Biology subjects

Kundu, S.

Publications and source records attributed to Kundu, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Comparative evaluation on the fermentative potential of single and assorted fruit and vegetable waste for production of bioethanol

Fruit and vegetable waste (FVW) constitute a large part of municipal solid waste (MSW) and create significant environmental challenges by emitting the greenhouse gas methane. In contrast, these are a big source of fermentable sugar to produce bioenergy. Therefore, in this study, the Scanning Electron Microscopy (SEM), Attenuated total reflectance-fourier-transform infrared spectroscopy (ATR-FTIR), thermogravimetric analysis (TGA), X-ray diffraction (XRD), and biochemical quantification of -cellulose and acid-insoluble lignin contents were carried out to compare fermentative potential between single (potato peel vs. orange peel) and assorted vegetable and fruit wastes. Collectively, the study indicates that assorted vegetable waste holds enormous biofuel potential by having low moisture content (6.78 %), moderately low decomposition temperature (Tmax: 299.36 {degrees}C), low crystallinity index (CrI: 19.12%), and high -cellulose contents (32.35 - 47.73 gm%) yielding high sugar (325.65 mg/g). The study also optimized several key fermentation parameters such as pH, sugar concentration and the time to produce bioethanol. It was observed that maximum production of ethanol (2.14%) was achieved at 28{degrees}C, at pH 6.3 with a higher initial sugar concentration of 200mg/g by using Saccharomyces cerevisiae strain NCIM 3594. This study supports the likely utility of unsorted vegetable waste to obtain bioethanol at a mass scale.

plant biology↗

bioRxiv2025

Self-amplifying mRNA expression is governed by mitochondrial machinery and ACSL4

RNA-based vaccines offer a superior efficacious and economic approach over traditional vaccines. However, current dose requirement and short half-life of conventional modified mRNA (modRNA) may hinder the development of more effective vaccines. Self-amplifying mRNA (saRNA) is a modality that has the potential to address these limitations by reducing delivery dosage and enhancing the duration of expression over modRNA. Despite marked success in preclinical studies, saRNA vaccines have thus far underperformed in most clinical trials. We hypothesized that non-optimal human cellular context limits saRNA expression, and that elucidating the factors underlying saRNA expression would be key in developing saRNA as a viable therapeutic modality. To identify factors involved in the regulation of saRNA, we performed a quasi-genome-wide CRISPR knockout screen, which revealed that saRNA expression in human cells is linked to mitochondrial function and governed by ACSL4. We validated these findings through pharmacological intervention and single gene editing, demonstrating that ACSL4-mediated regulation is unique to saRNA and does not impact modRNA. Moreover, we show that modulating ACSL4 leads to improved saRNA expression across multiple cell types. We demonstrate for the first time that mitochondrial function and ACSL4 play key roles in saRNA expression, providing insight for the development and implementation of saRNA as a therapeutic modality.

molecular biology↗

bioRxiv2025

Vascular smooth muscle cell atherosclerosis trajectories characterized at single cell resolution identify causal transcriptomic and epigenomic mechanisms of disease risk

Vascular smooth muscle cells (SMC) contribute to heritable coronary artery disease (CAD) risk and undergo complex cell state transitions to multiple disease related phenotypes. To investigate the genetic basis of SMC state trajectories that underlie the SMC component of CAD causality we have developed a dense timecourse single cell transcriptomic and epigenetic map of atherosclerosis in a murine disease animal model. Cellular trajectories were derived from the temporal data and probabilistic fate modeling with Waddington-Optimal Transport (WOT). We created transcription factor (TF) centered regulons mapped across the developmental timeline and through network-based prioritization with WOT predicted TFs and in silico TF perturbation, identified key drivers of cell state changes associated with EMT, vascular development, and circadian clock functions. Integration of mouse disease data with human CAD genetic findings identified transition SMC phenotypes that mediate disease risk and point to causal disease mechanisms. Parallel studies using knockout of the validated CAD gene Tcf21 revealed its impact on SMC transition cellular phenotypes and disease risk genes, due in part to a role regulating the transition of SMC precursor cells in the secondary heart field. Together, these studies characterize atherosclerosis trajectories at single cell resolution and identify genetic causal transcriptomic and epigenomic mechanisms of CAD risk.

