Biology subjects

Gupta, A.

Publications and source records attributed to Gupta, A..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Mosquito Ferritin: A Haem-Binding Iron Store Required for Egg Development and Targetable for Malaria Control

Iron metabolism is essential for both mosquito reproductive fitness and Plasmodium development within the vector, yet this axis remains insufficiently explored as a potential target for malaria control. Ferritin, the major iron-storage protein and a central regulator of iron homeostasis, has not previously been biochemically or functionally characterized in any hematophagous insect. Our computational and phylogenetic analyses reveal that mosquito ferritin represents a unique case, combining conserved iron-binding motifs with distinctive haem-coordinating residues that confer an exceptionally high haem-binding affinity exceeding that of bacterioferritin. In addition, through a screen of multiple iron chelators in Anopheles larvae, we identified potent candidates with potential utility for vector control. Together, these findings establish mosquito ferritin as an evolutionarily distinct and physiologically essential protein, and they highlight iron homeostasis as a promising dual-target axis for next-generation vector control strategies.

biochemistry↗

bioRxiv2025

BoltzGen: Toward Universal Binder Design

We introduce BoltzGen, an all-atom generative model for designing proteins and peptides across all modalities to bind a wide range of biomolecular targets. BoltzGen builds strong structural reasoning capabilities about target-binder interactions into its generative design process. This is achieved by unifying design and structure prediction, resulting in a single model that also reaches state-of-the-art folding performance. BoltzGens generation process can be controlled with a flexible design specification language over covalent bonds, structure constraints, binding sites, and more. We experimentally validate these capabilities in eight diverse design campaigns with functional and affinity readouts across 26 targets. In our experiments, binder modalities span from nanobodies to disulfide-bonded peptides, and targets from disordered proteins to small molecules. In particular, we identify nanobody binders for novel targets with low similarity to proteins with already known bound structures. We release model weights, data, and both inference and training code at: https://github.com/HannesStark/boltzgen.

bioengineering↗

bioRxiv2025

Cisplatin and Copper Induce Distinct Endocytic Pathways of Copper Transporter-1 and Elicits Unique Response on Copper Homeostasis Proteins

Cisplatin (CDDP), a widely used chemotherapy drug is imported in mammalian cells by copper transporter CTR1. We observed that CDDP cytotoxicity is significantly reduced in CTR1 deficient yeast and in copper loaded mammalian cells indicating its shared uptake pathway with copper. CTR1 resides on the plasma membrane undergoes endocytosis during CDDP import as in copper, involving its amino-terminal His-Met-motifs and the conserved 150MXXM154 pore motif. Unlike Copper, CDDP directs endocytosed CTR1 toward lysosomes rather than being sorted through VPS35-positive endosome. CDDP enhances lysosomal acidity and cathepsin activity. Furthermore, CDDP selectively downregulate cuproproteins, CCS and SOD1, disrupting copper homeostasis. This study highlights the unique aspects of copper versus CDDP uptake by CTR1 and its differential downstream ramification on lysosomes and cuproproteins.

cell biology↗

bioRxiv2025

Suppression of Glucosylceramide Synthase Reverses Drug Resistance in Cancer Cells Harbor Homozygous p53 Mutants

