioral deficits. These data indicate complementary roles between Foxp1 and Foxp2 in the D1-SPNs.

p38-MAPK is prerequisite for the synthesis of SARS-CoV-2 protein.

The inhibition of p38 mitogen-activated protein kinase (p38-MAPK) by small molecule chemical inhibitors was previously shown to impair severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, however, mechanisms underlying antiviral activity remains unexplored. In this study, reduced growth of SARS-CoV-2 in p38-α knockout Vero cells, together with enhanced viral yield in cells transfected with construct expressing p38α, suggested that p38-MAPK is essential for the propagation of SARS-CoV-2. The SARS-CoV-2 was also shown to induce phosphorylation (activation) of p38, at time when transcription/translational activities are considered to be at the peak levels. Further, we demonstrated that p38 supports viral RNA/protein synthesis without affecting viral attachment, entry, and budding in the target cells. In conclusion, we provide mechanistic insights on the regulation of SARS-CoV-2 replication by p38 MAPK.

FOXP1 regulates the development of excitatory synaptic inputs onto striatal neurons and induces phenotypic reversal with reinstatement.

Long-range glutamatergic inputs from the cortex and thalamus are critical for motor and cognitive processing in the striatum. Transcription factors that orchestrate the development of these inputs are largely unknown. We investigated the role of a transcription factor and high-risk autism-associated gene, FOXP1, in the development of glutamatergic inputs onto spiny projection neurons (SPNs) in the striatum. We find that FOXP1 robustly drives the strengthening and maturation of glutamatergic input onto dopamine receptor 2-expressing SPNs (D2 SPNs) but has a comparatively milder effect on D1 SPNs. This process is cell-autonomous and is likely mediated through postnatal FOXP1 function at the postsynapse. We identified postsynaptic FOXP1-regulated transcripts as potential candidates for mediating these effects. Postnatal reinstatement of FOXP1 rescues electrophysiological deficits, reverses gene expression alterations resulting from embryonic deletion, and mitigates behavioral phenotypes. These results provide support for a possible therapeutic approach for individuals with FOXP1 syndrome.

Compensation between FOXP transcription factors maintains proper striatal function.

Spiny projection neurons (SPNs) of the striatum are critical in integrating neurochemical information to coordinate motor and reward-based behavior. Mutations in the regulatory transcription factors expressed in SPNs can result in neurodevelopmental disorders (NDDs). Paralogous transcription factors Foxp1 and Foxp2, which are both expressed in the dopamine receptor 1 (D1) expressing SPNs, are known to have variants implicated in NDDs. Utilizing mice with a D1-SPN specific loss of Foxp1, Foxp2, or both and a combination of behavior, electrophysiology, and cell-type specific genomic analysis, loss of both genes results in impaired motor and social behavior as well as increased firing of the D1-SPNs. Differential gene expression analysis implicates genes involved in autism risk, electrophysiological properties, and neuronal development and function. Viral mediated re-expression of Foxp1 into the double knockouts was sufficient to restore electrophysiological and behavioral deficits. These data indicate complementary roles between Foxp1 and Foxp2 in the D1-SPNs.

Limiting mitochondrial plasticity by targeting DRP1 induces metabolic reprogramming and reduces breast cancer brain metastases.

Disseminated tumor cells with metabolic flexibility to utilize available nutrients in distal organs persist, but the precise mechanisms that facilitate metabolic adaptations remain unclear. Here we show fragmented mitochondrial puncta in latent brain metastatic (Lat) cells enable fatty acid oxidation (FAO) to sustain cellular bioenergetics and maintain redox homeostasis. Depleting the enriched dynamin-related protein 1 (DRP1) and limiting mitochondrial plasticity in Lat cells results in increased lipid droplet accumulation, impaired FAO and attenuated metastasis. Likewise, pharmacological inhibition of DRP1 using a small-molecule brain-permeable inhibitor attenuated metastatic burden in preclinical models. In agreement with these findings, increased phospho-DRP1 expression was observed in metachronous brain metastasis compared with patient-matched primary tumors. Overall, our findings reveal the pivotal role of mitochondrial plasticity in supporting the survival of Lat cells and highlight the therapeutic potential of targeting cellular plasticity programs in combination with tumor-specific alterations to prevent metastatic recurrences.

Evaluation of the safety, immunogenicity and efficacy of a new live-attenuated lumpy skin disease vaccine in India.

Lumpy skin disease (LSD) was reported for the first time in India in 2019 and since then, it has become endemic. Since a homologous (LSD-virus based) vaccine was not available in the country, goatpox virus (GPV)-based heterologous vaccine was authorized for mass immunization to induce protection against LSD in cattle. This study describes the evaluation of safety, immunogenicity and efficacy of a new live-attenuated LSD vaccine developed by using an Indian field strain, isolated in 2019 from cattle. The virus was attenuated by continuous passage (P = 50) in Vero cells. The vaccine (50th LSDV passage in Vero cells, named as Lumpi-ProVacInd) did not induce any local or systemic reaction upon its experimental inoculation in calves (n = 10). At day 30 post-vaccination (pv), the vaccinated animals were shown to develop antibody- and cell-mediated immune responses and exhibited complete protection upon virulent LSDV challenge. A minimum Neethling response (0.018% animals; 5 out of 26,940 animals) of the vaccine was observed in the field trials conducted in 26,940 animals. There was no significant reduction in the milk yield in lactating animals (n = 10108), besides there was no abortion or any other reproductive disorder in the pregnant animals (n = 2889). Sero-conversion was observed in 85.18% animals in the field by day 30 pv.