genomics↗

bioRxiv2025

Enhancing salt tolerance in a hybrid poplar (Populus nigra x maximowiczii) through exogenous putrescine application: Growth, physiological, and biochemical responses

IntroductionPutrescine, a polyamine involved in plant growth and stress responses, has shown potential in mitigating abiotic stress effects. However, little is known about the exogenous addition of putrescine regarding salt tolerance in trees. MethodsThis study was conducted to investigate whether exogenous putrescine application via foliar spray enhances growth in a hybrid poplar (Populus nigra x maximowiczii, clone NM6) under a short duration of salt stress. Salt stress was induced by irrigating roots with 100 mM and 200 mM NaCl, followed by foliar spraying of putrescine on several days. Measurement of growth including plant height and stem diameter for each plant were recorded in the greenhouse every 15 days throughout the experiment. Gas exchange, total chlorophyll, carotenoids, soluble sugars and proteins, amino acids, polyamines, and relative water content were analyzed in foliage collected 3, 6, 7, 13, 20, 35 days after treatment. ResultsPutrescine spray on the salt-treated plants caused a significant increase in plant growth. Putrescine application caused a significant increase in fructose, glucose, and galactose in all plants, but putrescine spray had a variable impact on the sucrose content of 100 mM NaCl-treated plants. Based on metabolic responses, plants treated with 100 mM NaCl fared better when sprayed with putrescine than those treated with 200 mM NaCl. DiscussionExogenous application of putrescine mitigated growth inhibition effects of salinity. These finding highlight the potential of putrescine as a practical approach to increase salt tolerance in young poplar trees, with implications for forestry and land reclamation in saline environment.

plant biology↗

bioRxiv2025

Physical principles of phase-separation action on chromatin looping associated to pathogenic gene activation

Phase-separation of chimeric proteins resulting from genetic mutations has been shown to trigger aberrant chromatin looping, contributing to disease development, including cancer. However, the physical mechanisms regulating these processes are not yet fully understood. In this study, we employ polymer physics models of chromatin and numerical simulations to investigate the relationship between phase-separation of proteins and chromatin structure. We demonstrate that a simple model, including only protein-protein and protein-chromatin interactions, effectively explains the aberrant looping observed around oncogenes, such as PBX3, in cells expressing the NUP98-HOXA9 chimeric protein, which is associated with leukemia. In this scenario, looping occurs through a switch-like mechanism controlled by the concentration of the chimera and its affinity with chromatin. Moreover, when incorporating the presence of extruding factors in a more complex model, similar results are observed, indicating a mild dependence of this looping mechanism on loop-extrusion activity. Finally, leveraging our numerical simulations, we propose potential strategies to inhibit the formation of enhancer-gene loops by directly targeting the chimeric protein with interfering molecules.

biophysics↗

bioRxiv2025

Fbxl10/Kdm2b is required for Kmt2b/Mll2 binding across thegenome and regulates H3K4 methylation on bivalent promoters

The presence of histone modifications associated with both transcriptional repression (H3K27me3) and activation (H3K4me3) on key developmental promoters in embryonic stem cells results from the co-localization of repressive Polycomb group (PcG) and activating Trithorax group (TrxG) protein complexes. Functional interactions between PcG and TrxG on these promoters are not fully understood. Here we focus on the relationships between Fbxl10/Kdm2b, a component of a PcG complex PRC1, and Kmt2b/Mll2, an essential component of TrxG at bivalent promoters. Computational analysis of previously published data revealed genome-wide correlation between chromatin occupancies of these two proteins, suggesting potential crosstalk between Kdm2b and Mll2 at both active and repressed promoters. We tested this hypothesis experimentally and found that loss of Kdm2b resulted in depletion of Mll2 at promoters genome-wide, suggesting that Kdm2b is required for Mll2 occupancy at both bivalent and active promoters. Loss of Kdm2b or the core PRC1 component Ring1b also resulted in the reduction of H3K4me3 specifically at bivalent promoters. These findings provide a direct pathway for cooperation between PcG and TrxG at bivalent promoters, suggesting an unexpected modification to the current model of bivalency. In addition, these findings reveal genome-wide role of Kdm2b independent of the full PRC1 complex.

