Biology subjects

Bisht, K.

Publications and source records attributed to Bisht, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Marrow-mimicry MEW scaffolds recapitulate erythroblastic islands for high-density cRBC biomanufacturing

Biomanufactured cell therapies are delivering a step-change in modern medicine, but their high costs have prevented widespread adoption. Cultured red blood cells (cRBCs) are one type of cell therapy in clinical trials for off-the-shelf blood transfusion but remain limited by the exorbitant costs of manufacturing trillions of RBCs required for a blood transfusion. These costs could be mitigated if culture platforms could grow cells at higher density, like those found in the bone marrow, requiring less media and lower concentrations of expensive growth factors per cell. We engineered melt electrowritten (MEW) polycaprolactone (PCL) scaffolds in geometries which imitate marrow trabeculae structures and were then plasma-treated to enable peripheral blood mononuclear cells (PB MNC) attachment. PB MNC monocytes first adhered to the scaffold and rapidly differentiated into CD68+ macrophage-like cells. These macrophage-loaded scaffolds adhered CD71+ erythroblasts, which then grew from 10 to 25% between days 4 and 8 and produced CD235a+ erythroid cells by day 20, under erythrogenic culture conditions. MEW PCL scaffolds in trabecular-like geometries had greater surface area, adhered more macrophages, and generated more erythroblasts than traditional MEW crosshatch geometries. The MEW scaffolds continuously released less-adherent cells to the supernatant, providing a 74% increase in supernatant cell numbers versus liquid suspension controls with identical cell seeding numbers and media volumes. High-cell-density scaffolds exhibited increased metabolism and endogenous production of self-supportive cytokines, which may offer cost-saving opportunities through future media optimisation. This study reports marrow trabeculae-mimetic MEW PCL designs capable of selectively adhering PB MN cells to develop macrophage-coated scaffolds, which provided physical and paracrine support for cRBC production. This high-cell-density culture platform may be useful for future cell therapy biomanufacturing protocols.

bioengineering↗

bioRxiv2026

Polymer Polydispersity and Lipid Composition Control Nanoplastic Disassembly in Membranes

Nanoplastic interactions with cell membranes can influence particle uptake, translocation, and biological effects, making their molecular description important for assessing the environmental and biological consequences of plastic pollution. However, molecular simulations generally represent nanoplastics using monodisperse polymer chains, whereas nanoplastics formed by environmental degradation are polydisperse. Here, we construct 4 nm polyethylene terephthalate (PET), polyethylene (PE), and polystyrene (PS) nanoparticles with lognormal molecular-weight distributions reported for degraded polymers, and with melt-like chain packing and entanglement. Using Martini coarse-grained molecular dynamics, we simulate their interactions for 10 microseconds with three lipid bilayers of increasing complexity. PE and PS insert rapidly and remain largely intact, whereas PET remains surface-associated and progressively releases individual chains. Fluid, polyunsaturated lipid environments undergo greater deformation and promote substantially more chain release, up to 56% of the chains for PET and 43% for PS. Release begins with the shortest chains and progressively extends to longer chains, while semicrystalline PE remains largely intact. In contrast, a size-matched monodisperse PS particle releases no chains and minimally perturbs the membrane. These results identify the molecular-weight distribution as a key determinant of nanoplastic-membrane interactions, and indicate that uniform model particles underestimate the release of polymer chains into biological membranes.

biophysics↗

bioRxiv2026

Temperature-dependent reprogramming of virulence traits during Pseudomonas aeruginosa biofilm formation

