Biology subjects

Singh, P. K.

Publications and source records attributed to Singh, P. K..

At least 19 recordsLinked to original sources

bioRxiv2026

Extracellular vesicle-mediated suppression of macrophage STING signaling promotes immune dysfunction in dedifferentiated liposarcoma

BackgroundDedifferentiated liposarcoma (DDLPS) is characterized by abundant immune cell infiltration yet derives limited benefit from immune checkpoint blockade and stimulator of interferon genes (STING) agonist-based strategies, suggesting tumor-mediated suppression of antitumor immunity. Tumor-associated macrophages are the most abundant immune populations in DDLPS, but the factors regulating their function remain incompletely understood. MethodsExtracellular vesicles (EVs) were isolated from two DDLPS cell lines and serum from 16 DDLPS patients and 13 healthy donors. EVs impact on cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) -induced macrophage activation was assessed by cytokine secretion, surface markers, functional assays and macrophage-T-cell coculture. Proteomics was performed in EV-treated and EV-untreated macrophages from three donors. Pathway and protein interaction analyses were integrated with The Cancer Genome Atlas (TCGA) DDLPS transcriptomic and survival data. ResultsWe show that EVs released by DDLPS cells suppress macrophage responsiveness to classic STING agonist cGAMP. EVs derived from DDLPS attenuated cGAMP-induced expression of type I interferon-associated cytokines and chemokines, reduced IFN-{beta} secretion, and impaired phosphorylation of STING, TBK1 and IRF3. Functionally, DDLPS EV exposure shifted macrophages toward an immunoregulatory phenotype, restrained phagocytic activity, and attenuated macrophage-dependent T-cell proliferation while promoting T-cell exhaustion. Proteomic profiling revealed extensive macrophage reprogramming characterized by suppression of STING-associated signaling, antigen processing and presentation associated pathways and proteins targeted by miR-16-5p. Consistent with these findings, STING expression was associated with prolonged overall survival in DDLPS, while reduced expression of miR-16-5p target proteins was associated with attenuated STING pathway activity and immunostimulatory macrophage signatures. ConclusionsThese findings identify EV-mediated suppression of macrophage STING signaling as a mechanism of immune dysfunction in DDLPS and provide a framework for understanding immune resistance in this disease.

cancer biology↗

bioRxiv2026

Comparative pangenomics of Trypanosoma brucei reveal host specialization is driven by targeted or segmental genome streamlining in human and equine-restricted lineages

How the eukaryotic parasite Trypanosoma brucei remodels its genome during host specialization remains a fundamental question in evolutionary biology. In contrast to the uniform, systemic gene loss seen in resident bacterial genomes, the structural variations driving niche restriction in extracellular pathogens are poorly defined. Here, we establish a high-resolution comparative structural pangenomic map of the T. brucei complex. By analyzing the human-restricted specialist DAL972 and the equine-restricted specialist Trypanosoma equiperdum IVM-t1 against the conserved TREU927 backbone, we determine how host-vector constraints dictate eukaryotic genome architecture. Our analyses reveal that host specialization is governed by a coordinated spatial blueprint that segregates structural variation into two distinct evolutionary pathways: targeted perimeter streamlining and systemic core erosion. The human specialist DAL972 preserves its core metabolic toolkit under intense purifying selection, restricting genomic variation to the compartmentalized trimming of its sub-telomeric variant surface glycoprotein (VSG) archives. Conversely, the equine specialist IVM-t1, which has completely abandoned cyclical tsetse fly transmission, displays multi-focal core erosion across internal domains. We demonstrate that vector abandonment forces a shift to strict asexual clonal population dynamics. This transition eases purifying selection, allowing obsolete, insect-stage flagellar and cytoskeletal structures to decay through neutral genetic drift. At the chromosomal perimeters, this structural allocation unmasks an unannotated sub-telomeric reservoir of 86 profile-validated retrotransposon hot spot (RHS) and catalytic DDE transposase domains organized into 23 discrete loci. Operating under adaptive positive selection in the human specialist, these silent, repetitive elements function as physical recombination anchors that stabilize strand exchange. This mechanism drives rapid antigenic diversification while insulating the core genome from dangerous macro-rearrangements. Importantly, we bridge this macro-scale chromosomal architecture to micro-scale proteomic execution through tertiary modeling of these unannotated specialized loci. This structural translation unmasks a biophysical mechanism of structural sequestration, where lineage-specific mutations and specificity-determining positions are deeply buried within hydrophobic protein cores to shield functional divergence from host immune surveillance. By pairing these rigid, hidden core packing networks with hyper-variable surface tiles and dynamic boundary hinges, the parasite deploys tiered molecular mimicry to selectively manipulate host cellular networks. Together, these findings redefine our understanding of structural and non-coding architecture as mechanical drivers of parasitic adaptability, uncovering a direct pipeline from pangenomic spatial variations to stable, structure-based allosteric drug targets.

