Biology subjects

Mukherjee, A.

Publications and source records attributed to Mukherjee, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Dual Targeting for Enhanced Tumor Immunity: Conditionally Active CD28xVISTA Bispecific Antibodies Promote Myeloid-Driven T-Cell Activation

Reinvigoration of tumor-reactive T-cells using co-stimulatory bispecific antibodies (bsAbs) targeting CD28 or CD137 is emerging as a promising therapeutic strategy. Conditional, tumor-specific recruitment can offer a necessary layer of control and specificity. We developed pH-selective CD28xVISTA bsAbs to act specifically within the acidic tumor microenvironment (TME), aiming for enhanced T-cell-mediated cancer cell killing while minimizing systemic T-cell activation and Cytokine Release Syndrome (CRS) risk. CD28 agonism by CD28xVISTA bsAbs relies on pH-selective engagement of VISTA, a protein robustly expressed on myeloid cells highly prevalent in most solid tumors. This modality avoids engagement of tumor-associated antigens (TAAs) with the potential to provide highly tumor specific activity with minimal on-target/off-tumor side effects. We report the identification of a lead candidate with pH-dependent simultaneous engagement of both targets, and VISTA-dependent CD28 signaling in a reporter cell line. CD28xVISTA avidly bound VISTA-positive cells, and co-stimulation was shown in vitro by its ability to activate and expand T-cells and enhance T-cell mediated cancer cell killing in co-cultures of human PBMCs and cancer cells in the presence of a TAA-targeted anti-CD3 T-cell engager. Interestingly, our findings support both signaling in cis (between T-cell and cell displaying peptide-MHC complex) and in trans with stimulation occurring through CD28 clustering outside of the immune synapse. Our lead candidate displayed efficient tumor growth inhibition of human VISTA-expressing MC38 cells in a humanized CD28 syngeneic mouse model in combination with PD-1 blockade. Importantly, our CD28xVISTA bsAb showed no signs of superagonistic properties in several in vitro assays geared towards revealing induction of CRS. Our data supports clinical development in combination with anti-PD-1 or any TAA-targeted anti-CD3 T-cell engagers developed for solid tumors.

cancer biology↗

bioRxiv2025

Transcription factor hubs exhibit gene-specific properties that tune expression

Transcription factors (TFs) form dynamic, high-concentration clusters, condensates, or hubs, proposed to increase TF binding frequency at target enhancers. However, how enhancer sequence shapes hub properties remains unclear. We developed a live-imaging based framework to quantify the spatiotemporal relationship between TF hubs and actively transcribed genes in live Drosophila embryos. Examining hubs formed by the TF Dorsal across enhancers with defined binding-site composition, we find that hub enrichment and persistence scale with the number of Dorsal binding motifs. However, these hub properties do not predict transcriptional bursts for a given enhancer. Combining quantitative imaging with computational modeling, we show that Dorsal hub formation can be explained by TF-DNA binding kinetics alone. These findings support a model in which TF hubs emerge from enhancer-encoded TF-DNA interactions rather than higher-order regulatory assemblies.

biophysics↗

bioRxiv2025

A cluster of RNA Polymerase II molecules is stably associated with an active gene

In eukaryotic nuclei, transcription is associated with the clustering of RNA Polymerase II (RNAPII) molecules. The mechanisms underlying cluster formation, their interactions with genes, and their impact on transcriptional activity remain heavily debated. Here, we take advantage of the naturally occurring increase in transcriptional activity during Zygotic Genome Activation (ZGA) in Drosophila melanogaster embryos to characterize the functional roles of RNAPII clusters in a developmental context. Using single-molecule tracking and lattice light-sheet microscopy, we find that RNAPII cluster formation depends on transcription initiation, and that cluster lifetimes depend on transcriptional activity when not constrained by interphase duration. We show that single clusters are stably associated with active gene loci during transcription and that cluster intensities are strongly correlated with transcriptional output. Collectively, our data and simulations on cluster formation kinetics show that RNAPII clusters reflect local accumulations of transcriptionally engaged polymerases and do not form through higher-order mechanisms such as phase separation.

