Biology subjects

Banerjee, A.

Publications and source records attributed to Banerjee, A..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Accurate Identification and Mechanistic Evaluation of Pathogenic Missense Variants with Rhapsody-2

Understanding the effects of missense mutations or single amino acid variants (SAVs) on protein function is crucial for elucidating the molecular basis of diseases/disorders and designing rational therapies. We introduce here Rhapsody-2, a machine learning tool for discriminating pathogenic and neutral SAVs, significantly expanding on a precursor limited by the availability of structural data. With the advent of AlphaFold2 as a powerful tool for structure prediction, Rhapsody-2 is trained on a significantly expanded dataset of 117,525 SAVs corresponding to 12,094 human proteins reported in the ClinVar database. Adopting a broad set of descriptors composed of sequence evolutionary, structural, dynamic, and energetics features in the training algorithm, Rhapsody-2 achieved an AUROC of 0.94 in 10-fold cross-validation when all SAVs of a particular test protein (mutant) were excluded from the training set. Benchmarking against a variety of testing datasets demonstrated the high performance of Rhapsody-2. While sequence evolutionary descriptors play a dominant role in pathogenicity prediction, those based on structural dynamics provide a mechanistic interpretation. Notably, residues involved in allosteric communication, and those distinguished by pronounced fluctuations in the high frequency modes of motion or subject to spatial constraints in soft modes usually give rise to pathogenicity when mutated. Overall, Rhapsody-2 provides an efficient and transparent tool for accurately predicting the pathogenicity of SAVs and unraveling the mechanistic basis of the observed behavior, thus advancing our understanding of genotype-to-phenotype relations. Significance StatementUnderstanding the impact of single amino acid variants (SAVs) on protein function is pivotal for detecting and treating genetic disorders. We present Rhapsody-2, a machine learning tool that leverages AlphaFold2-predicted protein structures to accurately predict the pathogenicity of SAVs. By integrating sequence, structure, and dynamics properties, Rhapsody-2 achieves a high performance in distinguishing pathogenic variants across multiple benchmark datasets. Our tool not only provides a robust framework for predicting the impact of SAVs but also allows for mechanistic interpretability of pathogenicity, offering a valuable resource for understanding genotype-to-phenotype relationships toward assisting in precision medicine.

bioinformatics↗

bioRxiv2025

Actin contractility and endocytosis create apico-basal tension gradient in HeLa cells

The functions of the plasma membrane of a cell are coupled to its mechanical state. To understand the players that contribute to the tight regulation of membrane mechanics with its local heterogeneities, the tension of the cell membrane requires to be mapped out from the basal to the apical membrane. In this work, optical-trap-based tension measurements performed at two axial (z) planes per cell reveal an apico-basal gradient, that is understood better on comparison with basal membrane mechanics and membrane compaction measurements by a combination of high resolution microscopy (IRM) and fluorescence lifetime (Flipper-TR) measurements, respectively. While the apico-basal gradient in the apparent tension (low to high) was found to depend on an intact actin cytoskeleton, analysing the colocalization of the cortical actin with the motor protein myosin and the actin-membrane linker ezrin suggested that cytoskeleton contractility is a major determinant of the patterned tension. Tension-sensitive lifetime of Flipper-TR, validated the gradient but failed to catch the dependence on actin due to the dual and opposite effects of tension on lifetime. Finally, measurements utilizing fluorescently tagged transferrin (Tf) demonstrated that the functional state of the membrane also showed similar height dependence. Thus, planes where tension maximized showed a peak in accumulation of Tf colocalizing with clathrin indicative of the presence of more pits. This enabled us to demonstrate that cells exist in tightly regulated patterned tension states that is coupled to their cytoskeleton as well membrane functions like endocytosis. Significance StatementThe cell membrane is protective but also highly dynamic - performing a diverse set of functions usually via membrane shape changes. These functions affect membrane mechanics but are also mechano-regulated. Mapping the state of cell surface mechanics or tension of live cells is therefore of fundamental importance. Interestingly, recent works have suggested that membrane tension can be non-uniform. However, no single tension-measuring technique can truly reach every part of the cell. Our work addresses this issue where we map tension in live HeLa cells using multifarious approaches including the measurement of tether forces, membrane fluctuations, and lipid compaction. We show that at the top/apical surface, the membrane-tension is lower than that at the midplane. The observed gradient resembles that of the state of endocytosis and controlled acto-myosin contractility.