Pseudomonas aeruginosa is an opportunistic pathogen that occupies diverse ecological niches, including soil, water, and the human host. Environmental cues encountered across these habitats trigger adaptive responses that promote survival and persistence through regulation of virulence-associated traits, including secreted factors, siderophores, and biofilm exopolysaccharides (EPS). One major change experienced during the transition from environmental reservoirs to the host is an increase in temperature. Although temperature is a key signal encountered during host transition, its global impact on P. aeruginosa physiology remains poorly understood. We therefore investigated the effects of temperature on planktonic and biofilm-populations, comparing both growth states at 23{degrees}C and 30{degrees}C, representing environmental temperatures, and at 37{degrees}C and 40{degrees}C, representing normal and febrile host temperatures. Transcriptomic and phenotypic analyses revealed extensive temperature-dependent regulation of virulence determinants in both growth states. Expression of the type VI secretion system was elevated at environmental temperatures, whereas pyoverdine biosynthesis and the type III secretion system were upregulated at host temperatures. Building on our previous finding that biofilms formed at environmental and host temperatures differ in architecture, biomass, and EPS composition, we next examined how these structural differences influence stress tolerance. Biofilms grown at 23{degrees}C and 30{degrees}C exhibited substantially greater tolerance to antibiotic stress than biofilms grown at 37{degrees}C and 40{degrees}C. Growth temperature therefore establishes biofilm properties that subsequently influence the stress-tolerance profile of the population. Collectively, our findings identify temperature as a major environmental cue that reprograms P. aeruginosa physiology in ways likely to support persistence across distinct ecological niches.

microbiology↗

bioRxiv2026

Endotoxemia and TLR4 via tissue resident macrophages triggers anemia in mouse model of colitis

Anemia is one of the most debilitating and frequent complication of inflammatory bowel diseases (IBD) and is often treated with iron supplementation, which has limited efficacy and causes additional co-morbidities. Damaged intestinal barrier function is a hallmark of IBD and causes the translocation of endotoxins from gut bacteria into the bloodstream. A previous study in mice reported that endotoxins suppress erythropoiesis by reprogramming erythroblastic island macrophages (EBI M{varphi}). Here, we show that IBD patients and mice with acute colitis developed endotoxemia associated with anemia. Endotoxemia in IBD patients was negatively correlated with blood erythrocyte counts. In line with this, mice with acute colitis caused by drinking water containing dextrin sodium sulphate (DSS) had endotoxemia together with anemia characterized by reduced red blood cell counts, hemoglobin content and hematocrit, and reduced medullary erythropoiesis which was in part compensated by increased extramedullary erythropoiesis. As the endotoxin receptor TLR4 is expressed by CD169+ gut-resident macrophages as well as erythroid island macrophages in the bone marrow, we tested the hypothesis that TLR4 expressed by these CD169+ macrophages mediate both inflammatory colitis and anemia. Indeed, mice with a conditional deletion of the Tlr4 gene specifically in CD169+ tissue-resident macrophages were protected from DSS-induced anemia, inflammation and colitis. In addition, treatment with the TLR4 inhibitor C34 abated inflammation and anemia in DSS mice. These results suggest that endotoxins leaking from the inflamed gut may play a crucial role in IBD and associated anemia via CD169+ tissue-resident macrophages and that drugs targeting TLR4 may protect against IBD-associated anemia.

immunology↗

bioRxiv2025

Endothelial protein C receptor CD201 is a better marker than SCA1 to identify mouse long-term reconstituting hematopoietic stem cells following septic challenge