Glucosylceramide synthase (GCS) catalyzes ceramide glycosylation in response to cell stress that produces glucosylceramide and other glycosphingolipids. GCS overexpression is a cause of drug resistance and enriches cancer stem cells (CSCs) during cancer chemotherapy. Previous studies showed that GCS modulates expression of p53 mutants and oncogenic gain-of-function (GOF) in heterozygous knock-in cell models (TP53 R273H-/+). However, it is unclear whether GCS can modulate the effects of homozygous p53 mutations, which are common in many cancer cases. We report herewith that inhibition of GCS, via UGCG-knockout and using new inhibitor (Genz-161), effectively re-sensitizes drug resistance and diminishes CSCs in colon cancer cells carrying the homozygous p53 R273H mutation. In aggressive WiDr cells carrying TP53 R273H mutation, knockout of UGCG gene using CRISPR/Cas9 editing or inhibition of GCS with Genz-161 sensitized cancer cells to oxaliplatin, irinotecan and paclitaxel. With decreased ceramide glycosylation in lipidomic profiling, both UGCG-knockout and Genz-161 treatments substantially decreased wound healing, and diminished CSCs and tumor growth under chemotherapy. Interestingly, inhibition of RNA m6A methylation by neplanocin A reactivated p53 function and reversed drug resistance. Mechanistic investigation revealed that GCS inhibition downregulated METTL3 expression and repressed RNA-m6A modification on mutant p53 R273H effects. Altogether, our findings demonstrate that ceramide glycosylation promotes METTL3 expression and RNA m6A methylation in response to drug-induced stress, thereby promoting mutant p53 expression and associated GOF. Conversely, inhibition of GCS can diminish CSCs and drug resistance via reduction of m6A modification and reactivation of p53 function. GCS inhibition is an achievable approach for mutant cancer treatment.

cancer biology↗

bioRxiv2025

Blind Prediction of Complex Water and Ion Ensembles Around RNA in CASP16

Biomolecules rely on water and ions for stable folding, but these interactions are often transient, dynamic, or disordered and thus hidden from experiments and evaluation challenges that represent biomolecules as single, ordered structures. Here, we compare blindly predicted ensembles of water and ion structure to the cryo-EM densities observed around the Tetrahymena ribozyme at 2.2-2.3 [A] resolution, collected through target R1260 in the CASP16 competition. 26 groups participated in this solvation cryo-ensemble prediction challenge, submitting over 350 million atoms in total, offering the first opportunity to compare blind predictions of dynamic solvent shell ensembles to cryo-EM density. Predicted atomic ensembles were converted to density through local alignment and these densities were compared to the cryo-EM densities using Pearson correlation, Spearman correlation, mutual information, and precision-recall curves. These predictions show that an ensemble representation is able to capture information of transient or dynamic water and ions better than traditional atomic models, but there remains a large accuracy gap to the performance ceiling set by experimental uncertainty. Overall, molecular dynamics approaches best matched the cryo-EM density, with blind predictions from bussilab_plain_md, SoutheRNA, bussilab_replex, coogs2, and coogs3 outperforming the baseline molecular dynamics prediction. This study indicates that simulations of water and ions can be quantitatively evaluated with cryo-EM maps. We propose that further community-wide blind challenges can drive and evaluate progress in modeling water, ions and other previously hidden components of biomolecular systems.

biophysics↗

bioRxiv2025

Motility-Driven Viscoelastic Control of Tissue Morphology in Presomitic Mesoderm

During development, embryonic tissues experience mechanical stresses ranging from cellular to supracellular length scales. In response, cells generate active forces that drive rearrangements, allowing the tissue to relax accumulated stresses. The nature of these responses depends strongly on the magnitude and duration of the deformation, giving rise to the tissues characteristic viscoelastic behavior. Although experiments have characterized tissue rheology in various contexts, simpler theoretical approaches that directly connect cellular activity to emergent rheological behavior are still limited. In this study, we employ a vertex-based model of epithelial tissue incorporating active force fluctuations in cell vertices to represent cell motility. We capture distinct rounding dynamics and motility-dependent timescales by benchmarking against experimental observations such as the bulging of presomitic mesoderm (PSM) explants driven by Fibroblast Growth Factor(FGF) gradients. Stress relaxation tests reveal rapid short-timescale relaxation alongside persistent longtimescale residual stresses that decrease from anterior to posterior (AP) region of the PSM. By applying oscillatory shear, we analyzed the resulting elastic and viscous responses, revealing motility dependence of storage and loss modulus. Finally, we introduce spatially patterned cues applied in a temporally pulsed manner, mimicking dynamic biochemical or mechanical signals during development. Our results show that while higher motility promotes tissue remodeling in response to these cues, this response is constrained by spatial scale; cellular-scale perturbations are relaxed irrespective of motility strength, preventing complete morphological adaptation.