genomics↗

bioRxiv2026

Macrophage Migration Inhibitory Factor (MIF)-CD74 Signaling Pathway Mediates Trabecular Meshwork Dysfunction in Glaucoma.

PurposeMacrophage migration inhibitory factor (MIF) is a pleiotropic cytokine implicated in many inflammatory and fibrotic diseases; however, its role in primary open-angle glaucoma (POAG) and trabecular meshwork (TM) dysfunction remains unknown. In this study, we investigated whether MIF-CD74 signaling regulates TM pathobiology through modulation of the transcription factor, Blimp-1, and downstream cytoskeletal reorganization and extracellular matrix (ECM) remodeling. MethodPrimary human TM cells (HTMC) were exposed to glaucomatous stressors, including TGF-{beta}2, rMIF, or a pro-inflammatory milieu. Expression of MIF, its receptor CD74, and Blimp-1 was measured by qPCR and immunoblotting. ECM proteins and phosphorylated myosin-light chain (pMLC) were evaluated by immunofluorescence staining. In vivo, MIF-CD74 and Blimp-1 expression were examined in the TM/anterior segment (AS) tissue of Tg.CreMYOCY437H and lentiviral (LV)-TGF-{beta}2-induced ocular hypertension (OHT) mouse models. Functional involvement of MIF signaling in TM pathobiology was examined using the irreversible MIF inhibitor 4-IPP and the immunomodulatory metabolites agmatine and thiamine. ResultsGlaucomatous stressors significantly upregulated MIF and CD74 expression with concomitant suppression of Blimp-1 in HTMC. Similarly, TM/AS tissue from both OHT models (Tg.CreMYOCY437H and LV-TGF-{beta}2) demonstrated increased MIF-CD74 expression accompanied by reduced Blimp-1 levels. Activation of MIF-CD74 signaling triggered pro-inflammatory and cell death pathways and promoted ECM remodeling, characterized by increased fibrotic protein expression and enhanced RhoA/ROCK-mediated MLC phosphorylation, indicating modulation of TM contractility. Pharmacological inhibition of MIF attenuated inflammatory signaling, reduced ECM deposition and cytoskeletal remodeling, and suppressed RhoA/ROCK/MLC activation, restoring a protective TM phenotype. ConclusionOur findings identify MIF-CD74 signaling as a previously unrecognized regulator of TM dysfunction in POAG. MIF-mediated suppression of Blimp-1 mechanistically links inflammatory signaling to cytoskeletal contractility and fibrotic ECM remodeling, key determinants of aqueous humor outflow resistance. Targeting the MIF-CD74/Blimp-1 axis may represent a novel therapeutic strategy to restore TM homeostasis and reduce intraocular pressure in glaucoma.

immunology↗

bioRxiv2026

Neuron-Specific WWOX Gene Therapy Produces Dose-Dependent, Durable Rescue in a Model of WWOX-Related Epileptic Encephalopathy