biophysics↗

bioRxiv2025

Quantifying the impact of genetic determinants of antibiotic resistance on bacterial lineage dynamics

The dynamics of antimicrobial resistance in bacterial populations are informed by the fitness impact of genetic determinants of resistance and antibiotic pressure. However, estimates of real-world fitness impact have been lacking. To address this gap, we developed a hierarchical Bayesian phylodynamic model to quantify contributions of resistance determinants to strain success in a 20-year collection of Neisseria gonorrhoeae isolates. Fitness contributions varied with antibiotic use, and genetic pathways to phenotypically identical resistance conferred distinct fitness effects. These findings were supported by in vitro and experimental infection competition. Quantifying these fitness contributions to lineage dynamics reveals opportunities for investigation into other genetic and environmental drivers of fitness. This work thus establishes a method for linking pathogen genomics and antibiotic use to define factors shaping ecological trends.

microbiology↗

bioRxiv2024

Structure of the Staphylococcus aureus bacteriophage 80a neck shows the interactions between DNA, tail completion protein and tape measure protein

Tailed bacteriophages with double-stranded DNA genomes (class Caudoviricetes) play an important role in the evolution of bacterial pathogenicity, both as carriers of genes encoding virulence factors and as the main means of horizontal transfer of mobile genetic elements (MGEs) in many bacteria, such as Staphylococcus aureus. The S. aureus pathogenicity islands (SaPIs), including SaPI1, are a type of MGEs are that carry a variable complement of genes encoding virulence factors. SaPI1 is mobilized at high frequency by "helper" bacteriophages, such as 80, leading to packaging of the SaPI1 genome into virions made from structural proteins supplied by the helper. 80 and SaPI1 virions consist of an icosahedral head (capsid) connected via a unique vertex to a long, non-contractile tail. At one end of the tail, proteins associated with the baseplate recognize and bind to the host. At the other end, a connector or "neck" forms the interface between the tail and the head. The neck consists of several specialized proteins with specific roles in DNA packaging, phage assembly, and DNA ejection. Using cryo-electron microscopy and three-dimensional reconstruction, we have determined the high-resolution structure of the neck section of SaPI1 virions made in the presence of phage 80, including the dodecameric portal (80 gene product (gp) 42) and head-tail-connector (gp49) proteins, the hexameric head-tail joining (gp50) and tail terminator (gp52) proteins, and the major tail protein (gp53) itself. We were also able to resolve the DNA, the tail completion protein (gp51) and the tape measure protein (gp56) inside the tail. This is the first detailed structural description of these features in a bacteriophage, providing insights into the assembly and infection process in this important group of MGEs and their helper bacteriophages.

microbiology↗

bioRxiv2024

Gut MicrobiotAware: how much do we know about gut microbiota? An international questionnaire.

Over the past two decades, our understanding of human gut microbiota composition and function has grown significantly. The surge in studies and data has unveiled the profound influence of gut microbiota on host nutrition, health, and behavior, bridging biology, medicine, and ecology. The dynamic interaction between daily choices, life events, and gut microbiota composition makes it a fascinating subject for scientists to communicate to diverse audiences. Effective scientific communication involves adapting the delivery of knowledge to different audiences, using precise language in academic settings and accessible concepts in public forums like science festivals or social media. With the growing interest in gut microbiota beyond academic circles, there is an increased risk of disseminating information lacking scientific rigor. This study aims to assess the general publics knowledge about gut microbiota and evaluate healthcare professionals understanding of its links to various health conditions, ultimately informing better communication strategies for both groups.