developmental biology↗

bioRxiv2025

Classification of Human Transcription Factors Based on Their Effector Domains via Unsupervised Learning

TFs combine DBDs, which anchor them to DNA, with EDs that regulate transcription through activation or repression, yet the sequence logic linking ED composition to function remains unclear. Here, we systematically define proxy regions--disordered segments adjacent to DBDs--to enable quantitative analysis of ED-like sequences across the human TF repertoire. Using a biophysically interpretable 22-feature classifier (FALK22) together with an embedding-based model (ESM), we map ED diversity and identify composition and charge-pattern signatures that correspond to regulatory activity along a disorder continuum, separating activation-from repression-associated regions. FALK22 identified classes align well with those identified from ESM while providing transparent, sequence-level features. Proxy regions near C-termini exhibit gradients that track DBD families, suggesting that EDs and DBDs might have co-evolved rather than evolved independently. These results establish proxy regions and FALK22 as a framework to connect sequence features with transcriptional activity and to generate testable hypotheses about effector-domain function and co-evolution with DNA-binding domains. HIGHLIGHTSO_LIWe define proxy regions as systematically identified disordered segments adjacent to DNA-binding domains (DBD), enabling quantitative analysis of effector domain (ED)-like sequences across the human transcription factor (TF) repertoire. C_LIO_LIWe develop FALK22, a 22-feature classification algorithm that classifies transcription factors based on simple sequence properties of their EDs and shows strong alignment with complex embedding-based representations from the Evolutionary Scale Model (ESM). C_LIO_LIFALK22 and ESM uncover distinct amino-acid composition and patterning signatures of EDs that correlate with transcriptional function, separating activation- and repression-associated regions along a disorder continuum. C_LIO_LIProxy regions located at the C-termini exhibit gradients that correspond to their DBD families, suggesting that EDs did not evolve as independent modular units but rather co-evolved with, or became selectively matched to, their DBD contexts. C_LI

bioinformatics↗

bioRxiv2025

H19 is a PERK-regulated long non-coding RNA that fine-tunes UPR signalling and inhibits endoplasmic reticulum stress-induced cell death

The endoplasmic reticulum (ER) responds to stimuli that disrupt its homeostasis by activating a signalling network known as unfolded protein response (UPR), that restores cellular balance and determines cell fate through three key sensors: IRE1, PERK, and ATF6. Emerging evidence suggests that the UPR regulates the expression of numerous long non-coding RNAs (lncRNAs), which play critical roles in modulating stress responses. Here we show that expression of lncRNA H19 is downregulated in response to ER stress in breast cancer cells. Using genetic and pharmacological approaches, we demonstrate that this downregulation is primarily mediated by the PERK arm of the UPR. Specifically, knockdown or chemical inhibition of PERK compromised the ER stress-mediated H19 repression, while PERK activation significantly reduced H19 expression. H19 overexpression promotes the optimal activation of ATF6 and PERK pathways, while it attenuates the signalling by IRE1-XBP1 axis of the UPR. Bioinformatic analyses across multiple breast cancer cohorts revealed that high H19 expression correlates with poor prognosis, particularly in basal-like subtypes. Furthermore, in triple-negative breast cancer (TNBC) cells, H19 was found to provide resistance to ER stress-induced apoptosis. Collectively, our findings establish H19 as a key regulatory node in the UPR network, where PERK-mediated repression of H19 shapes cellular outcomes under ER stress. These insights position H19 as a potential therapeutic target in breast cancer, especially in aggressive subtypes such as TNBC.

cancer biology↗

bioRxiv2025

Estrogen Signaling During Abrupt Involution Leads to Long-Term Metabolic Dysfunction Similar to Estrogen Receptor Negative Breast Cancer

