Biology subjects

Sawant, M.

Publications and source records attributed to Sawant, M..

7 recordsLinked to original sources

bioRxiv2026

Benchmarking three simple DNA staining-based image metrics for live-cell tracking of chromatin organization

Quantifying chromatin-state dynamics in living cells remains challenging, in part because most methods require fixation or cell lysis. Here, we benchmark and introduce three simple live-cell image-derived metrics computed from routine DNA staining - the coefficient of variation (CV), 1-Gini, and the Diffuse Signal Index (DSI), introduced here - as fixation-free readouts of chromatin organization. Using HL60-derived neutrophils (dHL-60) undergoing NETosis as a model system with a pronounced compact-to-decompact chromatin transition, we show that all three metrics track progressive chromatin reorganization in live-cell trajectories, with DSI providing the strongest trajectory-level discrimination between NETing and non-NETing cells. Comparison with Tn5-based chromatin accessibility measurements in fixed cells shows that all three metrics correlate with chromatin accessibility, supporting their biological interpretability. We further show that all three metrics track chromatin reorganization in a second, mechanistically distinct biological process, capturing mitotic chromatin compaction and post-mitotic chromatin decompaction in U2OS cells. Together, our results provide a practical framework for extracting chromatin-organization readouts from routine live-cell DNA staining and highlight that metric performance is task- and context-dependent. To make our metrics accessible to a broad audience, we built NucMetrics, an open-source ImageJ/Fiji macro toolset that computes all three metrics without requiring programming.

cell biology↗

bioRxiv2026

Hydrogel Fiber Endomicroscopy

Multimode fibers enable minimally invasive, high-resolution imaging through ultrathin probes, thereby enhancing diagnostic precision and facilitating real-time monitoring in delicate anatomical regions. In this work, we introduce HYFEN, a hydrogel-based endomicroscopic imaging platform for flexible, biocompatible, and subcellular-scale fluorescence microscopy. HYFEN leverages the unique properties of hydrogel materials, adaptive optics, and pixel-wise image enhancement to address challenges associated with silica-based fibers, including mode scrambling, limited field of view, and mechanical rigidity. The technique achieves precise mode threading, rapid diffraction-limited focusing at kilohertz speeds, and high-fidelity fluorescence signal acquisition with subcellular resolution. Notably, the approach extends fluorescence imaging under enhanced fiber dimensions and bending conditions that are unachievable with conventional modalities. Together, these advances establish HYFEN as a versatile platform for next-generation biointerfacing and minimally invasive imaging across biomedical and clinical settings.

bioengineering↗

bioRxiv2026

Investigation of Regulation and Binding Patterns of the Human Cathelicidin Peptide LL-37 in Complexation with Nucleic Acids, and its Impact on Neutrophil Extracellular Traps

The human cathelicidin host defense peptide LL-37 forms complexes with nucleic acids that can have either beneficial or detrimental health effects. We suggest that these differential impacts are directly connected to dsDNA binding by LL-37 and to complex formation between protomers. Here, we show using phage {lambda} DNA that LL-37 binds non-specifically to dsDNA, condensing it, followed by complex formation between LL-37 peptides. We find that complex formation is concentration-dependent, with low LL-37 amounts yielding loosely aggregated DNA structures, while higher LL-37 concentrations lead to well-defined, disc-like structures of about 150 nm in diameter. The condensation of the nucleic acids, which causes a loss of the characteristic B-DNA features, results from interactions of the phosphodiester backbone with protonated amino acid side chains of the peptide at physiological pH, predominantly in A-T rich sequences of the nucleic acid. However, in our studies, electrostatic interactions did not appear to be the driving force for complexation, but rather we found the -helical structure of the peptide with its amphipathic and hydrophobic surfaces to be essential. Further, we show that LL-37 also interacts with nucleic acids from neutrophil extracellular traps (NETs) in a concentration-dependent way, causing a reduction in NET aggregate area, which may offer new biophysical insights into diseases such as systemic lupus erythematosus (SLE), which involve slower-than-normal NET clearance. Our results indicate the key importance of LL-37 expression levels for regulation of the innate immune system for optimal human health, since the relative amounts of expressed LL-37 present to interact with extracellular DNA will determine the extent to which the DNA can be condensed, which in turn will affect the ability of the body to clear the NETs before they can cause inflammatory conditions.