scientific communication and education↗

bioRxiv2024

The interplay between Zfh1 and Nau orchestrates the myogenic stage-dependent expression of Rbfox1 and its target Stat92E via a mir-9a-mediated negative feedback loop in Drosophila melanogaster

RNA-binding Fox protein 1 (Rbfox1) controls gene expression at various levels: as a transcription co-factor, a splicing factor, and a regulator of mRNA stability and translation. Rbfox1 and its vertebrate orthologues have critical functions during development. Rbfox1 mRNA, during adult myogenesis in Drosophila, exhibits bimodal expression, suggestive of the presence of a negative feedback loop. However, there are no known negative regulators of Rbfox1 expression for this context. In this study, we show that the microRNA mir-9a functions as a repressor of Rbfox1 expression. Furthermore, the expression of mir-9a is regulated by the expression of the identity transcription factor Nau. Nau and its target mir-9a are expressed at mid myogenesis stage, and serve to restrict the expression of Rbfox1, and its targets Stat92E and zfh1, to the early and the late myogenesis stages, as their ectopic expression is detrimental to early myofibrillogenesis. Zfh1, in turn, represses mir-9a expression, completing the feedback loop. Therefore, our findings identify the mechanism by which the temporal expression of the developmental gene Rbfox1 and its targets are tightly regulated, essential for their functions during myogenesis. Summary statementUsing Drosophila, we show that a microRNA-mediated negative feedback loop controls the expression of genes critical for skeletal muscle development.

developmental biology↗

bioRxiv2024

Rbfox1 and mir-33 regulate pleiotropic roles of JAK/STAT signalling during adult myogenesis in Drosophila melanogaster

The JAK/STAT pathway is a conserved metazoan signal transduction system with roles in regulating growth, stem cells, and immune responses. Although its components are ubiquitously expressed during development, JAK/STAT signalling exerts cell-specific effects through mechanisms largely unknown. Here, we demonstrate that cell-specific effects of JAK/STAT signalling could be realised through post-transcriptional regulation. We show that JAK/STAT signalling in early adult myogenesis is critical for the patterning of the dorsal longitudinal muscles in Drosophila melanogaster. RNA-binding Fox protein 1 (Rbfox1) and the microRNA mir-33 regulate the transcript stability of Signal-transducer and activator of transcription protein at 92E (Stat92E), which encodes the sole Drosophila STAT orthologue. Moreover, Rbfox1 also controls the alternative splicing of Stat92E and its key downstream effector, Zn finger homeodomain 1 (zfh1). The coupling of Rbfox1 with Stat92E function, during early adult myogenesis, not only allows maintenance of stemness, but also mediates filamentous actin dynamics, and prevents apoptosis in myoblasts via some other Stat92E target genes. Given that the functions of the JAK/STAT pathway in cell proliferation and survival are conserved between Drosophila and vertebrates, our report presents a novel example for the context-dependant regulation of the developmental role of an important signalling pathway.

developmental biology↗

bioRxiv2024

Functional characterization of luciferase in a brittle star indicates parallel evolution influenced by genomic availability of haloalkane dehalogenase

Determining why convergent traits use distinct versus shared genetic components is crucial for understanding how evolutionary processes generate and sustain biodiversity. However, the factors dictating the genetic underpinnings of convergent traits remain incompletely understood. Here, we use heterologous protein expression, biochemical assays, and phylogenetic analyses to confirm the origin of a luciferase gene from haloalkane dehalogenases in the brittle star Amphiura filiformis. Through database searches and gene tree analyses, we also show a complex pattern of presence and absence of haloalkane dehalogenases across organismal genomes. These results first confirm parallel evolution across a vast phylogenetic distance, because octocorals like Renilla also use luciferase derived from haloalkane dehalogenases. This parallel evolution is surprising, even though previously hypothesized, because many organisms that also use coelenterazine as the bioluminescence substrate evolved completely distinct luciferases. The inability to detect haloalkane dehalogenases in the genomes of several bioluminescent groups suggests that the distribution of this gene family influences its recruitment as a luciferase. Together, our findings highlight how biochemical function and genomic availability help determine whether distinct or shared genetic components are used during the convergent evolution of traits like bioluminescence.