Epidemiological data links lack of breastfeeding with increased risk of breast cancer. Breast tissue undergoes remodeling to pre-pregnancy state after birth through involution. Long-term breastfeeding leads to gradual involution (GI). Lack of breastfeeding leads to abrupt involution (AI). While estrogen impacts repopulation of adipocytes, AI causes several precancerous changes in the mouse mammary gland. The impact of AI on adipocyte repopulation and metabolism is yet to be elucidated. ObjectivesTo investigate effects of AI on mammary gland metabolism and its potential link to breast cancer. MethodsAt partum (day 0), FVB/n dams were randomized to AI or GI and standardized to 6 pups. AI mice had pups removed on day 7 postpartum to mimic short-term breastfeeding. GI mice had 3 pups each were removed on day 28 and 31 postpartum to mimic gradual weaning. Mammary glands were harvested on day 28, 56, and 120 postpartum. Subset of AI mice had long-term sustained release tamoxifen placed subscapular on day 8 postpartum. Metabolic changes were assessed using: 1) transcriptional; 2) functional; 3) oxidative stress; and 4) metabolites analysis. ResultsDay 28 GI glands sustains/continues milk synthesis pathways impacting metabolic comparison with day28 AI glands. Day 28 AI when compared to day 56 GI showed upregulation of estrogen signaling, neutrophil degranulation, glucose metabolism, RNA synthesis, and down regulation of adipogenesis and glycolysis. At day 120, AI glands had downregulation of oxidative phosphorylation and upregulation of mitochondria dysfunction similar to pregnancy associated estrogen receptor negative breast cancer. Tamoxifen treatment of AI dams showed metabolic pathways and estrogen signaling similar to that of GI glands on day 28. ConclusionEarly metabolic phenotypes in AI and GI glands may be caused by differences in adipocyte repopulation related to estrogen signaling. Long-term metabolic effects of AI lead to similar metabolic effects found in breast cancer.

cancer biology↗

bioRxiv2025

Methionine metabolism shapes immune response against Mycobacterium tuberculosis

The methionine metabolism is central to epigenetic reprogramming to produce pro-inflammatory cytokines. Disruptions in methionine metabolism contribute to complex disorders, providing an important target for nutrient interventions. Here, Mycobacterium tuberculosis (Mtb) H37Rv-infected C57BL/6 mice showed functional heterogeneity between alveolar and non-alveolar macrophage (AMs/Non-AMs) with major metabolic reprogramming in Non-AMs. Global metabolite and proteome analysis of Mtb-infected bone marrow-derived macrophages (BMDMs) showed a diversion of flux from methionine metabolism toward increased nucleotide salvage and glutathione (GSH) production. Carbon units of 13C5-methionine via isotopomer analysis contributed to polyamine synthesis, fuelling the nucleotide salvage node in Mtb-infected BMDMs. Methionine supplementation increased mycobacterial clearance in C57BL/6 mice and in BMDMs by promoting a pro-inflammatory response, as evident by increased IL-1{beta} and IFN-{gamma}. Increased IL-1{beta} production is mainly contributed to by increased H3K4 trimethylation in macrophages. These findings reveal an important strategy by which methionine supplementation in macrophages and mice remodels host metabolism to enhance acute pro-inflammatory responses and exploit dietary supplementation to improve nutritional immunity in TB. HighlightsO_LIMycobacterium tuberculosis (Mtb) infection decreases intracellular methionine levels and increases purine levels in primary macrophages. C_LIO_LIMethionine carbon units contribute to nucleotide salvage, and their restriction lowers IL-1 response and mycobacterial clearance. C_LIO_LIEx vivo methionine supplementation in Mtb-infected macrophages increases H3K4me3 at the Il-1b gene promoter and gene body, leading to an increased pro-inflammatory response and improved mycobacterial clearance. C_LIO_LIIn vivo methionine supplementation improved pro-inflammatory response in lungs, spleen and bone marrow, leading to accelerated mycobacterial clearance upon Mtb-H37Rv infection. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/680834v2_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1bf5corg.highwire.dtl.DTLVardef@ba4dbdorg.highwire.dtl.DTLVardef@145f23dorg.highwire.dtl.DTLVardef@122b2a2_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Serum cytokines during acute respiratory infection and relationship to age