Stem cell antigen-1 (SCA1) is widely used to identify mouse hematopoietic stem cells (HSC) and multipotent progenitors (MPP) among lineage-negative KIT+ (LK) cells. However, SCA1 is expressed only in a few inbred mouse strains and becomes strongly upregulated on LK cells following in vivo challenge with interferons, lipopolysaccharide (LPS) or pathogens leading to incorrect analysis of HSC function subsets and delineation of HSC, MPP and lineage-restricted progenitor subsets. Endothelial protein C receptor CD201can be used as an alternative marker for mouse and even human HSC. However, whether CD201 expression changes following infectious challenge is unknown. Unlike SCA1, CD201 expression did not change on mouse LK cells in response to LPS in vivo. Long-term competitive transplantations with CD201+, CD201- or SCA1+ LK cells showed that most reconstituting HSCs are within the LK CD201+ population after LPS challenge. However long-term competitive repopulation potential of LK SCA1+ cells from LPS-treated mice was much more severely reduced than that of LK CD201+ cells from the same LPS-treated donors suggesting that the LK SCA1+ population in challenged donors becomes contaminated with CD201- progenitors devoid of long-term repopulation potential. Based on CD201 gating strategy, we re-assessed the effect of LPS on HSC and MPP cycling and mobilization, and their dependency on MY88 and TRIF adaptors. In conclusion, CD201 enables a more accurate analysis of mouse HSC and MPP subsets in all inbred strains in septic conditions or steady-state. HighlightsO_LISCA1 expression is markedly enhanced on MPPs and myeloid progenitors in vivo following LPS treatment whereas CD201 expression is not. C_LIO_LIOver 90% of the long-term competitive repopulation potential resides within LK CD201+ cells after LPS treatment. C_LIO_LILK CD201+ cells sorted from LPS-treated mice give 4.4-fold higher multi-lineage chimerism than LK SCA1+ cells. C_LIO_LISCA1 upregulation leads to overestimation of LT-HSC and MPP subsets, and underestimation of myeloid progenitors in femoral BM of LPS-treated mice. C_LI

immunology↗

bioRxiv2025

Rapid Screening and Validation of Intracellularly Active Antimycobacterial Compounds with Efficacy in Cellular and Murine Tuberculosis Models

The discovery of effective intracellular antimycobacterial agents is limited by the slow growth of Mycobacterium tuberculosis (Mtb) and a lack of scalable models that effectively replicate host-pathogen interactions. To address this, we established a robust ex vivo infection model using human monocyte-derived THP-1 macrophages and the fast-growing pathogenic surrogate Mycobacterium marinum (Mmar) which recapitulates key features of Mtb intracellular infection, including macrophage colonization and innate immune activation. This model supports medium-throughput screening workflows for assessing antibiotic-mediated control of intracellular mycobacteria. Using this platform, we screened FDA-approved antipsychotics and identified the phenothiazines- trifluoperazine (TFP) and fluphenazine (FFP), as potent adjuncts to frontline anti-tuberculosis therapy (ATT). Combinations of either phenothiazine with frontline TB drugs enhanced intracellular bacterial clearance more effectively than the previously reported antidepressant sertraline, initiating significant control within 12 hours of treatment in macrophages. In murine models of TB infection, TFP and FFP further potentiated bacterial clearance and tissue recovery when used in combination with standard anti-TB therapy (ATT). Together, our results validate the THP1-Mmar infection model as a rapid, robust, and scalable platform for identifying intracellular antimycobacterial agents and host-directed therapeutics, offering a valuable tool to accelerate TB drug discovery and development.

microbiology↗

bioRxiv2025

Macrophage Cholesterol Homeostasis Underpins the role of Sertraline as an Adjunctive Agent in Tuberculosis Therapy.

Cellular metabolism plays a deterministic role in the macrophage responses to intracellular pathogens such as Mycobacterium tuberculosis (Mtb) and infection control. Here, we demonstrate that the significant rewiring of the macrophage cholesterol metabolism by sertraline (SRT), an FDA-approved antidepressant, effectively enhances bacterial control. SRT, by virtue of its potent cationic amphiphilicity leads to the accumulation of lysosomal cholesterol in macrophages and activating the transcription factor SREBP2 and enhancing cholesterol biosynthesis. By specific gene silencing and biochemical inhibition assays, we further demonstrate that this metabolic reprogramming promotes lysosomal membrane permeabilization, mitochondrial reactive oxygen species (ROS) generation, heightened IL-1{beta} release, collectively enhancing macrophage bactericidal activity. Our results thus highlight a previously underappreciated link between cholesterol homeostasis and inflammasome activation, which contributes to improved Mtb clearance in the presence of adjunctive sertraline therapy. Statement of significanceTuberculosis remains a leading cause of global mortality, and treatment success is limited due to a long treatment regimen. This study identifies macrophage cholesterol homeostasis as a central pathway to enhance anti-tubercular therapy. We show that Sertraline, an FDA-approved antidepressant, a cationic amphiphilic drug (CAD), reprograms cholesterol trafficking through NPC1 inhibition. This drives the consequential processing of the master regulator SREBP2, accompanied by lysosomal membrane permeabilization. Collectively, these lead to mitochondrial ROS production and activation of NLRP3 signalling, boosting inflammasome-mediated bacterial clearance. By establishing cholesterol sequestration as the mechanistic basis of host-directed activity of sertraline and distinguishing it from ineffective CADs, our work provides a mechanistic framework for developing cholesterol-modulating host-directed adjuncts to improve TB treatment outcomes. Graphical abstract: SRT induced lysosomal cholesterol accumulation results in enhanced bacterial control via inflammasome activation O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/665474v3_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1019b4dorg.highwire.dtl.DTLVardef@7b0ae8org.highwire.dtl.DTLVardef@9297eorg.highwire.dtl.DTLVardef@1832bef_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Sertraline enhances bacterial control by improving the pharmacodynamic -pharmacokinetic properties of frontline TB drugs

