Biology subjects

Banerjee, B.

Publications and source records attributed to Banerjee, B..

10 recordsLinked to original sources

bioRxiv2026

Cell Mechanics Regulate Membrane Tubulation-Driven Trogocytosis in Adherent Cells

Trogocytosis is a widespread and physiologically important process in which one cell ingests fragments of another. It has been observed in diverse biological contexts, such as in Entamoeba histolytica, a human enteric parasite that nibbles host intestinal cells via trogocytosis, thereby invading the tissue and leading to lethal extra-intestinal disease, and in macrophages on interaction with adherent tumor monolayers. Despite its physiological relevance, knowledge of trogocytosis with adherent cells remains elusive. Here, we established a monolayer-based trogocytosis assay and uncovered a distinct membrane-tube-mediated trogocytic mechanism operative in adherent cells. We characterized it into four sequential events: contact, membrane tubulation, stretching, and scission. By targeting the proteic constituents of target cell stiffness, we show that trogocytic output exhibits a non-monotonic dependence on the viscoelastic properties of the target cell, with maximal uptake occurring in an intermediate mechanical regime. Further, we integrated live-cell trogocytosis observations with a viscoelastic model-based theoretical framework to propose a tube-breaking mechanism across distinct mechanical subtypes. Finally, using micropatterning, we demonstrated that target cell shape influences trogocytosis kinetics. This work suggests that trogocytosis is sensitive to the mechanical state of the target cell.

cell biology↗

bioRxiv2026

lncRNA MANCR isoforms selectively mediate multiple levels of epigenomic and P53-responsive transcriptional control in triple negative breast cancer

Long noncoding RNAs (lncRNAs) are important regulators of gene expression and are frequently dysregulated in cancer. The mitotically associated lncRNA MANCR is highly expressed in aggressive cancers and contributes to genomic instability in triple-negative breast cancer (TNBC), but the molecular mechanisms underlying its activity remain poorly defined. Here we integrate computational and experimental approaches to examine the structure and regulatory interactions of MANCR isoforms. Analysis of transcriptomic datasets revealed tumor-type-specific expression patterns for seven MANCR isoforms in breast cancer cell lines. Computational prediction of RNA secondary structures identified conserved structural features across isoforms, suggesting potential functional specialization. We identify p53 as a MANCR-interacting protein through computational docking and RNA immunoprecipitation sequencing (RIP-seq) and demonstrate that MANCR depletion reduces p53-dependent transcriptional activity. Chromatin isolation by RNA purification sequencing (ChIRP-seq) revealed 1, 250 genomic regions associated with MANCR, including enrichment of p53 consensus motifs and GC-rich sequence elements. Motif analysis further identified candidate sequence features associated with MANCR-occupied chromatin regions. Computational prediction of RNA-miRNA interactions identified multiple potential miRNA binding sites across MANCR isoforms, including miR-6756-5p, which targets the androgen receptor (AR). Consistent with this prediction, AR expression decreased following MANCR knockdown in TNBC cells. Together, these results suggest that MANCR isoforms may contribute to transcriptional regulation in TNBC through interactions with chromatin, p53 signaling pathways, and potential miRNA regulatory networks. One Sentence SummaryMitotically-associated lncRNA (MANCR) is prevalent in aggressive cancers interacting with DNA, P53, and miRNAs, to mediate multiple levels of epigenetic transcriptional control in triple negative breast cancer.

cancer biology↗

bioRxiv2026

Probing the role of residues lining the active site in the generation of glucose-tolerant variants of a fungal GH1 enzyme

The hydrolytic breakdown of cellobiose into glucose, catalysed by {beta}-glucosidases, is the last and rate-limiting step in cellulose saccharification for producing fermentable glucose in the bioethanol industry. This limitation arises because {beta}-glucosidase activity is inhibited by factors such as temperature, pH, and glucose accumulation in reactors. Enzyme inactivation leads to the buildup of cello-oligosaccharides, which, in turn, inhibit upstream cellulases. Therefore, glucose-tolerant {beta}-glucosidases are preferred for the formulation of industrial cellulase cocktails. In this study, we have recombinantly expressed, purified, and biochemically characterised a {beta}-glucosidase from the cellulolytic fungus Fusarium odoratissimum (FoBgl-WT). FoBgl-WT exhibits optimal cellobiose hydrolysis over a broad pH range (4.5-7.5), an important and industrially desirable property for its application in bioreactors. However, the glucose tolerance of FoBgl-WT was [~]0.56 M. Structure-based analyses were carried out to map the residues lining the active site of FoBgl, and their roles in stabilising the product glucose (or even the substrate, cellobiose) were elucidated through a series of site-specific mutations, followed by biochemical characterisation of the resulting FoBgl mutants. Among all the mutants generated, FoBgl-K256I-Y325F exhibits >2.5-fold greater glucose tolerance ([~]1.4 M) than FoBgl-WT. Further, we have observed that the FoBgl-K256W and FoBgl-K256I mutants exhibit improved kinetic properties, such as catalytic efficiencies. The structure-based rational engineering efforts improve glucose tolerance and the kinetic properties of FoBgl mutants, making it a useful and promising candidate enzyme for industrial cellulase cocktails.