Influenza and other seasonal respiratory viruses affect millions annually. While age is generally known to be correlated with risk and outcome, the mechanisms underlying these differences, especially in the infected, have been poorly defined. Previous studies have focused primarily on cell-subset shifts in older adults, leaving a gap in understanding of how cytokine responses vary across the full age spectrum. Cytokine data from Luminex assays were compiled from Stanford clinical studies and after batch control filtering, the dataset included 181 healthy individuals (ages 4-97) and 27 symptomatic individuals (mostly confirmed influenza, ages 14-77). Ordinary least squares regression was applied to assess cytokine differences due to infection status, age, and their interaction, with statistical significance defined as p < 0.05. The results illustrated that pro-inflammatory cytokines were found to be significantly elevated in infected individuals, with a trend of stronger effects in the young supported by comparison of intercepts between the regressions for the healthy and infected cohorts. The concept of inflammaging was also seen through several biomarkers with significant non-zero slopes in healthy cohorts that were not well-established in prior research. Our findings reveal both expected and novel cytokine behavior in influenza infection across a wide range of ages. By incorporating younger individuals and including age as a continuous variable, the study makes progress towards a deeper understanding of the changes in the immune system and its response to influenza across the lifespan.

immunology↗

bioRxiv2025

Splenic granulopoiesis and S100A9 drive resistance to checkpoint inhibitors conferred by liver metastases

Here, we investigate why liver metastases reduce the efficacy of immune checkpoint inhibitors (CPI). The poor prognosis of patients with liver metastases is associated with a systemic increase in neutrophils. Using experimental models, we confirm that mice with liver metastases respond poorly to CPI, have elevated neutrophils and suppress the response of subcutaneous lesions to CPI. We demonstrate that liver metastases, acting partly via IL-6, boost granulopoiesis in the spleen and promote the generation of immature S100A9hi neutrophils that suppress T-cell proliferation. Human liver metastases exhibit a similar increase in S100A9hi neutrophils. Neutrophil depletion attenuates the growth of liver metastases, but not subcutaneous metastases. Moreover, genetic deletion of S100A9 enables liver metastases to be effectively treated with CPI, and prevents liver metastases from suppressing the response in subcutaneous metastases. Thus, we document how liver metastases specifically change splenic granulopoiesis leading to changes in the microenvironment of non-hepatic lesions, and how targeting a key neutrophil protein restores the efficacy of CPI.

immunology↗

bioRxiv2025

Comprehensive Analysis of Multi-Omics Vaccine Response Data Using MOFA and Stabl Algorithms

FluPRINT is a multi-omics dataset that measures donors protein expression and cell counts across various assays. Donors were also assigned a binary value (0 or 1), being labeled as high responders (1) if they had a fold change [≥] 4 of the antibody titer for hemagglutinin inhibition (HAI) from day 0 to day 28, and low responders otherwise (0). In this project, we used the MOFA and Stabl algorithms to analyze FluPRINT, estimate the population structure from the data, and identify the most important features for predicting response to the vaccine. The preprocessing of the dataset included removing repeat features, scaling by assay, and removing outliers. Since Stabl does not directly address missing values, features with high amounts of missing values were removed and the remaining were ignored. MOFA identified the top feature in structure extraction as IL neg 2 CD4 pos CD45RA neg pSTAT5. MOFA explains well the variance of the data while also choosing features that have good significance, as illustrated by their significant p-values (p < 0.05). Stabl found the top feature for explaining the outcome to be CD33- CD3+ CD4+ CD25hiCD127low CD161+ CD45RA+ Tregs, which matched the top result of previously published analysis. MOFAs features achieved an AUROC of 0.616 (95% CI of 0.426-0.806), and Stabls achieved an AUROC of 0.634 (95% CI of 0.432-0.823). Our research addresses a key knowledge gap: understanding how these fundamentally different analytical approaches perform when analyzing the same complex dataset. Our exploration evaluates their respective strengths, limitations, and biological insights and provides guidance on using MOFA and Stabl to find the best predictive cell subsets and features for understanding large immunological multi-omics data. The code for this project can be found at https://github.com/aanya21gupta/fluprint.