Biallelic loss-of-function mutations in WWOX cause a spectrum of neurodevelopmental disorders, including the severe, early-onset WOREE syndrome, frequently associated with intractable epilepsy and premature mortality, and the milder SCAR12, characterized by subtler neurological manifestations. While neuronal replacement of WWOX has emerged as a potential therapeutic strategy, the parameters required for safe, durable, and clinically translatable gene delivery remain undefined. Here, we systematically delineate the determinants of effective WWOX gene therapy by evaluating promoter selection, cellular targeting, vector configuration, dose, and developmental timing in a severe Wwox-null mouse model. Neuron-restricted expression driven by the human Synapsin I promoter uniquely enabled sustained phenotypic correction, whereas ubiquitous or oligodendrocyte-restricted expression failed to confer durable benefit. To better regulate transgene expression, we generated a vector lacking the WPRE element, enabling dose calibration within a clinically relevant range. Comparative dose-response analyses identified an optimal therapeutic dose of AAV9-hSynI-WWOX that produced robust, dose-dependent rescue of survival and growth. Moreover, the rescued mice displayed glucose, behavioral function and fertility indistinguishable from WT mice, accompanied by long-term restoration of WWOX DNA, transcript, and protein across central and peripheral neural tissues without detectable hepatic expression. Neuronal WWOX reconstitution promoted widespread myelination and attenuated neuroinflammatory responses, including astrogliosis and microglial activation to levels indistinguishable from WT. Continuous electrocorticographic monitoring uncovered early postnatal neuronal hyperexcitability in Wwox-null mice, which was effectively suppressed by therapeutic WWOX delivery. Finally, our data define an early postnatal therapeutic window in Wwox-null mice, showing that treatment initiated between postnatal days 1 and 5 supports durable rescue. Together, these findings define a rigorously optimized, neuron-targeted AAV9-WWOX gene therapy framework and establish critical design and timing principles for translational treatment of WWOX-associated developmental and epileptic encephalopathies.

neuroscience↗

bioRxiv2026

Fast-ripples are emergent properties of neuronal networks

Fast-ripples (250-500 Hz) have been proposed as a promising biomarker in epilepsy, but their specificity remains unclear. In particular, it is uncertain whether they reflect chance coincident neural activity or distinctly generated pathological entities. We combined in silico simulations, neuronal cultures, the intra-hippocampal kainate rat model of hippocampal epilepsy, and human microwire recordings to investigate whether fast-ripples occur more frequently than expected by the chance aggregation of action potentials. Our simulations showed that chance aggregation can generate fast-ripples and that their incidence changes depending on brain state, an observation that we confirmed in our rodent data. The likelihood of exceeding chance depended on biological system complexity and vigilance state: fast-ripples in neuronal cultures did not surpass chance levels, whereas those in awake - but not sleeping - rodents did. Similarly, the incidence of fast-ripples in awake human recordings was ~30% greater than expected by chance. As such, our findings suggest that most fast-ripples reflect stochastic network activity rather than distinctly generated pathological entities. This work does not rule out FRs as potential indicators of epileptogenic tissue, but it does challenge prevailing assumptions about their generation and specificity. Their higher prevalence in epileptogenic tissue is likely primarily due to increased excitation and/or neural synchronization, rather than peculiar abnormalities in network behavior.

neuroscience↗

bioRxiv2025

Leaf microbiome assembly is linked to plant phylogeny

Background and AimsThe plant microbiome is considered as an extended part of the plant genome, and it provides key functions in regulating plant fitness, and stress tolerance. Plants and associated microbiomes have co-evolved over millennia, yet evidence for a strong influence of plant phylogeny in influencing their microbiomes is largely lacking. Our main aims was to identify key drivers of plant microbiome assembly. MethodsHere, we conducted a full factorial experiment that included three levels of soil microbial diversity, five plant species from three functional groups (C3, C4, and C3 nitrogen-fixing), and two moisture availability levels. ResultsOur results showed that host identity and plant functional group exerted the strongest effect on leaf microbial assembly, while root and soil microbiomes showed less sensitivity to host selection. The initial soil microbial diversity and community structure significantly impacted soil and root microbial composition, but not leaf microbiomes. Importantly, we observed significant positive linkage between host phylogeny distance and Bray-Curtis dissimilarity index in leaf microbiomes. This finding was further validated through analysis of microbiome data from seven plant species grown across different field and environmental conditions. Interestingly, there was no significant impact of short-term water stress on plant microbial communities. ConclusionsBy providing empirical evidence for important role of host selection in shaping plant microbiomes, this study advances our fundamental knowledge of plant-microbe interactions and their co-evolutionary relationships, and enhances our ability to develop future tools harnessing plant microbiome to improve plant health and productivity.

microbiology↗

bioRxiv2025

Cross-Fostering with control dams rescues Gut Dysbiosis and Chromatin-associated Transcriptional Changes in Offspring of Opioid-Exposed Dams