molecular biology↗

bioRxiv2025

M6A reader protein YTHDF3 regulates cardiomyocyte death and atrophy by modulating the alternative splicing program

BackgroundThe functional impact of m6A modifications on RNA is governed by reader proteins that read the m6A marks and process the RNA accordingly. Recent studies have highlighted the importance of m6A reader proteins in the heart. However, the function of a reader protein Ythdf3 in the heart remains completely unknown. ObjectiveWe aim to uncover the function of Ythdf3 in the hearts. Methods& ResultsHere, we show that Ythdf3 is indispensable for the heart, and its knockdown leads to cardiac cachexia evident by cardiomyocyte atrophy, death, and less dense myofibrils visualized by electron microscopy. We also found downregulation of Ythdf3 in response to doxorubicin, and its overexpression rescues doxorubicin-induced cardiomyocyte atrophy and death. Contrary to primarily cytoplasmic localization, we found that Ythdf3 localizes in the cardiomyocyte nucleus. Furthermore, using co-immunoprecipitation coupled with LC-MS/MS, we show that Ythdf3 interacts with splicing proteins like DDX5 and HNRNPU and regulates alternative splicing in the heart. Mechanistically, we found that Ythdf3 regulates the splicing of CaMKII{delta}, and its knockdown leads to an increase in CaMKII{delta}A and CaMKII{delta}C isoforms while a decrease in CaMKII{delta}9 isoform. ConclusionCollectively, Ythdf3 knockdown in the adult heart leads to cardiac cachexia due to the alteration of the splicing program.

cell biology↗

bioRxiv2025

Complement C3 Deficiency Enhances Renal Leptospiral Load and Inflammation While Impairing T Cell Differentiation During Chronic Leptospira interrogans Infection

Leptospirosis is a neglected zoonotic disease caused by pathogenic Leptospira spp., affecting an estimated of one million people annually and resulting in approximately 60,000 fatalities. The disease can lead to hepatic, renal, and pulmonary dysfunctions and may contribute to the development of chronic kidney disease. The Complement System plays a crucial role in eliminating bacteria by generating opsonins, anaphylotoxins, that degranulate mastocytes and basophils, and attract immune cells to the infection site, among other important functions. This study aimed to investigate the role of C3, the central protein of the Complement System, in vivo during chronic infection of L interrogans serovar Copenhageni strain FIOCRUZ L1-130 (LIC). C57BL/6 wild-type (WT) and C3 knockout (C3KO) mice were infected with 108 LIC and monitored at 15, 30, 60, 90 and 180 days post-infection (d.p.i.). LIC-infected C3KO mice exhibited a significantly higher leptospiral load in the kidneys compared to WT counterparts. Both groups showed local inflammation at 15 and 30 d.p.i., but only C3KO LIC-infected mice had a higher number of Leptospira DNA copies at 30 d.p.i. At the same time point, C3KO LIC-infected mice developed a larger fibrotic area than WT mice. Interestingly, independent of C3, mice pre-treated with a nephrotoxic drug increased the renal inflammatory response; however, this pretreatment did not affect the local leptospiral load in infected mice. Additionally, levels of specific IgG2b and IgG3 antibodies were significantly higher in LIC-infected C3KO mice compared to WT mice. Proteomic analysis showed lower levels of C5/C5a in WT and C3KO LIC-infected mice, as well as in C3KO control mice. M-CSF and SDF-1 cytokine levels were reduced in both LIC-infected groups. Furthermore, naive T lymphocytes count (both CD4+ and CD8+) were higher in LIC-infected C3KO mice, whereas effector CD8+ T lymphocyte numbers declined during LIC infection - a phenomenon more pronounced in C3KO mice. Overall, this study demonstrates that during LIC infection, the absence of C3 does not affect mouse survival but leads to increased renal leptospiral load and fibrosis. Additionally, it highlights the crucial role of C3 in supporting the maturation and differentiation of T lymphocytes into pre-effector cells, underscoring its importance as a key link between the innate and adaptive immune responses in leptospirosis. Author SummaryLeptospirosis is an infectious disease with approximately one million new cases annually and is responsible for about 5% of deaths, particularly in underdeveloped countries. Our objective is to understand the immune response in leptospirosis, specifically the role of the Complement C3 protein. In this study, we observed that C3 deficiency is associated with a higher renal leptospiral load and an increased incidence of renal fibrosis after one month of chronic infection. Notably, C3 also influences the differentiation of helper and cytotoxic T lymphocytes into effector cells, potentially contributing to the increased severity of chronic leptospirosis observed in C3-deficient animals.