evolutionary biology↗

bioRxiv2024

AlphaMut: a deep reinforcement learning model to suggest helix-disrupting mutations

1Helices are important secondary structural motifs within proteins and are pivotal in numerous physiological processes. While amino acids (AA) such as alanine and leucine are known to promote helix formation, proline and glycine disfavor it. Helical structure formation, however, also depends on its environment, and hence, prior prediction of a mutational effect on a helical structure is difficult. Here, we employ a reinforcement learning algorithm to develop a predictive model for helix-disrupting mutations. We start with a toy model consisting of helices with only 30 AA and train different models. Our results show that only a few mutations lead to a drastic disruption of the target helix. We further extend our approach to helices in proteins and validate the results using rigorous free energy calculations. Our strategy identifies amino acids crucial for maintaining structural integrity and predicts key mutations that could alter protein function. Through our work, we present a new use case for reinforcement learning in protein structure disruption.

bioinformatics↗

bioRxiv2024

Notch and LIM-homeodomain protein Arrowhead regulate each other in a feedback mechanism to play a role in wing and neuronal development in Drosophila

Notch pathway is an evolutionarily conserved signaling system that operates to influence an astonishing array of cell fate decisions in different developmental contexts. To identify novel effectors of Notch signaling, we analyzed the whole transcriptome of Drosophila wing and eye imaginal discs in which an activated form of Notch was overexpressed. A LIM homeodomain protein Arrowhead (Awh) was identified as a novel candidate which plays a crucial role in Notch mediated developmental events. Awh alleles show strong genetic interaction with Notch pathway components. Awh loss-of-function upregulates Notch targets Cut and Wingless. Awh gain-of-function downregulates Notch targets by reducing the expression of ligand, Delta. Consequently, the expression of Wingless effector molecule Armadillo and its downstream targets, Senseless and Vestigial, also gets downregulated. Awh overexpression leads to ectopicexpression of engrailed, a segment polarity gene in the anterior region of wing disc, leading to patterning defects. Additionally, Notch gain-of-function mediated neuronal defects get significantly rescued with Awh overexpression. Activated Notch inhibits Awh activity, suggesting a regulatory loop between Awh and Notch. Additionally, the defects caused by Awh gain-of-function were remarkably rescued by Chip, a LIM interaction domain containing transcriptional co-factor. The present study highlights the novel feedback regulation between Awh and Notch.

developmental biology↗

bioRxiv2024

A conserved cell-type gradient across the human mediodorsal and paraventricular thalamus

The mediodorsal thalamus (MD) and adjacent midline nuclei are important for cognition and mental illness, but their cellular composition is not well defined. Using single-nucleus and spatial transcriptomics, we identified a conserved excitatory neuron gradient, with distinct spatial mapping of individual clusters. One end of the gradient was expanded in human MD compared to mice, which may be related to the expansion of granular prefrontal cortex in hominids. Moreover, neurons preferentially mapping onto the parvocellular division MD were associated with genetic risk for schizophrenia and bipolar disorder. Midbrain-derived inhibitory interneurons were enriched in human MD and implicated in genetic risk for major depressive disorder.

neuroscience↗

bioRxiv2024

Dynamic and decay kinetics of H3 variants in live cells reveal the pivotal role of HIRA/NSD2 in maintaining the distinct H3.3 specific chromatin landscape