Prenatal opioid exposure disrupts gut homeostasis and causes gastrointestinal complications in offspring, but the mechanisms remain unclear. Here using a murine model of prenatal hydromorphone exposure, we examined gut microbiota, intestinal injury, transcriptomic signatures, and chromatin accessibility. Exposed pups displayed marked dysbiosis, epithelial damage, and upregulation of inflammatory gene programs accompanied by relaxed ileal chromatin. Cross-fostering to opioid-naive dams restored microbial diversity, reestablished metabolite-producing taxa, and reversed injury-associated transcriptional and chromatin changes. Fecal microbiota transplantation from exposed dams recapitulated intestinal injury, indicating a microbiome-driven mechanism. These findings reveal a novel gut-microbiome-epigenome axis underlying opioid-induced injury and highlight early microbial intervention as a potential strategy to mitigate developmental harm.

microbiology↗

bioRxiv2025

Inhibition of agr Quorum Sensing in Staphylococci by Novel Thiazole-Indole Hybrid Compounds

The rise of multidrug-resistant Staphylococcus aureus has prompted the search for innovative, non-bactericidal therapeutic strategies. Anti-virulence approaches targeting quorum sensing (QS) pathways offer a promising alternative by disarming pathogenicity, potentially without exerting selective pressure for resistance. In this study, we report the identification of two novel thiazole-indole hybrid compounds, namely 2-[(6-methoxy-1H-indol-2-yl)methyl]-5-cyano-1,3-thiazol-3-amine (designated C3) and 3-amino-5-cyano-2-(5-chloro-1H-indol-2-yl) thiazole (designated C4), as potent inhibitors of the S. aureus accessory gene regulator (agr) quorum sensing system. Building on two heterocyclic scaffolds commonly used in medicinal chemistry and associated with QS modulation, these synthetic small molecules act on the S. aureus AgrC QS receptor, attenuating virulence without affecting bacterial viability. Both compounds significantly reduced the production of secreted virulence factors without promoting biofilm formation, a known drawback of some other QS inhibitors. Furthermore, their activity extended to Staphylococcus lugdunensis, while sparing the commensal Staphylococcus epidermidis, indicating pathogen-selective QS interference. These findings establish novel thiazole-indole hybrids as a promising chemotype for anti-virulence intervention and provide valuable chemical tools for probing agr-mediated regulation in Staphylococcus species.

microbiology↗

bioRxiv2025

B12 promotes gut dysbiosis and an inflammatory microenvironment that potentiates Tet2-deficient hematopoiesis.

Recent studies have linked elevated vitamin B12 serum levels with the presence of clonal hematopoiesis (CH) and an increased risk of developing myeloid malignancy. High B12 supplementation increases serum levels, alters gut microbial composition, and reduces the production of short-chain fatty acids (SCFAs), which help maintain gut barrier function and mucosal integrity. TET2 mutation is a frequent driver of CH that progresses in a positive feedback loop in response to microbial signals suggesting that B12 may influence CH via the gut microbiome. We evaluated the microenvironmental effects of B12 supplementation in a Tet2-deficient model of CH and found that B12 enhances myelopoiesis, heightens the responses of myeloid cells to bacterial stimuli, and increases the levels of circulating inflammatory cytokines. B12 supplementation also induced gut dysbiosis and reduced the levels of SCFA-producing bacteria in both wild-type and Tet2-deficient mice. Importantly, the effects of excess B12 were reversible upon oral supplementation with the SCFA butyrate. These findings suggest that B12 may promote CH progression by disrupting microbiome-derived SCFA metabolism, highlighting a potential therapeutic role for SCFA supplementation in mitigating CH.

cancer biology↗

bioRxiv2025

Gut microbial metabolites butyrate and acetate limit Zika virus replication and associated ocular manifestations via the G-protein coupled receptor 43/FFAR2.