immunology↗

bioRxiv2025

A Phenotype-Driven Multi-Omic Atlas of Glioblastoma Invasion

BackgroundGlioblastoma (GBM) invasion is clinically decisive but difficult to model systematically. Existing patient-derived xenograft (PDX) resources rarely couple reproducible in vivo invasion phenotypes with matched multi-omic profiles at scale, limiting mechanistic insight and phenotype-informed therapeutic hypotheses. MethodsWe established the HGCC Phenobank, comprising 65 patient-derived GBM stem-like cultures with matched multi-omic profiling and orthotopic engraftment in 449 mice. Blinded histopathology quantified ten invasion traits per case. These phenotypes were integrated with RNA sequencing, DNA methylation, and mass-spectrometry-based proteomics. Multi-Omic Factor Analysis (MOFA) identified latent molecular programs. Phenotype-specific RNA signatures were matched to LINCS drug-perturbation profiles and validated in 3D gliomasphere and ex vivo brain-slice assays. ResultsTwo dominant, reproducible invasion modes emerged across models: diffuse parenchymal infiltration and perivascular/condensed growth. Proneural cultures formed more aggressive tumors in immunodeficient mice, and mouse survival showed a modest correlation with patient survival in matched cases (Pearson r = 0.1832, p = 0.045). MOFA identified 15 latent factors; Factor 1, enriched for ASCL1/OLIG1/OLIG2 programs and associated with TP53/DCHS2/WNK2 alterations, was linked to increased tumor formation, diffuse invasion, and shorter mouse survival, and stratified GBM patients in TCGA and in our matched patient cohort. Drug-signature matching separated mechanisms targeting diffuse versus perivascular invasion. Experimental validation confirmed phenotype-selective sensitivities, and inhibitors PIK-75 and buparlisib suppressed invasion dynamics across representative models in 3D and brain-slice assays. ConclusionsThe HGCC Phenobank provides the first openly available PDX resource that systematically links GBM invasion phenotypes to multi-omic programs and therapeutic predictions. This framework enables reproducible model selection, mechanistic dissection of invasion modes, and phenotype-guided therapeutic discovery. Key PointsO_LIDiffuse and perivascular invasion define orthogonal GBM axes C_LIO_LIASCL1/OLIG factor links initiation, diffuse growth, and survival C_LIO_LIPhenotype-matched drugs validated; PIK-75 and buparlisib curb invasion dynamics C_LI Importance of the StudyGlioblastoma invasion varies substantially between patients, yet existing patient-derived xeno-graft resources rarely combine reproducible in vivo phenotyping with matched multi-omic profiling at scale. The HGCC Phenobank addresses this gap with standardized, blinded scoring of ten invasion traits across 449 orthotopic xenografts from 65 molecularly characterized GBM stem-like cultures, integrated with transcriptomic, methylomic, and proteomic data. We identify two dominant, reproducible invasion modes and a cross-modal neurodevelopmental program, the ASCL1/OLIG1/2-associated Factor 1, that links tumor initiation, diffuse growth, and survival in mice, and stratifies GBM patients in TCGA and in our matched patient cohort. In a spatially resolved xenograft section, Factor 1 signal localizes to the invasive tumor periphery. By matching phenotype-specific RNA signatures to drug-induced transcriptional responses, we show that invasion phenotypes nominate selective vulnerabilities, exemplified by PIK-75. This openly shared resource enables reproducible model selection, mechanistic dissection of invasion programs, and phenotype-guided therapeutic discovery.