biophysics↗

bioRxiv2025

Chromatin is a long-range force generator that regulates plasma membrane tension and cell integrity independently of gene expression

Primarily studied for its role in gene expression, chromatin organization is emerging as an important regulator of nuclear mechanics. Although the nucleus is in mechanical equilibrium with the cell, we do not know whether and how chromatin reorganization actively regulates the mechanical properties and downstream behaviors of cells. Here, we tested the hypothesis that as a dynamic crosslinked polymer, chromatin directly impacts cell mechanics independently of transcription by studying NETosis: a transcription-independent process where chromatin decompacts and the plasma membrane (PM) ruptures. Using high resolution microscopy and ATAC-see, we found that chromatin accessibility progressively increases during NETosis suggesting that chromatin binding proteins (CBPs) dissociate from chromatin during NETosis. To determine the identity and dynamics of these dissociated CBPs, we used fluorescent recovery after photobleaching to measure the mobility and localization of the linker histone H1, the nucleosomal histone H3 and the heterochromatin binding protein HP1. We found that the mobile fraction of nuclear H1 increases during NETosis while fractions of HP1 and H3 diffuse outside of the nucleus suggesting that they become cytosolic osmolytes and potentially alter the mechanical state of cells. Consistently, we found that plasma membrane tension and cell volume increase as chromatin decompacts during NETosis. In non-NETing U2OS cells, we found that inducing chromatin decompaction increases plasma membrane tension, independently of the cytoskeleton, indicating a causal relationship between chromatin organization, cell volume and plasma membrane tension. Our findings reveal a novel non- genetic role of chromatin in cellular biophysics: regulating cell volume, PM tension, and thus, overall cell mechanics. Considering the critical role of cell mechanics in biological processes such as cell migration, proliferation and pathogen killing, our work broadens our understanding of how chromatin regulates cell physiology and pathology. Significance StatementChromatin organizes our DNA inside the nucleus and is important for gene expression. However, chromatin is also a polymer which can passively regulate the rigidity of the nucleus, but whether and how chromatin can actively regulate the mechanical properties of the whole cell remains unknown. Here, we leverage the immune process of NETosis to show that the organization of chromatin inside the nucleus actively regulates the volume and tension of cells. Our work establishes chromatin as a long-range force generator in cells, broadening our understanding of the roles of this crucial polymer network in cells and opening the door to new strategies for controlling the mechanical properties of cells as needed by their physiology.

cell biology↗

bioRxiv2025

A gut-brain axis for aversive interoception drives innate and anticipatory emesis in Drosophila.

Signals from the gut are increasingly recognized as modulators of brain function and behavior. However, the pathways through which the gut conveys adverse or unpleasant information to the brain are still not well understood. In this study, we identify an aversive gut-brain axis in Drosophila melanogaster that detects toxin-induced gut damage and triggers both innate and learned anticipatory emesis (vomiting). After toxin ingestion, reactive oxygen species are produced by midgut enterocytes and detected by the transient receptor potential channel TrpA1 on nearby enteroendocrine cells. This sensing stimulates the release of neuropeptides from enteroendocrine cells, likely representing the gastric malaise flies experience after eating. We show that these neuropeptides act on specific serotonergic and dopaminergic neurons in the brain. These neurons interact with each other and signal to the downstream memory-related mushroom bodies to promote emesis. This circuit not only drives an immediate emetic response but also represents a malaise-driven aversive signal. The signal manifests as the persistent activity of dopaminergic neurons, which reinforces aversive valence to odor cues in the mushroom bodies. Thus, the flies learn that a specific odor predicts the presence of a toxin in food and exhibit anticipatory emesis upon re-exposure to the same odor. Taken together, we have identified an interoceptive signaling pathway that may be conserved for detecting harmful gut conditions and for remembering how to avoid them. Our work offers a mechanistic framework for studying aversive gut-brain communication involved in feeding, metabolism, depression, brain injury, and neurodegenerative diseases.