Given the need for innovative interventions for tackling the burden of TB, host directed therapies have emerged as a promising alternative in recent times. The combination of sertraline with frontline TB drugs has shown excellent promise in the murine models of infection in imparting better bacterial control and increasing host survival. We tested if the addition of sertraline worked to increase bacterial clearance in the random bred guinea pig model of TB infection that mimics the inter individual heterogeneity observed in the human response to infection. The combination of sertraline and frontline TB drugs effectively reduced bacterial burdens in the tissues of guinea pigs significantly better than the drugs alone with a marked betterment of lung histopathology. In order to evaluate the effect of sertraline on the pharmacodynamic properties of TB drugs, concentrations of the drugs were estimated in tissues at different time intervals over a period of 24h of administration to rats. Overall, addition of sertraline did not alter TB drug distribution or clearance from the animals, although enriching drug amounts transiently between 3-6 h in the different tissues. We thus highlight the advantage of an adjunct TB therapy with the inclusion of sertraline, an FDA approved antidepressant, in improving the PKPD of TB drugs and imparting better infection control in diverse models of infection.

microbiology↗

bioRxiv2025

Temperature-dependent modulation of aberrant influenza A virus RNA synthesis affects innate immune signaling

Fever during influenza A virus (IAV) infection is triggered by the innate immune response. Various factors contribute to this response, including IAV mini viral RNAs (mvRNA), which trigger RIG-I signaling when their replication and transcription are dysregulated by template loops (t-loop). It is presently not well understood whether the fever response to IAV infection impacts subsequent viral replication and innate immune activation. Here we show that IAV infection at temperatures that simulate fever leads to increased antiviral signaling in H1N1 and H3N2 infections. Mathematical modeling and experimental analyses reveal that differential IAV nucleoprotein and RNA polymerase production increase mvRNA and interferon production. Moreover, at the higher infection temperature, mvRNAs with dysregulating t-loops contribute most to the innate immune activation. We propose that fever during IAV infection can establish a positive feedback loop in which elevated aberrant RNA synthesis and innate immune activation can contribute to the dysregulation of cytokine production.

microbiology↗

bioRxiv2025

Inflammasome activation dictates the efficacy of antimycobacterial activity of frontline TB drugs

Recent developments in TB treatment have identified an enormous potential of host targeting entities (host directed therapies-HDT) in achieving better and faster control of infection. We have previously demonstrated the synergistic effect of sertraline with frontline TB drugs in clearing infection in murine tissues. We surmised that understanding the mechanism of sertraline mediated enhancement of antibiotic efficacy would help identify novel pathways of pathogen control. Here, we have used sertraline as a probe to identify host signaling pathways that are important for bacterial control. We identify a role for sertraline mediated modulation of macrophage mitochondrial physiology in this process. We show that the resultant ROS generated in cells acts as the secondary signal for host cell inflammasome activation catalyzing the greater release of IL1beta; and significant efflux of K+ from the SRT treated macrophages. We thus highlight an important relationship between mitochondrial physiology in regulating the activation of the inflammasome to achieve better control of Mtb by infected macrophages.