cancer biology↗

bioRxiv2025

Reconstructing the Single-Cell Spatiotemporal Dynamics of Glioblastoma Invasion

Glioblastoma invasion into healthy brain tissue remains a major barrier to effective treatment, yet current models fail to capture its full complexity in a scalable and patient-specific manner. Here, we introduce GlioTrace, a novel ex vivo imaging and AI-based analytical framework that enables real-time, spatiotemporal tracking of glioblastoma invasion dynamics in patient-derived glioma cell culture xenograft (PDCX) brain slices. By integrating whole-specimen confocal microscopy, vascular counterstaining, and an advanced computational pipeline combining convolutional neural networks and Hidden Markov Models, GlioTrace identifies distinct invasion modes, including dynamic morphological switching, vessel-guided migration, and immune cell interactions and quantifies patient-specific variations in invasion plasticity. Using GlioTrace, we demonstrate that targeted therapies can selectively modulate invasion phenotypes, revealing spatially and temporally distinct drug responses. This scalable platform provides an unprecedented window into glioblastoma progression and treatment response, offering a powerful tool for precision oncology and anti-invasion therapeutic development.

cancer biology↗

bioRxiv2025

Maternal Transfer of Vaccine-Induced Anti-OspA Antibodies in Peromyscus spp Protects Pups from Tick-Transmitted Borrelia burgdorferi

The efficacy and duration of passive immunity protection depends on maternal antibody levels and transfer efficiency. We investigated whether oral vaccination of Peromyscus leucopus dams with recombinant OspA-expressing E. coli could induce maternal transfer of anti-OspA antibodies and protect pups from Borrelia burgdorferi challenge. Dams were vaccinated until breeding pairs were created (i), until parturition (ii), and until pups were 2 weeks old (iii). Pups were challenged with nymphal I. scapularis-transmitted B. burgdorferi at [~] 4 weeks of age. Anti-OspA IgG were quantified in dams and pups, and anti-B. burgdorferi IgG were quantified in pups. B. burgdorferi burden was assessed by flaB qPCR in pups tissues [~] 4 weeks after tick challenge and viability of B. burgdorferi was assessed by culture of heart tissue. P. leucopus pups born to dams vaccinated until breeding had low serologic anti-OspA antibody and were not protected from tick transmitted B. burgdorferi infection. However, when dams vaccination extended until parturition and until pups were two weeks old, significant anti-OspA antibody transfer and protection from B. burgdorferi infection occurred. This was evidenced by absence of antibody to B. burgdorferi PepVF, absence of B. burgdorferi flaB DNA in heart and bladder tissues, and absence of flaB in culture from heart tissues from pups euthanized >9 weeks after birth. We show that transfer of anti-OspA antibodies from vaccinated P. leucopus dams to offspring prevents tick transmission and infection dynamics of B. burgdorferi in the major reservoir host of this spirochete in the United States. ImportanceThis study contributes to our understanding of how interventions based in reservoir targeted OspA vaccines designed to block transmission of B. burgdorferi from infected I. scapularis ticks may disrupt the enzootic cycle of this spirochete and reduce incidence of Lyme disease.

microbiology↗

bioRxiv2025

Mapping the regulatory effects of common and rare non-coding variants across cellular and developmental contexts in the brain and heart

Whole genome sequencing has identified over a billion non-coding variants in humans, while GWAS has revealed the non-coding genome as a significant contributor to disease. However, prioritizing causal common and rare non-coding variants in human disease, and understanding how selective pressures have shaped the non-coding genome, remains a significant challenge. Here, we predicted the effects of 15 million variants with deep learning models trained on single-cell ATAC-seq across 132 cellular contexts in adult and fetal brain and heart, producing nearly two billion context-specific predictions. Using these predictions, we distinguish candidate causal variants underlying human traits and diseases and their context-specific effects. While common variant effects are more cell-type-specific, rare variants exert more cell-type-shared regulatory effects, with selective pressures particularly targeting variants affecting fetal brain neurons. To prioritize de novo mutations with extreme regulatory effects, we developed FLARE, a context-specific functional genomic model of constraint. FLARE outperformed other methods in prioritizing case mutations from autism-affected families near syndromic autism-associated genes; for example, identifying mutation outliers near CNTNAP2 that would be missed by alternative approaches. Overall, our findings demonstrate the potential of integrating single-cell maps with population genetics and deep learning-based variant effect prediction to elucidate mechanisms of development and disease-ultimately, supporting the notion that genetic contributions to neurodevelopmental disorders are predominantly rare.