Short-chain fatty acids (SCFAs) are gut microbial metabolites produced by gut microbiota from dietary fiber. SCFAs have shown both pro- and anti-viral roles among different viruses, and are known to regulate immune functions during infections. However, their role against the Zika virus (ZIKV) in general and ocular infection, in particular, has never been investigated. In the present study, we aimed to examine the role of three SCFA derivatives: phenylbutyrate (PBA), sodium butyrate (NaB), and sodium acetate (NaAC), on ZIKV replication and associated ocular complications using primary human trabecular meshwork cells (HTMCs) and an IFNAR1-deficient mouse model of ocular infection. Our findings reveal that PBA and NaAc treatment dramatically suppressed the ZIKV replication in HTMCs. NaB showed a slightly less effect than PBA and NaAc. PBA and NaAc treatment significantly attenuated the ZIKV-induced inflammatory cytokine, interferons, and interferon-stimulated genes response via antagonizing the RIG-I/NF{kappa}B/MAPKs/STAT1-3 signaling pathways. We discovered that ZIKV induces the expression of free fatty acid receptor 2 (FFAR2)/ GPR43 in HTMCs, which is further potentiated by PBA/NaAc. Pharmacological inhibition of FFAR2 abrogated the protective abilities of PBA/NaAc and significantly increased viral replication. Blocking FFAR2 receptors promoted ZIKV-induced cell death, which was suppressed by PBA and NaAc. Mechanistically, butyrate and acetate inhibited ZIKV binding and cellular entry and inactivated the virus before internalization. PBA and NaAc treatment in mice attenuated the ZIKV-induced ocular manifestations (intraocular pressure, RPE/retinal atrophy, and anterior segment inflammation), which was abrogated by FFAR2 inhibition. Collectively, our findings indicate that SCFA treatment is an effective approach to limit ZIKV replication and associated ocular damage and may be worth exploring as a means to treat or prevent ZIKV-induced ocular complications in humans. ImportanceZIKV is known to cause severe ocular manifestations in in-utero exposed infants; however, the molecular mechanisms of ZIKV-induced ocular complications remain unknown. SCFAs have demonstrated both pro- and anti-viral roles against different viruses; however, their role against ZIKV is unknown. We showed that SCFAs butyrate and acetate suppress ZIKV transmission and associated ocular complications. The anti-ZIKV activity of these SFACs is mediated via FFAR2, and pharmacological inhibition of FFAR2 promotes ZIKV-induced inflammatory and cell death responses, as well as ocular malformations.

microbiology↗

bioRxiv2025

Astroglial TNFR2 signaling regulates hippocampal synaptic function and plasticity in a sex dependent manner

Astrocytes participate in synaptic transmission and plasticity through tightly regulated, bidirectional communication with pre- and post-synaptic neurons, as well as microglia and oligodendrocytes. A key component of astrocyte-mediated synaptic regulation is the cytokine tumor necrosis factor (TNF). TNF signals via two cognate receptors, TNFR1 and TNFR2, both expressed in astrocytes. While TNFR1 signaling in astrocytes has been long demonstrated to be necessary for physiological synaptic function, the role of astroglial TNFR2 has never been explored. Here, we demonstrate that astroglial TNFR2 is essential for maintaining hippocampal synaptic function and plasticity in physiological conditions. Indeed, GfapcreERT2:Tnfrsf1bfl/fl mice with selective ablation of TNFR2 in astrocytes exhibited dysregulated expression of neuronal and glial proteins (e.g., SNARE complex molecules, glutamate receptor subunits, glutamate transporters) essential for hippocampal synaptic transmission and plasticity. Hippocampal astrocytes sorted from GfapcreERT2:Tnfrsf1bfl/fl mice displayed downregulation of genes and pathways implicated in synaptic plasticity, as well as astrocyte-neuron and astrocyte-oligodendrocyte communication. These alterations were accompanied by increased glial reactivity and impaired astrocyte calcium dynamics, and ultimately translated into functional deficits, specifically impaired long-term potentiation (LTP) and cognitive functions. Notably, male GfapcreERT2:Tnfrsf1bfl/fl mice exhibited more pronounced hippocampal synaptic and cellular alterations, suggesting sex-dependent differences in astroglial TNFR2 regulation of synaptic function. Together, these findings indicate that TNFR2 signaling in astrocytes is essential for proper astrocyte-neuron communication at the basis of synaptic function, and that this is regulated in a sex-dependent manner.