biochemistry↗

bioRxiv2026

Dual-mode ClfA-targeting DARPin biologics protect against diverse methicillin-resistant Staphylococcus aureus strains

Staphylococcus aureus uses the adhesin clumping factor A (ClfA) to bind fibrinogen and promote invasive infection through two distinct interfaces: an exposed, low-afinity site on the N3 head domain and a buried, high-afinity "dock, lock, and latch" (DLL) trench that is exposed only under shear. This dual-interface architecture allows limited antibody penetration, as antibodies typically block only the exposed site. Here, we establish a dual-mode inhibition strategy that overcomes this constraint by combining a high-afinity ClfA-binding designed ankyrin repeat protein (DARPin) with a fibrinogen {gamma}-chain peptide capable of occupying the DLL trench. Using cell-free click display and kinetics-guided afinity maturation, we engineer DARPin-{gamma}-peptide fusion biologics that simultaneously block both fibrinogen-binding interfaces. These molecules inhibit ClfA-fibrinogen interactions, prevent methicillin-resistant S. aureus agglutination in human plasma, neutralize major clinical ClfA variants, and confer Fc-independent protection in a lethal murine bacteremia model. This work provides a strategy for targeting antibody-intractable force-activated staphylococcal adhesins.

molecular biology↗

bioRxiv2025

Hydrolysis-dependent severing tunes internal monomeric heterogeneity to shape actin length distributions

The coordination of actin-binding proteins (ABPs) is essential for forming a wide range of actin structures that drive vital cellular processes. Many of the ABPs, like the severing protein cofilins, exhibit affinities dependent on the nucleotide state of actin monomers. Experiments have shown that cofilins interact with actin filaments cooperatively in a concentration-dependent manner, where it severs less at both low and high concentrations. However, how this nucleotide state-dependent severing influences actin filament distributions is poorly understood. Here, we propose a computational model of actin filament dynamics that combines stochastic polymerization, phosphate release, hydrolysis-dependent cooperative binding of cofilins, and severing of actin monomers. This model simultaneously reproduces key experimentally observed features: The non-monotonic change of the filament length and the monotonic decay in actin cap size with the increasing cofilin concentration, as well as the linear increase of the cap size with the actin concentration. We elucidate how both these emerge from the tuning of the heterogeneity in the arrangement of cofilin decorated and undecorated monomers within a filament. We also predict that, with varying cofilin concentrations, steady-state length distributions can change from symmetric bell-shaped to long-tailed skewe d shapes. We show that, in some regimes, our model simplifies to a coarse-grained model enabling us to mathematically predict that the distribution of undecorated monomers is exponential with a decay exponent that depends on cofilin and actin concentration. This study highlights the importance of non equilibrium processes that can modulate structural heterogeneity within a filament to shape actin length variation.

biophysics↗

bioRxiv2025

Inhibitory activities of monoclonal antibodies against Staphylococcus aureus Clumping factor A

Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. Clumping factor A (ClfA) displayed on the bacterial surface plays a key role in promoting S. aureus replication during invasive disease. Decades of research have pointed to a wide array of ligands engaged by ClfA. The sum of these interactions supports the unique ability of this pathogen to survive and replicate in the blood stream. One such ligand is fibrin. ClfA acts as the key agglutinating factor of S. aureus by promoting the shielding of bacteria in fibrin cables and their physical escape from phagocytes. Here, we compare a series of monoclonal antibodies elicited against the ligand binding domain of ClfA following immunization of mice. We analyze these antibodies for their ability to neutralize ClfA interactions and to promote the uptake of staphylococci in whole blood. We find that while all the antibodies promoted opsonophagocytic uptake, only those that also inhibited ClfA interactions with ligands, reduced bacterial burdens in animals following blood stream challenge with S. aureus. IMPORTANCEAntibody-based approaches to fight bacterial pathogens have been modeled on toxin-producing or encapsulated pathogens for which correlates of protection can be reduced to measuring antibody neutralization or complement-fixing activities using tissue cultured cells. Such approaches have failed against Staphylococcus aureus raising uncertainty about the value of antibodies. Here, we use a series of mouse monoclonal antibodies directed against ClfA, a surface protein that allows S. aureus to thrive in the blood stream, to query how antibodies may be exploited against a pathogen endowed with a formidable array of virulence factors.

microbiology↗

bioRxiv2024

Molecular insights into the oligosaccharide binding, recognition and hydrolysis by a fungal exo-beta-(1,3)-glucanase