genomics↗

bioRxiv2025

C3H-HeN as a TLR4 competent mouse model of sublethal leptospirosis

AbstractMice are slowly being accepted as alternative models for investigation of leptospiral infection. The strain most used to analyse sublethal disease (C3H-HeJ) expresses a tlr4 gene in its immune cells that is hyporesponsive to leptospiral LPS and thus the model is deemed immunocompromised. To help resolve this valid scientific concern we did a study in which we compared infection with Leptospira interrogans serovar Copenhageni Fiocruz in mice expressing fully competent tlr4 (C3H-HeN, C57BL6) versus tlr4 hyporesponsive mice (C3H- HeJ) over a period of two weeks. We found that the C3H-HeJ mouse was the only strain that sustained lethal infection, thus confirming the association between tlr4 function and acute disease. We found that the two mouse strains with a fully functional tlr4 gene (C3H-HeN and C57BL/6) also developed clinical and molecular signs of sublethal leptospirosis nearly on par with C3H-HeJ, as quantified by weight loss, survival curves, presence of Leptospira 16S rRNA in blood and urine, and burden of viable spirochetes in kidney. Regarding kidney markers of inflammation and fibrosis, C3H-HeN and C57BL/6 produced less IL-1 beta, iNOS and ColA1 than C3H-HeJ, which is consistent with increased resilience to infection expected of the tlr4 competent strains. Regarding Leptospira-specific antibody, all mouse strains produced IgM and T-cell independent IgG3. Interestingly, in contrast to C57BL/6, both C3H strains produced circulating IgG1 but only C3H-HeN produced IgG2a to L. interrogans two weeks post infection. Thus, TLR4-competent C3H-HeN and C57BL/6 may be more appropriate mouse models of sublethal leptospirosis, whereas C3H-HeJ can be used to further establish a mouse model of lethal leptospirosis. Our data strongly suggests that TLR4 function is important but not sufficient to cause susceptibility to leptospirosis. Author summaryPrevious studies by different laboratories have shown C3H-HeJ to be a susceptible mouse strain to sublethal and lethal leptospirosis. The goal of our study was to evaluate if low tlr4 responsiveness to leptospiral LPS in C3H-HeJ mice is the factor that drives susceptibility to infection with pathogenic Leptospira. We did a comparative study using mouse strains immunocompetent and hyporesponsive to tlr4. The data shows that although tlr4 does affect the virulence of leptospirosis (only C3H-HeJ succumbed to infection), the tlr4 competent strains (C3H-HeN and C57BL/6) also developed measurable sublethal leptospirosis. Thus, competent recognition of leptospiral-LPS by murine TLR4 plays a role but it is not sufficient to determine susceptibility to leptospirosis. Furthermore, the data suggests that T-cell dependent responses could be involved in susceptibility to leptospirosis in the C3H models of infection.

microbiology↗

bioRxiv2025

Threshold effects of prenatal stress on striatal microglia and relevant behaviors

