Biology subjects

Banerjee, S.

Publications and source records attributed to Banerjee, S..

At least 163 records · Page 9Linked to original sources

bioRxiv2023

CRISPR based programmable RNA editing in primary neurons

Investigating the RNA regulation landscape primarily relies on our understanding of how RNA-protein interactions are governed in various cell types, including neurons. Analysis of RNA-protein interactions in physiological environments warrants the development of new tools that rely on RNA manipulation. Recently, A CRISPR-based RNA-editing tool (dCas13b-ADAR2DD) was developed to mitigate disease associated point mutations in cell lines. Here, we have explored the targeted sequence editing potential of the tool (dCas13b-ADAR2DD system) by adapting it to manipulate RNA function with an aim to visualize RNA editing in primary hippocampal neurons. This is a two-component system that includes a programmable guide RNA (gRNA) complementary to the target RNA, and a catalytically dead version of the Cas13b enzyme fused to ADAR. The RNA editing protocol outlined in this manuscript relies on using the gRNA-dependent targeting of dCas13b-ADAR fusion protein to the mutant form of mDendra transcript. We first abrogated the fluorescence of Dendra2 by introducing a nonsense mutation that precludes the formation of the functional protein. To visualize the efficacy of the RNA editing in neurons, we used the dCas13b-ADAR2DD system to edit specific nucleotides within the Dendra2 mRNA to restore the amino acid codes critical for Dendra2 fluorescence. This method therefore lays the foundation to future studies on the dynamicity of activity-induced RNA-protein interactions in neurons and can be extended to manipulate the endogenous RNome in diverse neuronal subtypes. Furthermore, this methodology will enable investigators to visualize the spatial and temporal resolution of RNA-protein interactions without altering the genomes via conventional methods. HighlightsO_LICRISPR-Cas13 based application that enables site specific A-to-I in target RNAs directed by gRNA; optimized in neurons. C_LIO_LIEnables the temporal mapping of developmentally relevant RNAs and their cis-interacting RNA binding proteins. C_LI

neuroscience↗

bioRxiv2023

A novel role of macrophage PIST protein in regulating Leishmania major infection

PDZ protein interacting specifically with Tc10 or PIST is a mammalian trans-Golgi resident protein that regulates subcellular sorting of plasma membrane receptors. PIST has recently been found to play an important role in regulating viral pathogenesis. Alteration in PIST expression is linked to the reprogramming of cell surface receptors which is crucial in determining herpes simplex virus1 infection. In this context, PIST is crucial in triggering autophagy via Beclin 1 -PI3KC3 pathway. However, there is complete lack in our knowledge on the role of this protein in any parasitic infection. Leishmania parasites infect their host macrophage cells via phagocytosis and their survival within the parasitophorous compartment has recently been found to be dependent on host autophagy by a yet to be identified mechanism. Using Leishmania major (L. major)-macrophage infection model system we, for the first-time report here that in infected macrophages Golgi resident PIST protein migrates towards the parasite containing compartment. We have also found that PIST associates with Beclin 1, however, not with LC3 within L. major parasite containing compartment of infected macrophages. Further, we performed genetic ablation of PIST by siRNA and observed that knockdown of macrophage PIST in turn helps in parasite replication. Contrary to this, overexpression of PIST in macrophages restricted the multiplication of L. major. Collectively, our study for the first time reveals that PIST is essential in regulating intracellular parasite, L. major infection within macrophage cells. Summary StatementMammalian PIST protein plays a crucial role in regulating cellular trafficking events. Here, we showed that PIST status is altered within Leishmania major parasite infected macrophages. Further, we confirmed that PIST is essential in restricting parasite growth. Additionally, a potential interacting axis between PIST and Beclin 1 is identified during infection.

cell biology↗

bioRxiv2023

Colocalization of Cancer Associated Biomarkers on Single Extracellular Vesicles for Early Cancer Detection