Carbohydrate hydrolysing enzymes assume special industrial and commercial interest as a source for yielding fermentable glucose especially for the biofuel industry. Among these enzymes, the exo-{beta}-(1,3) glucanases are promising for industrial use as they hydrolyze sugars such as laminarin, a major constituent of the algal cell wall. Exploring the structure and function of these enzymes is of particular interest for the improvement of their functional properties for industrial use. We report the structural and biochemical characterizations of Aspergillus oryzae exo-{beta}-(1,3) glucanase (AoBgl). We have expressed, purified, and performed biochemical characterizations of the recombinant AoBgl. Purified AoBgl is found to hydrolyse {beta}-(1,3)-glycosidic linkages present in the oligosaccharide laminaritriose and the polysaccharide, laminarin effectively while retaining >50% activity at glucose concentrations of around 1.5M. We have determined three high-resolution structures of AoBgl: (a) apo form at 1.75 [A], (b) complexed form with bound disaccharide at 1.73 [A] and (c) glucose-bound form at 1.20 [A]. Sequence analysis and structural comparison indicate that AoBgl belongs to the GH5 sugar hydrolase family. The sugar-bound structures reveal the mode of substrate binding and interactions at the active site of AoBgl. Further, molecular dynamics simulation and mutational studies indicate that AoBgl can effectively bind trisaccharides and higher oligosaccharides. Our biochemical and structural data provide detailed molecular insights into the active site of this GH5 enzyme and would be helpful in the rational engineering of glycosyl hydrolases belonging to similar families for industrial use.

biochemistry↗

bioRxiv2021

Automated Hyperspectral Vegetation Index Derivation Using A Hyperparameter Optimization Framework for High-Throughput Plant Phenotyping

O_LIHyperspectral vegetation indices (VIs) are widely deployed in agriculture remote sensing and plant phenotyping to estimate plant biophysical and biochemical traits. However, existing VIs consist mainly of simple 2-band indices which limits the net performance and often do not generalize well for other traits than they were originally designed for. C_LIO_LIWe present an automated hyperspectral vegetation index (AutoVI) system for the rapid generation of novel 2- to 6-band trait-specific indices in a streamlined process covering model selection, optimization and evaluation driven by the tree parzen estimator algorithm. Its performance was tested in generating novel indices to estimate chlorophyll and sugar contents in wheat. C_LIO_LIResults show that AutoVI can rapidly generate complex novel VIs ([≥]4-band index) which correlated strongly (R2 > 0.8) with measured chlorophyll and sugar contents in wheat. AutoVI-derived indices were used as features in simple and stepwise multiple linear regression for chlorophyll and sugar content estimation, and outperformed results achieved with existing 47 VIs and those provided by partial least squares regression. C_LIO_LIThe AutoVI system can deliver novel trait-specific VIs readily adoptable in high-throughput plant phenotyping platforms and should appeal to plant scientists and breeders. A graphical user interface of AutoVI is herein provided. C_LI

plant biology↗

bioRxiv2020

Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay

A key mechanism in cellular regulation is the ability of the transcriptional machinery to physically access DNA. Pioneer transcription factors interact with DNA to open chromatin, which subsequently enables changes to gene expression during development, disease, or as a response to environmental stimuli. However, the regulation of DNA accessibility via the recruitment of transcription factors is difficult to understand in the context of the native genome because every genomic site is distinct in multiple ways. Here we introduce the Multiplexed Integrated Accessibility Assay (MIAA), a multiplexed parallel reporter assay which measures changes to genome accessibility as a result of the integration of synthetic oligonucleotide phrase libraries into a controlled, natively inaccessible genomic context. We apply MIAA to measure the effects of sequence motifs on cell type-specific DNA accessibility between mouse embryonic stem cells and embryonic stem cell-derived definitive endoderm cells, screening a total of 7,905 distinct phrases. MIAA is able to recapitulate differential accessibility patterns of 100-nt sequences derived from natively differential genomic regions, identifying the presence of E-box motifs common to epithelial-mesenchymal transition driver transcription factors in stem cell-specific accessible regions that become repressed during differentiation to endoderm. We further present causal evidence that a single binding motif for a key regulatory transcription factor is sufficient to open chromatin, and classify sets of stem cell-specific, endoderm-specific, and shared pioneer factor motifs. We also demonstrate that over-expression of two definitive endoderm transcription factors, Brachyury and FoxA2, results in changes to accessibility in phrases containing their respective DNA-binding motifs. Finally, we use MIAA results to explore the order of motif interactions and identify preferential motif ordering arrangements that appear to have an effect on accessibility.

genomics↗

bioRxiv2018

Non-histone human chromatin protein, PC4 is critical for genomic integrity and negatively regulates autophagy

Multifunctional human transcriptional positive co-activator 4 (PC4) is a bonafide non-histone component of the chromatin and plays a pivotal role in the process of chromatin compaction and functional genome organization. Knockdown of PC4 expression leads to a drastic open conformation of the chromatin and thereby altered nuclear architecture, defects in chromosome segregation and changed epigenetic landscape. Interestingly, these defects do not induce cellular death but result in enhanced cellular proliferation possibly through enhanced autophagic activity. PC4 depletion confers significant resistance to gamma irradiation. Exposure to gamma irradiation further induced autophagy in these cells. Inhibition of autophagy by small molecule inhibitors as well as by silencing of a critical autophagy gene drastically reduces the survivability PC4 knockdown cells. On the contrary, complementation with wild type PC4 could reverse this phenomenon, confirming the process of autophagy as the key mechanism for radiation resistance in absence of PC4. These data connect the unexplored role of chromatin architecture in regulating autophagy during a stress condition such as radiation.

molecular biology↗