The incorporation of variant histone H3.3 into the genome is tightly linked with transcriptional activity, yet its precise regulatory mechanisms remain elusive. Traditional methods like Chromatin Immunoprecipitation offer static views of H3.3 distribution, lacking dynamic insights. Here, using the SNAP tag system, we employed Fluorescence Recovery After Photobleaching (FRAP) and live-cell imaging to investigate H3.3 mobility and decay kinetics in live mouse embryonic fibroblast cells. Our focus on interferon-induced transcriptional activation revealed rapid H3.3 exchange, indicative of its transcriptional regulatory role. Transcription inhibition hindered H3.3 mobility, emphasizing its involvement in transcription. Additionally, we probed into turnover dynamics(decay) of H3.1-SNAP and H3.3-SNAP variants, uncovering differential decay rates influenced by transcriptional activity and histone modifiers such as NSD2 and HIRA. Live-cell imaging showed faster decay of H3.3 compared to H3.1, further exacerbated upon NSD2/HIRA loss. Notably, HIRA and NSD2, regulators of H3.3 dynamics, proved crucial for both H3.3 mobility and decay, underscoring their pivotal role. These findings deepen our understanding of epigenetic regulation, emphasizing the dynamic nature of histone turnover in cellular function and its implications for disease pathogenesis. Taken together, this study sheds light on the dynamic behavior of H3.3 and its regulatory mechanisms, providing valuable insights into epigenetic regulation in cellular processes and disease contexts.

cell biology↗

bioRxiv2024

Impact of Species-Specific Plant Growth-Promoting Rhizobacteria (PGPR) on Maize (Zea mays) Phenotypic and Biochemical Diversity

Maize (Zea mays) is a vital cereal crop used as a staple diet in many countries. In contemporary farming practises, inoculation with plant-growth-promoting rhizobacteria (PGPR) can be promoted as a sustainable alternative to chemical fertilizers and pesticides in cereal crops including maize. For experimental verification of the above-mentioned hypothesis, four PGPR inoculants Pseudomonas fluorescens, P. putida, Azospirillum lipoferum and Bacillus subtilis were applied to three maize genotypes (AXE*, PMH1 and PMH10) and their effects were studied by measurement of various morphological and biochemical paramters. Substantial increase in the amount of chlorophyll a (45%), chlorophyll b (100%), total chlorophyll (95%), nitrate reductase (22%), superoxide dismutase (52%), protein content (16%), methionine content (31.8%), grain protein content (69%) were recorded over the control (non-treated or mock) plants. Morphological parameters also exhibited an increase in leaf number (53%), fresh weight (41%) and dry weight (62%) in test plants. Comparable outcome was observed for whole cob weight showing an increase of 42%, cob weight (60%), hundred-grain weight (25.9%), maize grain yield m-2 (18%) and yield ha-1 (18%) over the control. This study highlights the efficacy of the aforementioned four PGPR species as the most effective for maize crops. Bacillus subtilis and Azospirillum lipoferum may be considered species-specific PGPRs due to their superior performance compared to other strains. The considerable growth-promoting advantages observed in maize plants treated with bacterial inoculation indicated that PGPRs have the potential to be employed as sustainable solutions in maize production.

plant biology↗

bioRxiv2024

A fine kinetic balance of interactions directs transcription factor hubs to genes

Eukaryotic gene regulation relies on the binding of sequence-specific transcription factors (TFs). TFs bind chromatin transiently yet occupy their target sites by forming high-local concentration microenvironments (hubs and condensates) that increase the frequency of binding. Despite their ubiquity, such microenvironments are difficult to study in endogenous contexts due to technical limitations. Here, we use live embryo light-sheet imaging, single-molecule tracking, and genomics to overcome these limitations and investigate how hubs are localized to target genes to drive TF occupancy and transcription. By examining mutants of a hub-forming TF, Zelda, in Drosophila embryos, we find that hub formation propensity, spatial distributions, and temporal stabilities are differentially regulated by DNA binding and disordered protein domains. We show that hub localization to genomic targets is driven by a finely-tuned kinetic balance of interactions between proteins and chromatin, and hubs can be redirected to new genomic sites when this balance is perturbed.