immunology↗

bioRxiv2025

TIP60, a key lysine acetyltransferase, acts as a wound-induced factor essential for efficient wound response in planaria

Chromatin modifiers are essential regulators of gene expression, DNA repair, replication, and cell division. Among them, histone acetyltransferases (HATs) such as TIP60 play a central role in modulating chromatin dynamics through acetylation of histone and non-histone proteins. TIP60, a member of the MYST family of HATs, is known to regulate key cellular processes, including transcriptional activation, DNA damage response, and cell cycle progression. Although TIP60s role in stem cell maintenance and differentiation is well established, its function in stem cell-driven regeneration has remained unexplored. In this study, we characterize the role of the TIP60 homolog, SMED-TIP60, in the planarian Schmidtea mediterranea, a model organism renowned for its regenerative capacity. Biochemical assays confirmed SMED-TIP60s acetyltransferase and auto-acetylation activity. RNAi-mediated knockdown of Smed-tip60 resulted in severe defects in tissue homeostasis, survival, and regeneration, including impaired blastema formation and failure to regenerate tissues. In situ hybridization and immunofluorescence analyses revealed a marked reduction in stem cell populations and mitotic activity. Western blotting showed a peak in SMED-TIP60 expression at 5 days post-amputation, suggesting a role during regeneration. RNA-seq analysis revealed widespread dysregulation of gene expression at both anterior and posterior wounds, correlating with increased TIP60 expression post-injury. Notably, wound-response gene expression was aberrant in Smed-tip60 RNAi animals, indicating TIP60s essential role in initiating wound responses and resetting positional cues. Together, these findings establish TIP60 as a critical regulator of stem cell-mediated regeneration and wound healing in planarians.

cell biology↗

bioRxiv2025

Single-Nucleus Analysis of Human White Adipose Tissue Reveals Adipocyte Subsets with Distinct Metabolic Profiles

Anatomic location of white adipose tissue is a determinant of cardiometabolic risk. To understand differences within/between adipose depots, we generated 65,668 single-nucleus transcriptomes from human subcutaneous or intraabdominal adipose tissue (SAT/IAT). Unsupervised analysis revealed 26 adipose-resident cell clusters including two subpopulations of mature adipocytes, characterized by high vs. low expression of adipocyte maturation genes (ADIPOMAThi vs. ADIPOMATlo). ADIPOMATlo adipocytes demonstrate a low-differentiation, pro-inflammatory, and pro-fibrotic transcriptome. IAT-resident ADIPOMATlo were more abundant in higher BMI donors, while SAT-resident ADIPOMATlo associated with impaired glycemia. TSHZ3 was identified as a candidate regulator of ADIPOMATlo transcriptome. TSHZ3 knockdown in adipogenic progenitors inhibited differentiation, with downregulation of early adipogenic regulators (e.g. CEBPA/B, PPARG) and mature adipocyte genes. Heterozygous deletion of Tshz3 in mice reduced SAT and IAT weight. Here, we show that adipocyte subsets with distinct transcriptomic signature reside in human WAT; altered TSHZ3-mediated transcriptional regulation may contribute to low-maturation subpopulation linked to metabolic disease.

cell biology↗

bioRxiv2025

Repurposing Melatonin in dual-mode for Wilson disease therapy as a Copper Chelator and an antioxidant agent