neuroscience↗

bioRxiv2025

CPSF6 Promotes HIV-1 Preintegration Complex Function

Cleavage and polyadenylation specificity factor 6 (CPSF6) is part of the cellular cleavage factor I mammalian (CFIm) complex that regulates mRNA processing and polyadenylation. CPSF6 also functions as a HIV-1 capsid (CA) binding host factor and promotes viral DNA integration targeting into gene dense regions of the host genome. However, the effects of CPSF6 on the activity of the HIV-1 preintegration complex (PIC) - the machinery that carries out viral DNA integration to establish infection - is unknown. To study CPSF6s role in HIV-1 PIC function, we extracted PICs from cells depleted of CPSF6 or expressing a CPSF6 mutant that cannot bind to CA. These PICs exhibited significantly lower integration activity when compared to the control PICs. Addition of recombinant CPSF6 restored the integration activity of PICs extracted from the mutant cells, suggesting a direct role of CPSF6 in PIC function. To solidify CPSF6s effect on PIC function, we inoculated CPSF6-depleted and CPSF6-mutant cells with HIV-1 particles and measured viral DNA integration into the host genome. A significant reduction in viral integration in these cells was detected and this defect was not a consequence of reduced reverse transcription or nuclear entry. Additionally, mutant viruses deficient in CA-CPSF6 binding showed no integration defect in CPSF6 mutant cells. Finally, sequencing analysis revealed that HIV-1 integration in the CPSF6 mutant cells was significantly redirected from the gene dense regions of the host genome. Collectively, these results suggest that CPSF6-CA interaction regulates PIC function both in vitro and in infected cells. IMPORTANCEHIV-1 infection is dependent on the interaction of the virus with host factors. However, the molecular details of virus-host factor interactions are not fully understood. For instance, HIV-1 capsid provides binding interfaces for several host factors. CPSF6 is one such capsid-binding host factor, whose cellular function is to regulate mRNA processing and polyadenylation. Initial work identified a truncated cytosolic form of CPSF6 that restricted HIV infection by blocking viral nuclear entry. However, it is now established that the full-length CPSF6 primarily promotes integration targeting into gene dense regions of the host genome. Here we report that CPSF6-CA interaction promotes the activity of HIV-1 preintegration complexes (PICs). We also observed that disruption of CPSF6-CA binding in target cells significantly reduced viral integration and directed integration targeting away from gene-dense regions. These findings demonstrate a critical role for the CPSF6-CA interaction in PIC function and integration targeting.

biochemistry↗

bioRxiv2025

Timed chromatin invasion during mitosis governs prototype foamy virus integration site selection and infectivity

Selection of a suitable chromatin environment during retroviral integration is a tightly regulated and multilayered process that involves interplay between viral and host factors. However, whether intrinsic chromatin dynamics during mitosis modulate retroviral genome invasion is currently poorly described. Direct interaction between the spumaretrovirus prototype foamy virus (PFV) Gag protein and cellular chromatin has been described as a major determinant for integration site selection. A previous Gag chromatin-binding site (CBS)-nucleosome co-crystal structure revealed an interaction with the histone H2A-H2B acidic patch via a highly conserved arginine anchor residue. Yet, the molecular mechanisms regulating Gag-chromatin capture during PFV infection remain obscure. Here, we investigated the kinetics of Gag-chromatin interactions during mitosis and proviral integration of PFV-infected synchronized cells. Using Gag CBS variant viruses, we showed that alteration of Gag affinity for nucleosome binding induced untimely chromatin tethering during mitosis, decreased infectivity and redistributed viral integration sites to markers associated with late replication timing of host chromosomes. Mutant Gag proteins were moreover defective in their ability to displace the histone H4 tail from the nucleosome acidic patch of highly condensed mitotic chromatin. These data indicate that the mitotic chromatin landscape during Gag-nucleosome interactions hosts PFV integration site selection determinants and that spumaretroviruses evolved high-affinity chromatin binding to overcome early mitosis chromatin condensation for optimal viral DNA tethering, integration and infection.

microbiology↗

bioRxiv2024

Adaptation to cystine limitation stress promotes PDAC tumor growth and metastasis through translational upregulation of OxPPP