microbiology↗

bioRxiv2025

A two-step mechanism for RIG-I activation by influenza virus mini viral RNAs

Influenza A virus (IAV) non-canonical replication products can be bound by host pathogen sensors, such as retinoic acid-inducible gene I (RIG-I). However, innate immune activation is infrequent in cell culture infection, in particular by adapted strains. Moreover, it is not understood why non-canonical IAV RNAs activate RIG-I in a sequence- or RNA structure-dependent manner. We therefore hypothesized that multiple errors need to occur before influenza virus RNA synthesis activates innate immune signaling. To test this idea, we investigated whether RIG-I activation is stimulated by the non-canonical or aberrant transcription of mini viral RNAs (mvRNA), a <125 nt long RNA that is overexpressed in pandemic and highly pathogenic IAV infections. Using mvRNA sequences identified in tissue culture and ferret infections, we find that mvRNAs can cause non-canonical transcription termination through a truncated 5' polyadenylation signal or a 5' transient RNA structure that interrupts polyadenylation. The resulting capped complementary RNAs (ccRNA) can stimulate the release of a template mvRNA in vitro. Finally, we find that both mvRNA and ccRNA sequences can be bound by RIG-I in cell culture and that blocking mvRNA transcription with baloxavir reduces IFN promoter activity. Overall, our findings indicate that sequential rounds of non-canonical or aberrant viral replication and transcription are needed before mvRNAs trigger innate immune signaling in a sequence-dependent manner.

microbiology↗

bioRxiv2024

Dark Microglia Are Abundant in Normal Postnatal Development, where they Remodel Synapses via Phagocytosis and Trogocytosis, and Are Dependent on TREM2

This study examined dark microglia--a state linked to central nervous system pathology and neurodegeneration--during postnatal development in the mouse ventral hippocampus, finding that dark microglia interact with blood vessels and synapses and perform trogocytosis of pre-synaptic axon terminals. Furthermore, we found that dark microglia in development notably expressed C-type lectin domain family 7 member A (CLEC7a), lipoprotein lipase (LPL) and triggering receptor expressed on myeloid cells 2 (TREM2) and required TREM2, differently from other microglia, suggesting a link between their role in remodeling during development and central nervous system pathology. Together, these results point towards a previously under-appreciated role for dark microglia in synaptic pruning and plasticity during normal postnatal development.

neuroscience↗

bioRxiv2024

Global stress response in Pseudomonas aeruginosa upon malonate utilization

Versatility in carbon source utilization assists Pseudomonas aeruginosa in its adaptation to various niches. Recently, we characterized the role of malonate, an understudied carbon source, in quorum sensing regulation, antibiotic resistance, and virulence factor production in P. aeruginosa. These results indicate that global responses to malonate metabolism remain to be uncovered. We leveraged a publicly available metabolomic dataset on human airway and found malonate to be as abundant as glycerol, a common airway metabolite and carbon source for P. aeruginosa. Here, we explored and compared adaptations of P. aeruginosa UCBPP-PA14 (PA14) in response to malonate or glycerol as a sole carbon source using transcriptomics and phenotypic assays. Malonate utilization activated glyoxylate and methylcitrate cycles and induced several stress responses, including oxidative, anaerobic, and metal stress responses associated with increases in intracellular aluminum and strontium. Some induced genes were required for optimal growth of P. aeruginosa in malonate. To assess the conservation of malonate-associated responses among P. aeruginosa strains, we compared our findings in strain PA14 with other lab strains and cystic fibrosis isolates of P. aeruginosa. Most strains grew on malonate as a sole carbon source as efficiently as or better than glycerol. While not all responses to malonate were conserved among strains, formation of biomineralized biofilm-like aggregates, increased tolerance to kanamycin, and increased susceptibility to norfloxacin were the most frequently observed phenotypes. Our findings reveal global remodeling of P. aeruginosa gene expression during its growth on malonate as a sole carbon source that is accompanied by several important phenotypic changes. These findings add to accumulating literature highlighting the role of different carbon sources in the physiology of P. aeruginosa and its niche adaptation. ImportancePseudomonas aeruginosa is a notorious pathogen that causes local and systemic infections in immunocompromised individuals. Different carbon sources can uniquely modulate metabolic and virulence pathways in P. aeruginosa, highlighting the importance of the environment that the pathogen occupies. In this work, we used a combination of transcriptomic analysis and phenotypic assays to determine how malonate utilization impacts P. aeruginosa, as recent evidence indicates this carbon source may be relevant to certain niches associated within the human host. We found that malonate utilization can induce global stress responses, alter metabolic circuits, and influence various phenotypes of P. aeruginosa that could influence host colonization. Investigating the metabolism of malonate provides insight into P. aeruginosa adaptations to specific niches where this substrate is abundant, and how it can be leveraged in the development of much-needed antimicrobial agents or identification of new therapeutic targets of this difficult-to-eradicate pathogen.