AbstractPrenatal stress, a risk factor for neurodevelopmental disorders (NDDs), leads to immune alterations, including offspring neuroimmune cells. Differences in offspring outcomes may arise from whether the extent of prenatal stress crosses "thresholds" for effects on specific outcomes. Therefore, we sought to determine offspring outcomes using models with different extents of prenatal stress. We focused on striatal outcomes, because of their relevance for NDDs. Pregnant CD1 mice were assigned to four groups (each: N=6): no stress ("NoS") or one of the following stressors administered three times daily: i.p. saline injections (low prenatal stress, LoS), Interleukin-6 injections as a component of prenatal stress (immune prenatal stress; ImS), or restraint stress + saline injections (high prenatal stress, HiS), embryonic day 12-18. In adult offspring, HiS altered striatal-dependent behavior across males and females, while ImS induced fewer behavioral changes, and LoS did not affect behavior. Adult striatal microglia morphologies were mostly unchanged across groups, with only HiS leading to altered striatal density of minimally ramified cells. However, embryonic striatal microglia were affected by all models of stress, albeit in distinct ways. The HiS model, and to a lesser extent LoS, also influenced immune components of the maternal-fetal interface: placental macrophages. In conclusion, high and immune stress affected adult striatal-dependent behavior, exceeding the threshold necessary for persistent impacts, but all stress models affected embryonic microglia, suggesting that early neuroimmune outcomes had a lower threshold for impacts. Distinct severities and aspects of prenatal stress may therefore underlie different outcomes relevant to NDDs.

neuroscience↗

bioRxiv2025

A single mutation in the DSL motif of the acyl carrier protein can prevent its in vivo modification by E. coli Holo-acyl carrier protein synthase (AcpS)

E. coli expression system is the method of choice to obtain high yields of a pure protein. However, there is always a chance that an overexpressed protein shares structural or sequence homology with the substrate of an E. coli enzyme. In such cases, the expressed protein may be partially or fully converted into the product. A notable example is the expression of acyl carrier proteins (ACP) in E. coli. Since most type II ACPs of the fatty acid synthesis pathway (FAS) have a conserved helix II, the carrier proteins are recognized as a substrate by Holo-acyl carrier protein synthase (AcpS). Thus, most ACPs express as partially or fully loaded proteins in E. coli. This undesirable modification is a concern when the objective is to obtain milligram amounts of apo-ACP. Here, using an approach combining mutagenesis, enzyme activity, and NMR, we probed for residues in ACP that can prevent this in vivo modification, without affecting Sfp (Surfactin synthetase activating enzyme) function. Taking cues from the E. coli ACP-AcpS structure (PDB 5VCB), charge neutralization mutations were designed at five different positions in EcACP that participate in ion-pair interaction with AcpS. Three of the mutants expressed solely as apo-ACP in E. coli viz. D35N, E41A and E47A/E48A. However, only the D35N mutant could be converted into holo-/acyl-ACP using Sfp in vitro, establishing mutagenesis as a viable strategy to prevent undesired modifications in vivo. As proof of principle, the mutation was applied to two unrelated ACPs that express primarily as modified proteins in E. coli -Mus musculus mitochondrial FAS ACP (MmACP) and Salmonella Typhimurium invasion acyl carrier protein (IacP). Single D35N mutation of the ACPs prevented their in vivo modification by AcpS, and the mutants were efficiently converted into holo-ACP by Sfp in vitro. These results demonstrate that D35N mutagenesis is a useful strategy to express apo-ACP in E. coli and is applicable across all type II ACPs. Furthermore, we show that holo-IacP and holo-MmACP are not recognized as substrates by AcpH (E. coli Acyl carrier protein hydrolase), and therefore they express predominantly as modified proteins in E. coli.

biochemistry↗

bioRxiv2025

Physical confinement regulates transition in nematode motility

How do worms navigate their complex natural surroundings? Undulatory microswimmers such as nematodes typically inhabit environments such as soil, vegetable matter, and host tissues. While the natural habitats of nematodes are often three-dimensional granular niches with spatiotemporally varying visco-elasto-plastic material properties that impose physical constraints on their motion, current knowledge about nematode motility patterns broadly comes from investigating model organisms such as Caenorhabditis elegans either inside liquid cultures or the surface of soft agar pads. How nematodes move through 3D granular niches across different degrees of physical confinement remains poorly understood due to a lack of optically transparent 3D granular matrices. We bridge this gap by engineering an optically transparent granular matrix to directly visualise and quantitatively analyse nematode motion. Importantly, nematodes can freely move through this matrix by generating a minimal yield stress; once the nematode moves away, the matrix self-heals to ensure the material properties remain invariant. Using these platforms, we observe that the propulsive speed of nematodes shows a non-monotonic relation with the yield stress of their microenvironment. This non-monotonicity emerges as nematodes optimize for efficient navigation at higher yield stress, wherein, their forward propulsive speed matches the wave speed along their body. This regulation of locomotory behaviour is purely dictated by the physical interaction of the nematode with its environment without involving soft-touch sensory neurons. Remarkably, predictions from a slender body theory of undulatory motion exactly capture the scaling behaviour for both efficiency and mode of motion as obtained from the experimental data. Finally, in a phase space described by non-dimensional propulsive efficiency and a non-dimensional time scale of motility, we capture a gait transition from poorly efficient thrashing under low confinement to more efficient crawling under high confinement. Thus, our work establishes a new regulatory paradigm describing how distinct modes of undulatory motion emerge under different degrees of physical confinement.