Cystine/cysteine is critical for antioxidant response and sulfur metabolism in cancer cells and is one of the most depleted amino acids in the microenvironment of pancreatic ductal adenocarcinoma (PDAC). The effects of cystine limitation stress (CLS) on PDAC progression are poorly understood. Here we report that adaptation to CLS (CLSA) promotes PDAC cell proliferation and tumor growth through translational upregulation of the oxidative pentose phosphate pathway (OxPPP). OxPPP activates the de novo synthesis of nucleotides and fatty acids to support tumor growth. On the other hand, CLSA-mediated lipidomic reprogramming depends on triacylglycerides synthesis and lipid droplet formation to mitigate lipotoxicity. Through drug screening, we identify lomitapide as an inhibitor of CLSA PDAC tumor growth and a potent sensitizer of chemotherapy. Lomitapide inhibits triacylglycerides synthesis to interfere with CLSA and chemotherapy-induced lipidomic reprogramming. Taken together, we demonstrate that CLSA promotes PDAC tumor growth through metabolic reprogramming and lomitapide could be used to target the dysregulated lipid metabolism in PDAC.

cancer biology↗

bioRxiv2024

Structural and Mechanistic Bases for Resistance of the M66I Capsid Variant to Lenacapavir

Lenacapavir (LEN) is the first in class viral capsid protein (CA) targeting antiretroviral for treating multi-drug-resistant HIV-1 infection. Clinical trials and cell culture experiments have identified resistance associated mutations (RAMs) in the vicinity of the hydrophobic CA pocket targeted by LEN. The M66I substitution conferred by far the highest level of resistance to the inhibitor compared to other RAMs. Here we investigated structural and mechanistic bases for how the M66I change affects LEN binding to CA and viral replication. The high-resolution X-ray structure of the CA(M66I) hexamer revealed that the {beta}-branched side chain of Ile66 induces steric hindrance specifically to LEN thereby markedly reducing the inhibitor binding affinity. By contrast, the M66I substitution did not affect binding of Phe-Gly (FG)-motif-containing cellular cofactors CPSF6, NUP153, or SEC24C, which engage the same hydrophobic pocket of CA. In cell culture the M66I variant did not acquire compensatory mutations or replicate in the presence of LEN. Analysis of viral replication intermediates revealed that HIV-1(M66I CA) predominantly formed correctly matured viral cores, which were more stable than their wildtype counterparts. The mutant cores stably bound to the nuclear envelope but failed to penetrate inside the nucleus. Furthermore, the M66I substitution markedly altered HIV-1 integration targeting. Taken together, our findings elucidate mechanistic insights for how the M66I change confers remarkable resistance to LEN and affects HIV-1 replication. Moreover, our structural findings provide powerful means for future medicinal chemistry efforts to rationally develop second generation inhibitors with a higher barrier to resistance. IMPORTANCELenacapavir (LEN) is a highly potent and long-acting antiretroviral that works by a unique mechanism of targeting the viral capsid protein. The inhibitor is used in combination with other antiretrovirals to treat multi-drug-resistant HIV-1 infection in heavily treatment-experienced adults. Furthermore, LEN is in clinical trials for preexposure prophylaxis (PrEP) with interim results indicating 100 % efficacy to prevent HIV-1 infections. However, one notable shortcoming is a relatively low barrier of viral resistance to LEN. Clinical trials and cell culture experiments identified emergent resistance mutations near the inhibitor binding site on capsid. The M66I variant was the most prevalent capsid substitution identified in patients receiving LEN to treat muti-drug resistant HIV-1 infections. The studies described here elucidate the underlying mechanism by which the M66I substitution confers a marked resistance to the inhibitor. Furthermore, our structural findings will aid future efforts to develop the next generation of capsid inhibitors with enhanced barriers to resistance.

microbiology↗

bioRxiv2024

Interplay between the cyclophilin homology domain of RANBP2 and MX2 regulates HIV-1 capsid dependencies on nucleoporins