molecular biology↗

bioRxiv2024

Non-Electrostatic Basis for an Artificial Metalloenzyme Catalysis

The artificial metalloenzyme (referred to as Ir-Q) reported by Hartwig and coworkers presented an important milestone in merging the extraordinary efficiency of biocatalyst with the versatility of small molecule chemical catalyst in catalyzing a new-to-nature carbene insertion reaction. The artificial enzyme results from formal replacement of the Fe by an Ir(Me) moiety along with four C317G, T213G, L69V, V254L mutations in a natural Cytochrome enzyme CYP119 by directed evolution method. Importantly, this is a show-stopper enzyme as it exhibits a catalytic rate enhancement similar to that of natural enzymes. Despite this remarkable discovery, there is no mechanistic understanding as to why it displays extraordinary efficiency, so far been intractable to experimental methods. In this study, we have deciphered the catalytically active conformation of Ir-Q using large-scale molecular dynamics simulations and rigorous quantum chemical calculations. Our study reveals how directed evolution mutations precisely position the cofactor-substrate in an unusual orientation within a reshaped active site that emerged during evolution and fostered by C-H...{pi} interactions from more ordered mutated L69V and V254L residues. This productive conformation correctly reproduces the experimental barrier height and the catalytic effect of 2.7 kcal/mol, in excellent agreement with observed rate enhancement. Moreover, the active conformation features an unprecedented bonding interaction in a metal-carbene species that preferentially stabilizes the rate determining formation of an Iridium-Porphyrin Carbene intermediate to render the observed high catalytic rate acceleration. While the electrostatic criteria are widely established, this study suggests a new design paradigm towards realization of fully programmable protein catalysis.

biophysics↗

bioRxiv2024

A dual-gene reporter-amplifier architecture for enhancing the sensitivity of molecular MRI by water exchange

The development of genetic reporters for magnetic resonance imaging (MRI) is essential for investigating biological functions in intact animals. However, current MRI reporters have low sensitivity, making it challenging to create significant contrast against the tissue background, especially when only a small percentage of cells express the reporter. To overcome this limitation, we developed an approach that amplifies signals by co-expressing an MRI reporter gene, Oatp1b3, with a water channel, aquaporin-1 (Aqp1). We first show that the expression of Aqp1 amplifies the paramagnetic relaxation effect of Oatp1b3 by facilitating transmembrane water exchange. This mechanism provides Oatp1b3-expressing cells with access to a larger water pool compared with typical exchange-limited conditions. We further demonstrated that our methodology allows dual-labeled cells to be detected using approximately 10-fold lower concentrations of contrast agent than that in the Aqp1-free scenario. Finally, we show that our approach enables the imaging of mixed-cell populations containing a low fraction of Oatp1b3-labeled cells that are otherwise undetectable based on Oatp1b3 expression alone.

bioengineering↗

bioRxiv2024

Exploring the potential of water channels for developing MRI reporters and sensors without the need for exogenous contrast agents

Genetically encoded reporters for magnetic resonance imaging (MRI) offer a valuable technology for making molecular-scale measurements of biological processes within living organisms with high anatomical resolution and whole-organ coverage without relying on ionizing radiation. However, most MRI reporters rely on contrast agents, typically paramagnetic metals and metal complexes, which often need to be supplemented exogenously to create optimal contrast. To eliminate the need for contrast agents, we previously introduced aquaporin-1, a mammalian water channel, as a new reporter gene for the fully autonomous detection of genetically labeled cells using diffusion-weighted MRI. In this study, we aimed to expand the toolbox of diffusion-based genetic reporters by modulating aquaporin membrane trafficking and harnessing the evolutionary diversity of water channels across species. We identified a number of new water channels that functioned as diffusion-weighted reporter genes. In addition, we show that loss-of-function variants of yeast and human aquaporins can be leveraged to design first-in-class diffusion-based sensors for detecting the activity of a model protease within living cells.

bioengineering↗