biophysics↗

bioRxiv2025

An evolutionarily conserved mechanism for reformatting odor concentration in early olfactory circuits

Understanding how stimuli from the sensory periphery are progressively reformatted to yield useful representations is a fundamental challenge in neuroscience. In olfaction, assessing odor concentration is key for many behaviors, such as tracking and navigation. Initially, as odor concentration increases, the average response of first-order sensory neurons also increases. However, the average response of second-order neurons remains flat with increasing concentration - a transformation that is believed to help with concentration-invariant odor identification, but that seemingly discards concentration information before it could be sent to higher brain regions. By combining neural data analyses from diverse species with computational modeling, we propose strategies by which second-order neurons preserve concentration information, despite flat mean responses at the population level. We find that individual second-order neurons have diverse concentration response curves that are unique to each odorant -- some neurons respond more with higher concentration and others respond less -- and together this diversity generates distinct combinatorial representations for different concentrations. We show that this encoding scheme can be recapitulated using a circuit computation, called divisive normalization, and we derive sufficient conditions for this diversity to emerge. We then discuss two mechanisms (spike rate vs. timing based) by which higher order brain regions may decode odor concentration from the reformatted representations. Since vertebrate and invertebrate olfactory systems likely evolved independently, our findings suggest that evolution converged on similar algorithmic solutions despite stark differences in neural circuit architectures. Finally, in land vertebrates a parallel olfactory pathway has evolved whose second-order neurons do not exhibit such diverse response curves; rather neurons in this pathway represent concentration information in a more monotonic fashion on average, potentially allowing for easier odor localization and identification at the expense of increased energy use.

neuroscience↗

bioRxiv2025

Pathobiont-triggered induction of epithelial IDO1 drives regional susceptibility to Inflammatory Bowel Disease

The structure and function of the mammalian gut vary by region, yet why inflammatory diseases manifest in specific regions and not others remains unclear. We use a TNF-overexpressing Crohns disease (CD) model (Tnf{Delta}ARE/+), which typically presents in the terminal ileum (TI), to investigate how environmental factors interact with the hosts immune susceptibility to drive region-specific disease. We identified Chlamydia muridarum, an intracellular bacterium and murine counterpart to the human sexually transmitted C. trachomatis, as necessary and sufficient to trigger disease manifestation in the ascending colon (AC), another common site of human CD. Disease manifestation in the AC depends on indoleamine 2,3-dioxygenase (IDO1) activity induced by hypersensitive surface secretory cells in genetically susceptible hosts. Single-cell and microbial analyses of human specimens also implicates this pathobiont-epithelial IDO1 pathway in patients with a history of CD in the AC. Our findings demonstrate that genetic and microbial factors can independently drive region-specific disease and provide a unique model to study CD specific to the AC.

cell biology↗

bioRxiv2024

High-speed 10-plex DNA-PAINT with a larger sequence repertoire

DNA-Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) enables multiplexed super-resolution imaging of biological samples. We expand the repertoire of speed-optimized DNA sequences for DNA-PAINT imaging to drive visualization of as many as twelve targets in a sequential manner with molecular resolution. By implementing Exchange-PAINT protocol, we demonstrate 12-plex super-resolved imaging of docking strand patterned DNA origami nanostructures within four hours with a localization precision of 3 to 5 nm. Using these sequences, we demonstrate 9-plex super-resolution imaging of diverse nuclear targets within four hours. Further, we present a comprehensive analysis pipeline to quantify nanoscale chromatin in single cells. The combination of multiplexed imaging and analysis pipeline enabled us to reveal the loss of chromatin contacts with nuclear speckles upon global transcription inhibition. This work highlights the versatility of our approach to simultaneously image multiple targets at accelerated speeds while maintaining precise spatial localization for each target, enabling in depth mapping of the nuclear landscape. These speed-optimized imager sequences for high multiplexed super-resolution imaging will drive its further adoption for diverse cellular imaging applications.

biophysics↗

bioRxiv2024

Time restricted feeding with or without ketosis influences metabolism-related gene expression in a tissue-specific manner in aged rats