Loss-of-function mutations in copper-ATPase ATP7B underlie Wilson disease (WD), a disorder characterized by hepatic copper accumulation and severe hepato-neuropathology. Existing chelation therapeutics remove excess copper but lack intrinsic antioxidant capacity and frequently cause systemic toxicity. Here we evaluate melatonin, an FDA-approved indoleamine with antioxidant and putative metal-chelating activity, as a candidate therapeutic for WD. In ATP7B-/- hepatocytes, melatonin restored copper-induced reactive oxygen species (ROS) to basal levels, reduced apoptosis twofold, and attenuated Nrf2 nuclear translocation leading to reduction of Hemoxygenase-1 abundance. Live-cell ratiometric analysis of GSSG/GSH using GRX1-roGFP2 expressed in melatonin-treated ATP7B-/- hepatocytes revealed a significant reduction in intensity-ratio, indicating an effective mitigation of copper-induced glutathione oxidation. Isothermal calorimetric titration revealed moderate Cu2+ affinity (K ITC=4.54 x 103 M-{superscript 1}), rationalized by MD-simulations showing an interaction energy of 18.5 x 10-3 kcal{middle dot}mol-{superscript 1} via amide-Cu{superscript 2} coordination. In-cellulo studies also revealed that copper-induced vesicularized ATP7B reinstates to Golgi in melatonin-treated hepatocytes. In vivo, melatonin treatment reduced copper-induced oxidative stress in zebrafish embryos and lowered copper burden in Caenorhabditis elegans WD model. Our studies revealed that encapsulation of melatonin within an engineered polymeric nanocapsules having dithiol linkers, susceptible to cleavage by GSH, extended melatonins circulatory half-life ten-fold and enhanced its ROS-scavenging efficacy three-fold relative to free melatonin. This work introduces a unique dual-function therapeutic strategy that integrates antioxidant activity with copper chelation, simultaneously addressing copper overload and redox imbalance. Repurposing melatonin, with its established clinical safety, offers rapid and cost-effective translational pathway toward WD-therapy while providing a generalizable platform for redox- and metal-associated disorders.

cell biology↗

bioRxiv2025

CRISPR/Cas9-mediated identification of human macrophage SphK1 as druggable target for development of anti-leishmanial chemotherapeutics

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates apoptosis, autophagy, inflammation, and intracellular pathogen survival. While S1P signaling has been implicated in Leishmania donovani infection, the specific roles of its biosynthetic enzymes Sphingosine kinases SphK1 and SphK2 in host macrophage remains poorly defined. Here, we delineate the role of SphK1 in modulating host-pathogen interactions using CRISPR/Cas9-mediated knockout and pharmacological inhibition in THP-1 macrophages. We generated and validated CRISPR constructs targeting SphK1 (promoter, SBS, exon 6) and SphK2 (exon 3). SphK1 Knockout was confirmed at transcript and protein levels, accompanied by a marked reduction in SphK1 enzymatic activity and S1P levels. Functionally, SphK1 knockout macrophages exhibited decreased intracellular L. donovani burden, elevated TNF-, and reduced IL-10, and increased autophagic and apoptotic markers, suggesting a pro-inflammatory, cell-death-prone state. Pharmacological inhibition using the selective SphK1 inhibitor, PF-543 recapitulated these findings, showing reduced phosphorylated SphK1, enhanced p38MAPK activation, and augmented autophagy and apoptosis. Conversely, the SphK2 inhibitor ABC294640 had minimal effect, reinforcing the predominant role of SphK1. Together, our study identifies SphK1 as a critical host factor that facilitates L. donovani survival by modulating lysosomal stress, immune evasion, and cell fate pathways. Targeting SphK1-S1P signalling may offer a novel therapeutic approach for visceral leishmaniasis.

molecular biology↗

bioRxiv2025

Ultrasound-activated drug release with extracellular vesicles

Precise, noninvasive targeting and controlled drug release to the nervous system could transform both basic research and clinical therapy. In this work, we developed a focused ultrasound-responsive delivery system based on extracellular vesicles (EVs), which are naturally occurring and biocompatible phospholipid nanocarriers. To enable the ultrasound responsiveness, a lipid-gas phase transition material Perfluoropentane (PFP) was co-encapsulated with therapeutic agents into EVs via low-temperature sonication. The resulting EVs had a size of 100-200 nm and demonstrated efficient, on-demand release upon ultrasound stimulation. In primary cultured neurons, EVs loaded with lidocaine successfully suppressed calcium activity and showed minimum cytotoxicity. Finally, we demonstrated in vivo application of this system for modulation pain sensitivity in rats. Our EVs-drugs platform demonstrated a safe, and ultrasound-activated drug delivery with high spatiotemporal control, showing strong potential therapeutic applications.

bioengineering↗