microbiology↗

bioRxiv2023

Cas13-based amplification-free quantification of aberrant RNAs during influenza virus infection

Influenza A virus RNA synthesis produces full-length and aberrant RNA molecules, which include defective viral genomes (DVG) and mini viral RNAs (mvRNA). Sequencing approaches have shown that aberrant RNA species may be present during infection, and that they can vary in size, segment origin, and sequence. Moreover, a subset of aberrant RNA molecules can bind and activate host pathogen receptor retinoic acid-inducible gene I (RIG-I), leading to innate immune signaling and the expression of type I and III interferons. Understanding the kinetics and distribution of these immunostimulatory aberrant RNA sequences is important for understanding their function in IAV infection. Here, we use an amplification-free LbuCas13a-based detection method to quantify mvRNA amplification dynamics and subcellular distributions. We show that our assay can quantify the copy numbers of specific mvRNA sequences in infected tissue culture cells, ferret upper and lower respiratory tract tissue infected with two different pandemic H1N1 IAV strains, or clinical nasopharyngeal swab extracts of hospitalized patients infected with seasonal H1N1 or H3N2 strains. In addition, we find dynamic differences between immunostimulatory and non-immunostimulatory mvRNAs, as well as among mvRNAs derived from different segments, during IAV infection. Overall, our results reveal a hitherto hidden diversity in the behavior of IAV mvRNAs and suggest that individual aberrant RNAs are not produced stochastically.

microbiology↗

bioRxiv2023

Ketogenic diet alters microglial morphology and changes the hippocampal lipidomic profile distinctively in stress susceptible versus resistant male mice upon repeated social defeat

Psychological stress confers an increased risk for several diseases including psychiatric conditions. The susceptibility to psychological stress is modulated by various factors, many of them being modifiable lifestyle choices. The ketogenic diet (KD) has emerged as a dietary regime that offers positive outcomes on mood and health status. Psychological stress and elevated inflammation are common features of neuropsychiatric disorders such as certain types of major depressive disorder. KD has been attributed anti-inflammatory properties that could underlie its beneficial consequences on the brain and behavior. Microglia are the main drivers of inflammation in the central nervous system. They are known to respond to both dietary changes and psychological stress, notably by modifying their production of cytokines and relationships among the brain parenchyma. To assess the interactions between KD and the stress response, including effects on microglia, we examined adult male mice on control diet (CD) versus KD that underwent 10 days of repeated social defeat (RSD) or remained non-stressed (controls; CTRLs). Through a social interaction test, stressed mice were classified as susceptible (SUS) or resistant (RES) to RSD. The mouse population fed a KD tended to have a higher proportion of individuals classified as RES following RSD. Microglial morphology and ultrastructure were then analyzed in the ventral hippocampus CA1, a brain region known to present structural alterations as a response to psychological stress. Distinct changes in microglial soma and arborization linked to the KD, SUS and RES phenotypes were revealed. Ultrastructural analysis by electron microscopy showed a clear reduction of cellular stress markers in microglia from KD fed animals. Furthermore, ultrastructural analysis showed that microglial contacts with synaptic elements were reduced in the SUS compared to the RES and CTRL groups. Hippocampal lipidomic analyses lastly identified a distinct lipid profile in SUS animals compared to CTRLs. These key differences, combined with the distinct microglial responses to diet and stress, indicate that unique metabolic changes may underlie the stress susceptibility phenotypes. Altogether, our results reveal novel mechanisms by which a KD might improve the resistance to psychological stress. HighlightsO_LIKetogenic diet tends to promote resistance to psychological stress C_LIO_LIHippocampal microglia show morphological adaptations to stress and diet C_LIO_LIMicroglia of stress-susceptible mice make less synaptic contacts C_LIO_LIMicroglia of ketogenic diet-fed mice show less signs of cellular stress C_LIO_LILipids are differentially regulated in the hippocampi of susceptible mice C_LI