Many of the hallmarks of aging involve alterations in cellular and organismal metabolism. One pathway with the potential to impact several traditional markers of impaired function with aging is the PI3K/AKT metabolic pathway. Regulation of this pathway includes many aspects of cellular function, including protein synthesis, proliferation and survival, as well as many downstream targets, including mTOR and FOXOs. Importantly, this pathway is pivotal to the function of every organ system in the human body. Thus, we investigated the expression of several genes along this pathway in multiple organs, including the brain, liver and skeletal muscle, in aged subjects that had been on different experimental diets to regulate metabolic function since mid-life. Specifically, rats were fed a control ad lib diet (AL), a time restricted feeding diet (cTRF), or a time restricted feeding diet with ketogenic macronutrients (kTRF) for the majority of their adult lives (from 8-25 months). We previously reported that regardless of macronutrient ratio, TRF-fed rats in both macronutrient groups required significantly less training to acquire a biconditional association task than their ad lib fed counterparts. The current experiments expand on this work by quantifying metabolism-related gene expression across tissues and interrogating for potential relationships with cognitive performance. AKT expression was significantly reduced in kTRF fed rats within liver and muscle tissue. However, AKT expression within the perirhinal cortex (PER) was higher in kTRF rats with the best cognitive performance. Within CA3, higher levels of FOXO1 gene expression correlated with poorer cognitive performance in ad libitum fed rats. Together, these data demonstrate diet- and tissue-specific alterations in metabolism-related gene expression and their correlation with cognitive status.

neuroscience↗

bioRxiv2024

Mycobacterial methionine aminopeptidase type 1c moonlights as an anti-association factor on the 30S ribosomal subunit

Methionine aminopeptidase (MetAP), an essential metalloprotease, binds the 70S ribosome near the exit site of peptide tunnel and cleaves the N-terminal methionine from the nascent polypeptide chains. While E. coli expresses a single subclass of type1 MetAP, mycobacteria possesses two subclasses, namely, MetAP1a and MetAP1c, differentiated by the presence of a 40 amino acid long N-terminal tether in the later. Here we reveal a yet unknown moonlighting role of MetAP1c in mycobacteria. We observe stationary phase-specific enhancement of expression and a unique 30S subunit affinity of MetAP1c. A 5[A] cryo-EM map of Mycobacterium smegmatis (Msm) MetAP1c-30S subunit complex manifests for the first time the binding of MetAP1c at the inter-subunit side in the vicinity of proteins S13 and S19. MetAP1c binding and resulting conformational changes make the 30S subunit unfit for association with the 50S subunit, thereby conferring an anti-association property on MetAP1c. We further constructed two N-terminal mutants to establish the pivotal role of the N-terminal extension of MetAP1c in its moonlighting function.

biochemistry↗

bioRxiv2024

Signatures of glassy dynamics in highly ordered lipid bilayers with emergence of soft dynamic channels

Over the last few decades, extensive investigations on spatial and dynamic heterogeneity have been performed on carefully reconstituted biological lipid membranes. Characterising the molecular features in heterogeneous membranes is extremely challenging due to experimentally inaccessible time- and length-scales of these emergent systems. In this context, simulations can provide important insights into molecular-level interactions leading to membrane heterogeneity and associated functions. To that end, we use the non-affine displacement (NAD) framework (a concept borrowed from Physics of granular material) to faithfully capture molecular-scale local membrane order in simulated heterogeneous bilayers. In our latest application of NAD, we investigate the temperature-dependent spatial and temporal organisation on microseconds trajectories of liquid-ordered bilayer systems at all-atom resolution (DPPC/DOPC/CHOL: 0.55:0.15:0.30; 40 nm x 40 nm with a total of 5600 lipids and 2 million atoms). Lateral organisation in these large bilayer patches show noticeable dynamic heterogeneity despite their liquid-ordered nature. Moreover, our NAD analyses reveal soft fluid channels within the tightly packed membrane reminiscent of the classical two-component Kob-Andersen glass-forming binary mixture. Hence, we characterised these systems using classical glass physics markers for dynamic heterogeneities such as Overlap, Four-point Susceptibility, van Hove, and Intermediate Scattering functions to quantify the multiple time scales underlying the lipid dynamics. Our analyses reveal that highly ordered membrane systems can have glass-like dynamics with distinct soft fluid channels inside them. Biologically, these dynamic channels could act as conduits for facilitating molecular encounters for biological functions even in highly ordered phases such as lipid nanodomains and rafts. SignificanceLiquid-ordered lipid membrane offers a reliable representation of the cholesterolrich outer leaflet of the plasma membrane. We show that dynamic heterogeneity is present even in highly ordered phases of lipid membrane, which arise at equilibrium merely from thermal fluctuations. Membranes in this state act as strong glass formers, and are hence less susceptible to temperature fluctuations. The dynamics is faster than actin or proteinmediated reorganisation and presence of passive but dynamic "soft channels" amidst the highly ordered lipid environment suggests a new exciting possibility that these channels could act as fast conduits for molecular encounters on the robust membrane surface.