neuroscience↗

bioRxiv2025

Autoantibody hotspots reveal origin and impact of immunogenic XIST ribonucleoprotein complex

Four out of five patients with autoimmune diseases are women. The XIST ribonucleoprotein (RNP) complex, comprising the female-specific long noncoding RNA XIST and over 100 associated proteins, may drive several autoimmune diseases that disproportionately affect women, who have elevated levels of autoantibodies against the XIST RNP. However, the structural distribution, potential origin, and clinical significance of XIST RNP autoantibodies remained unexplored. Here, we find that XIST RNP is associated with autoantigens associated with six female-biased autoimmune conditions. Mapping autoantibody targets to their occupancy sites on XIST shows that these autoantigens are concentrated at discrete "hotspots" along the XIST lncRNA, notably the A-repeat. Cell type-specific protein expression data nominated neutrophils as a predominant source of hotspot antigens, and we confirmed the presence of both XIST and hotspot antigens in neutrophil extracellular traps during NETosis, an immunogenic programmed cell death pathway triggered by neutrophil activation upon which neutrophils extrude their nuclear content. Furthermore, we found that levels of autoantibodies against a top hotspot antigen, SPEN, that binds the A-repeat, correlate with severe digital ischemia in systemic sclerosis in two independent cohorts. Together, these data show a plausible mechanism for the origin of AXA, guided by RNA structure and RNA-protein interactions, and show that antibodies to XIST RNP holds promise for disease endotyping and prognostication in female-biased autoimmune conditions. One Sentence SummaryNovel autoantibodies target hotspots on XIST ribonucleoprotein complex in female-biased autoimmune diseases.

immunology↗

bioRxiv2022

Putative SET-domain methyltransferases in Cryptosporidium parvum and histone methylation during infection

Cryptosporidium parvum is a major cause of an intestinal pathology called cryptosporidiosis which affects humans and other vertebrates. Despite being declared as a public health problem by World Health Organization (WHO) since 2006, pathogenesis caused by this parasite remains poorly understood. More recently, C. parvum has been linked with oncogenesis. In particular, the mechanisms involved in the processes of gene expression regulation are completely unexplored in Cryptosporidium. In the current study, we took the opportunity to investigate a dynamic epigenetic modification called histone lysine methylation during the life cycle of the parasite. We successfully identified putative SET-domain containing proteins, lysine methyltransferases (KMTs), which catalyze the methylation of different lysine residues. Phylogenetic analysis classified them into distinct subfamilies namely CpSET1, CpSET2, CpSET8, CpKMTox and CpAKMT. Structural analysis further characterized CpSET1, CpSET2 and CpSET8 to be histone lysine methyltransferases (HKMTs). Their functional significance was predicted by using site-specific methyl-lysine antibodies during development of the parasite (CpSET1:H3K4; CpSET2:H3K36; CpSET8:H4K20). In particular, the SET domain of CpSET8 showcased methyltransferase activity confirming the existence of functional HKMTs in Cryptosporidium. Moreover, the consequence of C. parvum infection on the host lysine methylation events highlights the inherit potential of the parasite to exploit the host epigenetic regulation to its advantage. Thus, this study is the first one to provide insights on epigenetics mechanisms occurring throughout the parasites life cycle and during the interaction with its host. As Cryptosporidium is a protozoan that significantly affects the health of both humans and animals, a better understanding of its developmental processes within the definitive host may highlight novel infection control strategies. Author SummaryCryptosporidium species have a very compact genome (~9.2 Mb) unlike its apicomplexan homologs such as Toxoplasma (~63 Mb). Moreover, the lack of large families of transcriptional factors requires them to heavily rely on chromatin remodeling components for its gene regulation. Thus, study and identification of novel elements which contribute to chromatin dynamics could assist a better understanding of the biology of this parasite. In the current study we investigated histone lysine methylation, a dynamic epigenetic modification which regulates gene activation as well as repression. More importantly, characterizing the enzymes which bring about this regulation, provides potential new druggable targets to attack the parasite.

microbiology↗