neuroscience↗

bioRxiv2023

Phospholamban Inhibits the Cardiac Calcium Pump Through Reversing the Allosteric Enhancement of Calcium Affinity by ATP

Phospholamban (PLB) is a transmembrane micropeptide that regulates the Ca2+ pump SERCA in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca2+ sensitivity of active SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca2+ affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows the Ca2+-binding step in the SERCA enzymatic cycle, altering the Ca2+ dependence of cycling without affecting the true affinity of the Ca2+ binding sites. Here, we consider another hypothesis, that measurements of Ca2+ binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca2+ binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca2+ affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca2+ affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays versus Ca2+ binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca2+-binding sites. We propose this pathway is interrupted by PLB binding. Thus, PLB reduces the true Ca2+ affinity of SERCA by reversing allosteric activation of the pump by ATP. O_TEXTBOXSignificance StatementThe micropeptide phospholamban (PLB) serves a vital role as the "adrenaline trigger" for the heart. PLB inhibits the calcium pump SERCA at rest, and relief of this inhibition increases cardiac performance during exercise. Still, the physical mechanism for PLB inhibition of SERCA remains poorly understood. Here, our results reveal that PLB regulates SERCA by reversing an allosteric effect of ATP that enhances SERCAs calcium affinity. We mapped a novel allosteric pathway that structurally couples SERCAs ATP- and calcium-binding sites. PLB binds residues along this pathway to disrupt communication between the ligand-binding sites. The data reveal an allosteric mechanism that may be conserved in other ATP-dependent ion pumps. This allosteric pathway may provide new opportunities for pharmacological targeting of SERCA. C_TEXTBOX

biochemistry↗

bioRxiv2023

Amyloid β Induces Lipid Droplet-Mediated Microglial Dysfunction in Alzheimer's Disease

Several microglia-expressed genes have emerged as top risk variants for Alzheimers disease (AD). Impaired microglial phagocytosis is one of the main proposed outcomes by which these AD-risk genes may contribute to neurodegeneration, but the mechanisms translating genetic association to cellular dysfunction remain unknown. Here we show that microglia form lipid droplets (LDs) upon exposure to amyloid-beta (A{beta}), and that their LD load increases with proximity to amyloid plaques in brains from human patients and the AD mouse model 5xFAD. LD formation is dependent on age and disease progression and is prominent in the hippocampus in mice and humans. Despite differences in microglial LD load between brain regions and sexes in mice, LD-laden microglia exhibited a deficit in A{beta} phagocytosis. Unbiased lipidomic analysis identified a decrease in free fatty acids (FFAs) and a parallel increase in triacylglycerols (TGs) as the key metabolic transition underlying LD formation. DGAT2, a key enzyme for converting FFAs to TGs, promotes microglial LD formation and is increased in 5xFAD and human AD brains. Inhibition or degradation of DGAT2 improved microglial uptake of A{beta} and drastically reduced plaque load in 5xFAD mice, respectively. These findings identify a new lipid-mediated mechanism underlying microglial dysfunction that could become a novel therapeutic target for AD.

neuroscience↗

bioRxiv2023

Evolution of transient RNA structure-RNA polymerase interactions in respiratory RNA virus genomes

RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consists of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the IBV genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population.

microbiology↗