biophysics↗

bioRxiv2024

Design and synthesis of triazine-based hydrogel for combined targeted doxorubicin delivery and PI3Kinhibition

Melanoma, an aggressive skin cancer originating from melanocytes, presents substantial challenges due to its high metastatic potential and resistance to conventional therapies. Hydrogels, three-dimensional networks of hydrophilic polymers with high water-retention capacities, offer significant promise for controlled drug delivery applications. In this study, we report the synthesis and characterization of hydrogelators based on the triazine molecular scaffold, which self-assemble into fibrous networks conducive to hydrogel formation. Rheological analysis confirmed their hydrogelation properties, while microscopic techniques including FE-SEM and FEG-TEM provided insights into their morphological networks. The drug delivery capability of these hydrogelators was evaluated using doxorubicin, a widely employed anticancer agent, demonstrating enhanced biocompatibility and reduced side effects compared to free doxorubicin. Additionally, the hydrogelators exhibited inhibitory activity against phosphoinositide 3-kinase (PI3K), a key enzyme frequently mutated in cancer, and also involved in melanoma progression. The dual functionality of this delivery system - controlled drug release and PI3K inhibition - highlights the potential of triazine-based hydrogelators as innovative therapeutic platforms for melanoma treatment.

cancer biology↗

bioRxiv2024

Viral susceptibility and innate immune competency of Carollia perspicillata bat cells produced for virological studies

Multiple viruses that are highly pathogenic in humans are known to have evolved in bats. How bats tolerate infection with these viruses, however, is poorly understood. As viruses engage in a wide range of interactions with their hosts, it is essential to study bat viruses in a system that resembles their natural environment like bat-derived in vitro cellular models. However, stable and accessible bat cell lines are not widely available for the broader scientific community. Here, we generated in vitro reagents for the Sebas short-tailed bat (Carollia perspicillata), tested multiple methods of immortalization, and characterized their susceptibility to virus infection and response to immune stimulation. Using a pseudotyped virus library and authentic virus infections, we show that these C. perspicillata cell lines derived from a diverse array of tissues are susceptible to viruses bearing the glycoprotein of numerous orthohantaviruses, including Andes and Hantaan virus and are also susceptible to live hantavirus infection. Furthermore, stimulation with synthetic double-stranded RNA prior to infection with VSV and MERS-CoV induced a protective antiviral response, demonstrating the suitability of our cell lines to study the bat antiviral immune response. Taken together, the approaches outlined here will inform future efforts to develop in vitro tools for virology from non-model organisms and these C. perspicillata cell lines will enable studies on virus-host interactions in bats.

microbiology↗

bioRxiv2024

Modeling brain signaling as Markovian helps explain its structure-function relationship

Structure determines function. However, this universal theme in biology has been surprisingly difficult to observe in human brain neuroimaging data. Here, we link structure to function by hypothesizing that brain signals propagate as a Markovian process on an underlying structure. We focus on a metric called commute time: the average number of steps for a random walker to go from region A to B and then back to A. Commute times based on white matter tracts from diffusion MRI exhibit an average {+/-} standard deviation Spearman correlation of -0.26 {+/-} 0.08 with functional MRI connectivity data across 434 UK Biobank individuals and -0.24 {+/-} 0.06 across 400 HCP Young Adult brain scans. The correlation increases to -0.36 {+/-} 0.14 and to -0.32 {+/-} 0.12 when the principal contributions of both commute time and functional connectivity are compared for both datasets. The correlations are stronger by 33% compared to broadly used communication measures such as search information and communicability. The difference further widens to a factor of 5 when commute times are correlated to the principal mode of functional connectivity from its eigenvalue decomposition. Overall, the study points to the utility of commute time to account for the role of polysynaptic (indirect) connectivity underlying brain function by assuming that signals randomly traverse along the underlying brain structure.