biophysics↗

bioRxiv2024

Inhibition of the extracellular matrix protein fibulin-3 reduces immunosuppressive signaling in tumor stem cells and increases macrophage activation against glioblastoma

Glioblastoma tumors remain a formidable challenge for immune-based treatments because of their molecular heterogeneity, poor immunogenicity, and growth in the largely isolated and immunosuppressive neural environment. As the tumor grows, GBM cells change the composition and architecture of the neural extracellular matrix (ECM), affecting the mobility, survival, and function of immune cells such as tumor-associated microglia and infiltrated macrophages (TAMs). We have previously described the unique expression of the ECM protein EFEMP1/fibulin-3 in GBM compared to normal brain and demonstrated that this secreted protein promotes the growth of the GBM stem cell (GSC) population. Here, we describe a novel immunomodulatory role of fibulin-3 and the immuno-boosting effects of targeting this ECM protein. Mice carrying fibulin-3-deficient intracranial tumors showed increased myeloid infiltration and reduced expression of TAM pro-tumoral markers (Arginase, CD206) compared to controls. The opposite was observed in orthotopic tumors overexpressing fibulin-3. In silico dataset analysis of clinical datasets revealed positive correlation of fibulin-3 with an immunosuppressive signature, which was validated in GSCs and in vivo. We further demonstrated that fibulin-3 regulates the expression of immunosuppressive signals (CSF-1, TGF{beta}) and the innate immune checkpoint CD47 in GSCs via autocrine activation of NF-{kappa}B signaling. Accordingly, immunosuppressive signals were downregulated in GSCs by knockdown of fibulin-3 or inhibition of this protein with an anti-fibulin-3 antibody. Co-culture of GBM cells with syngeneic macrophage lines or primary macrophages in presence of anti-fibulin-3 antibody increased macrophage phagocytosis and antibody-dependent killing of the tumor cells, Furthermore, locoregional delivery of anti-fibulin-3 antibody in mice carrying intracranial GBM increased the infiltration of TAMs expressing pro-inflammatory markers, reducing tumor viability. Our findings show that anti-fibulin-3 approaches, which impact the pericellular ECM surrounding tumor cells and TAMs, can diminish immunosuppression in GBM and boost innate immune responses against the tumor.

cancer biology↗

bioRxiv2024

ChromBPNet: bias factorized, base-resolution deep learning models of chromatin accessibility reveal cis-regulatory sequence syntax, transcription factor footprints and regulatory variants

Despite extensive mapping of cis-regulatory elements (cREs) across cellular contexts with chromatin accessibility assays, the sequence syntax and genetic variants that regulate transcription factor (TF) binding and chromatin accessibility at context-specific cREs remain elusive. We introduce ChromBPNet, a deep learning DNA sequence model of base-resolution accessibility profiles that detects, learns and deconvolves assay-specific enzyme biases from regulatory sequence determinants of accessibility, enabling robust discovery of compact TF motif lexicons, cooperative motif syntax and precision footprints across assays and sequencing depths. Extensive benchmarks show that ChromBPNet, despite its lightweight design, is competitive with much larger contemporary models at predicting variant effects on chromatin accessibility, pioneer TF binding and reporter activity across assays, cell contexts and ancestry, while providing interpretation of disrupted regulatory syntax. ChromBPNet also helps prioritize and interpret regulatory variants that influence complex traits and rare diseases, thereby providing a powerful lens to decode regulatory DNA and genetic variation.

genetics↗