Interlinked interactions between the viral capsid (CA), nucleoporins (Nups), the antiviral protein myxovirus resistance 2 (MX2/MXB) influence HIV-1 nuclear entry and the outcome of infection. Although RANBP2/NUP358 has been repeatedly identified as a critical player in HIV-1 nuclear import and MX2 activity, the mechanism by which RANBP2 facilitates HIV-1 infection is not well understood. To explore the interactions between MX2, the viral CA, and RANBP2, we utilized CRISPR-Cas9 to generate cell lines expressing RANBP2 from its endogenous locus but lacking the C-terminal cyclophilin (Cyp) homology domain, and found that both HIV-1 and HIV-2 infection were reduced significantly in RANBP2{Delta}Cyp cells. Importantly, although MX2 still localized to the nuclear pore complex in RANBP2{Delta}Cyp cells, antiviral activity against HIV-1 was decreased. By generating cells expressing specific point mutations in the RANBP2-Cyp domain we determined that the effect of the RANBP2-Cyp domain on MX2 anti-HIV-1 activity is due to direct interactions between RANBP2 and CA. We further determined that CypA and RANBP2-Cyp have similar effects on HIV-1 integration targeting. Finally, we found that the Nup requirements for HIV infection and MX2 activity were altered in cells lacking the RANBP2-Cyp domain. These findings demonstrate that the RANBP2-Cyp domain affects viral infection and MX2 sensitivity by altering CA-specific interactions with cellular factors that affect nuclear import and integration targeting. Significance StatementHIV-1 entry into the nucleus is an essential step in viral replication that involves complex interactions between the viral capsid and multiple cellular proteins, including nucleoporins such as RANBP2. Nups also mediate the function of the antiviral protein MX2, however determining the precise role of Nups in HIV infection has proved challenging due to the complex nature of the nuclear pore and significant pleiotropic effects elicited by Nup depletion. We have used precise gene editing to assess the role of the Cyp domain of RANBP2 in HIV-1 infection and MX2 activity. We find that this domain affects viral infection, nucleoporin requirements, MX2 sensitivity, and integration targeting in a CA-specific manner, providing detailed insights into how RANBP2 contributes to HIV-1 infection.

microbiology↗

bioRxiv2024

Revolutionizing Molecular Cloning: Introducing FastCloneAssist, a Streamlined Python Tool for Optimizing Primer Design in Restriction & Ligation-Independent PCR Cloning

FastCloning, a paradigm shift in PCR cloning, has streamlined the process by eliminating laborious, multi-step traditional methods. This innovative technique, pioneered by Li et al. (2011), utilizes overlapping PCR primers and DpnI digestion for seamless integration of insert DNA into any desired vector position, regardless of restriction sites. This versatility makes FastCloning ideal for constructing fusion proteins, chimeric cDNAs, and manipulating genes with unparalleled ease. However, efficient primer design remains a critical hurdle, particularly for newcomers, as errors can lead to failed cloning attempts. To address this bottleneck, we present FastCloneAssist, a user-friendly Python program that automates FastCloning primer design with minimal user input. Users simply provide vector and insert sequences, along with the desired melting temperature (Tm), and FastCloneAssist provides best primer pairs after calculating optimal primer parameters for efficient PCR amplification and seamless DNA integration using established bioinformatics libraries. This open-source, freely available tool simplifies and accelerates cloning, making this powerful technique accessible to researchers of all levels and expediting scientific discovery.

bioengineering↗

bioRxiv2024

The role of LEDGF in transcription is exploited by HIV-1 to position integration

HIV-1 integration occurs across actively transcribed genes due to the interaction of integrase with host chromatin factor LEDGF. Although LEDGF was originally isolated as a co-activator that stimulates promoter activity in purified systems, this role is inconsistent with LEDGF-mediated integration across gene bodies and with data indicating LEDGF is a histone chaperone that promotes transcriptional elongation. We found LEDGF is enriched in pronounced peaks that match the enrichments of H3K4me3 and RNA Pol II at transcription start sites (TSSs) of active promoters. Our genome-wide chromatin mapping revealed that MLL1 had a dominant role in recruiting LEDGF to promoters and the presence of LEDGF recruits RNA Pol II. Enrichment of LEDGF at TSSs correlates strongly with levels of integration across the transcribed sequences, indicating that LEDGF at TSSs contributed to integration across gene bodies. Although the N-terminal Pro-Trp-Trp-Pro (PWWP) domain of LEDGF interacts with nucleosomes containing H3K36me3, a modification thought to recruit LEDGF to chromatin, we found H3K36me3 does not contribute to gene specificity of integration. These data support a dual role model of LEDGF where it is tethered to promoters by MLL1 and recruits RNA Pol II. Subsequently, LEDGF travels across genes to effect HIV-1 integration. Our data also provides a mechanistic context for the contribution made by LEDGF to MLL1-based infant acute leukemia and acute myeloid leukemia in adults.

genomics↗