neuroscience↗

bioRxiv2024

Hierarchy of prediction errors shapes the learning of context-dependent sensory representations

How sensory information is interpreted depends on context, yet the neural mechanisms by which context shapes sensory processing remain poorly understood. To address this question, we developed a computational model constrained by in vivo functional imaging of cortical neurons in mice during reversal learning of a tactile sensory discrimination task. During learning, layer 2/3 somatosensory neurons enhanced their response to reward-predictive stimuli. The model accounted for these observations through selective top-down gain amplification of apical dendritic inputs, accompanied by reduced reward-prediction errors and increased confidence in outcome predictions. Upon rule-reversal, the lateral orbitofrontal cortex, through disinhibitory VIP interneurons, encoded a context-prediction error signaling a loss of confidence. The hierarchy of reward- and context-prediction errors across cortical areas is mirrored in top-down signals modulating apical activity of simulated pyramidal neurons in the primary sensory cortex. The model explains how contextual changes are detected and how reward- and context-prediction error signals, originating in different cortical regions, interact to reshape the sensory representation.

neuroscience↗

bioRxiv2024

Selective expansion of motor cortical projections in the evolution of vocal novelty

Deciphering how cortical architecture evolves to drive behavioral innovations is a long-standing challenge in neuroscience and evolutionary biology. Here, we leverage a striking behavioral novelty in the Alstons singing mouse (Scotinomys teguina), compared to the laboratory mouse (Mus musculus), to quantitatively test models of motor cortical evolution. We used bulk tracing, serial two-photon tomography, and high-throughput DNA sequencing of over 76,000 barcoded neurons to discover a specific and substantial expansion ([~]200%) of orofacial motor cortical (OMC) projections to the auditory cortical region (AudR) and the midbrain periaqueductal gray (PAG), both implicated in vocal behaviors. Moreover, analysis of individual OMC neurons projection motifs revealed preferential expansion of exclusive projections to AudR. Our results imply that selective expansion of ancestral motor cortical projections can underlie behavioral divergence over short evolutionary timescales, suggesting potential mechanisms for the evolution of enhanced cortical control over vocalizations--a crucial preadaptation for human language.

neuroscience↗

bioRxiv2024

Mitochondrial metabolism is a key determinant of chemotherapy sensitivity in Colorectal Cancer

Therapy resistance is attributed to over 80% of cancer deaths per year emphasizing the urgent need to overcome this challenge for improved patient outcomes. Despite its widespread use in colorectal cancer (CRC) treatment, resistance to 5-fluorouracil (5FU) remains poorly understood. Here, we investigate the transcriptional responses of CRC cells to 5FU treatment, revealing significant metabolic reprogramming towards heightened mitochondrial activity. Utilizing CRC models, we demonstrate sustained enhancement of mitochondrial biogenesis and function following 5FU treatment, leading to resistance in both in vitro and in vivo settings. Furthermore, we show that targeting mitochondrial metabolism, specifically by inhibiting Complex I (CI), sensitizes CRC cells to 5FU, resulting in delayed tumour growth and prolonged survival in preclinical models. Additionally, our analysis of patient data suggests that oxidative metabolism signatures may predict responses to 5FU-based chemotherapy. These findings shed light on mechanisms underlying 5FU resistance and propose a rational strategy for combination therapy in CRC, emphasizing the potential clinical benefit of targeting mitochondrial metabolism to overcome resistance and enhance patient outcomes.

cancer biology↗

bioRxiv2024

Childhood brain morphometry in children with persistent stunting and catch-up growth

BackgroundEarly childhood stunting affects around 150 million young children worldwide and leads to suboptimal human potential in later life. However, there is limited data on the effects of early childhood stunting and catch-up growth on brain morphometry. MethodsWe evaluated childhood brain volumes at nine years of age in a community-based birth-cohort follow-up study in Vellore, south India among four groups based on anthropometric assessments at two, five, and nine years namely Never Stunted (NS), Stunted at two years and caught up by five years (S2N5), Stunted at two and five years and caught up by nine years (S2N9), and Always Stunted (AS). T1-weighted magnetic resonance imaging (MRI) images were acquired using a 3T MRI scanner, and brain volumes were quantified using FreeSurfer software. FindingsAmongst 251 children from the overall cohort, 178 children with a mean age of 9.54 were considered for further analysis. The total brain volume, subcortical volume, bilateral cerebellar white matter, and posterior corpus callosum showed a declining trend from NS to AS. Regional cortical brain analysis showed significant lower bilateral lateral occipital volumes, right pallidum, bilateral caudate, and right thalamus volumes between NS and AS. InterpretationTo the best of our knowledge, this first neuroimaging analysis to investigate the effects of persistent childhood stunting and catch-up growth on brain volumetry indicates impairment at different brain levels involving total brain and subcortical volumes, networking/connecting centres (thalamus, basal ganglia, callosum, cerebellum) and visual processing area of lateral occipital cortex.

neuroscience↗

bioRxiv2024

Structure-function relationship of PE11 esterase of Mycobacterium tuberculosis with respect to its role in virulence

The lipolytic enzymes of Mycobacterium tuberculosis play a critical role in immunomodulation and virulence. Among these proteins, PE11 which also belongs to the PE/PPE family, is the smallest ([~]10.8 kDa) and play a significant role in cell wall remodelling and virulence. PE11 is established to be an esterase, but its enzymatic and structural properties are not yet characterized. In this study, using homology modelling we deduced the putative structure which shows the presence of both -helix and {beta}-sheet structures which is in close agreement with that observed by CD spectra of the purified protein. PE11 was found to contain a GX3SX4G motif homologous to canonical GxSxG motif present in many serin hydrolases. The catalytic triad appears to be located within this motif as substitution of Serine26 and Glycine31 residues abrogated its enzymatic activity. Gel-filtration chromatography data indicate that PE11 possibly exists as dimer and tetramer showing positive cooperativity for binding its substrates. In addition, PE11 esterase activity was found to be critical for cell wall remodelling, antibiotic resistance and conferring survival advantages to M. tuberculosis. Our data suggest that PE11 can be targeted for designing potential therapeutic strategies.

microbiology↗

bioRxiv2024

Age associated alterations in the cortical representation of cardiac signals: A heartfelt tale

The brain continuously tracks signals from the body, including the heartbeat, which influence internal states such as the perception of time, well-known to accelerate with age and disrupted in disorders such as Parkinsons disease and dementia. The present study examined whether the representation of cardiac events onto spontaneous neural oscillations maps to regions responsible for processing time-perception, show age-related differences, and uncovered the mechanisms that may underlie any such reorganization. From a large cohort (N = 620), cortical heartbeat-evoked responses (HERs) were characterized and their sources were pinned to frontotemporal regions. Phase-based analyses demonstrated that cardiac signals affect phase of ongoing neural oscillations in the theta frequency band, rather than altering overall power, with this effect consolidating in older adults. Advanced statistical analysis further indicated that these changes are primarily driven by enhanced bottom-up heart-to-brain influences, revealing that altered interoceptive signaling can shape the age-related changes in time perception.

neuroscience↗

bioRxiv2024

Hemispheric specialization of functions are tuned by conduction velocities of neuronal propagation in large-scale brain networks

Analogous to the notion of pleiotropy where one gene can influence two or three unrelated traits, shared structural connectome encompassing both hemispheres of human brain gets functionally segregated along left vs right hemispheres during speech and melody processing, respectively. Here, the underlying neural mechanism is explored with a combined empirical and computational approach wherein, context-specific causal outflow of information emerging from primary auditory cortices (PAC) is shown to be guiding the hemispheric specialization of speech and melody processing while human volunteers listened to naturalistic music while their electroencephalogram (EEG) were recorded. Using a detailed whole-brain connectome model guided by diffusion MRI, individual-specific functional lateralization were predicted at cortical sources representing an extended auditory processing network. High levels of accuracy in prediction can be achieved after optimizing conduction velocity - controller of transmission delays of neural information processing. Thus, a tuning-by-delay mechanism is presented, which when triggered by the spectro-temporal complexity of the task context, guides the geometry of lateralization.

neuroscience↗