Detection of cancer early, when it is most treatable, remains a significant challenge due to the lack of diagnostic methods sufficiently sensitive to detect nascent tumors. Early-stage tumors are small relative to their tissue of origin, heterogeneous, and infrequently manifest in clinical symptoms. Detection of their presence is made more difficult by a lack of abundant tumor-specific indicators (i.e., protein biomarkers, circulating tumor DNA, etc.) that would enable detection using a non-invasive diagnostic assay. In addition, many benign conditions manifest in a similar manner, thus discriminating an early-stage cancerous lesion from a benign tumor can present additional challenges and result in unnecessary medical procedures. To overcome these obstacles, we have developed a liquid biopsy assay that interrogates circulating extracellular vesicles (EVs) to detect tumor-specific biomarkers colocalized on the surface of individual EVs. Extracellular vesicles from all cell types, including early-stage tumors, are known to be abundant in blood, are remarkably stable, and serve as a biopsy of their cell of origin. The detection of a colocalized combination of cancer associated biomarkers that provide tumor specificity on the surface of extracellular vesicles enables the discrimination of early- and late-stage cancer from non-malignant conditions.

cancer biology↗

bioRxiv2022

GOThresher: a program to remove annotation biases from protein function annotation datasets

MotivationAdvances in sequencing technologies have led to a surge in genomic data, although the functions of many gene products coded by these genes remain unknown. While in-depth, targeted experiments that determine the functions of these gene products are crucial and routinely performed, they fail to keep up with the inflow of novel genomic data. In an attempt to address this gap, high-throughput experiments are being conducted in which a large number of genes are investigated in a single study. The annotations generated as a result of these experiments are generally biased towards a small subset of less informative Gene Ontology (GO) terms. Identifying and removing biases from protein function annotation databases is important since biases impact our understanding of protein function by providing a poor picture of the annotation landscape. Additionally, as machine learning methods for predicting protein function are becoming increasingly prevalent, it is essential that they are trained on unbiased datasets. Therefore, it is not only crucial to be aware of biases, but also to judiciously remove them from annotation datasets. ResultsWe introduce GOThresher, a Python tool that identifies and removes biases in function annotations from protein function annotation databases. Implementation and AvailabilityGOThresher is written in Python and released via PyPI https://pypi.org/project/gothresher/ and on the Bioconda Anaconda channel https://anaconda.org/bioconda/gothresher. The source code is hosted on GitHub https://github.com/FriedbergLab/GOThresher and distributed under the GPL 3.0 license. Contact{idoerg@iastate.edu | parnal@iastate.edu}

bioinformatics↗

bioRxiv2022

Configurational entropy-based screening and selection of hydrophilic polymers using the tripartite split green fluorescent protein

Measuring the entropic properties of polymers such as proteins is critical to accurate prediction of their functional properties. However, the measurement of configurational entropy is possible only by low throughput techniques such as calorimetry, NMR and CD spectroscopy. Moreover, to our knowledge no system exists that allows molecular selection/enrichment based on the molecules configurational entropy. We tested the ability of the scalable tripartite GFP system to offer fine resolution of differences in configurational entropy in molecules and to isolate molecules based on their configurational entropy. The system was able to both finely resolve molecules with different configurational entropies, as well as capture them for isolation. We were able to tune the sensitivity of the system by using different mutations of the protein components. Lastly, we were able to apply the system to polypeptoid molecules and posit that the system may be applied to any other hydrophilic polymer of up to 10^3 repeating units.

biophysics↗

bioRxiv2022

Identification of potential antifibrinolytic compounds against kringle-1 and serine protease domain of plasminogen and kringle-2 domain of tissue-type plasminogen activator using combined virtual screening, molecular docking, and molecular dynamics simulation approaches.

The zymogen protease Plasminogen (Plg) and its active form plasmin (Plm) carry out important functions in the blood clot disintegration (breakdown of fibrin fibres) process. Inhibition of plasmin effectively reduces fibrinolysis to circumvent heavy bleeding. Currently, available Plm inhibitor tranexamic acid (TXA) that is used to treat severe hemorrhages is associated with an increased incidence of seizures which in turn were traced to gamma-aminobutyric acid antagonistic activity (GABAa) in addition to having multiple side effects. Fibrinolysis can be suppressed by targeting the three important protein domains: kringle-1 and serine protease domain of plasminogen and kringle-2 domain of tissue plasminogen activator. In the present study, combined approaches of structure-based virtual screening and molecular docking using Schrodinger Glide, AutoDock Vina, and ParDock/BAPPL+ were employed to identify potential hits from the ZINC database. Thereafter, the drug-likeness properties of the top three leads for each protein target were evaluated using Discovery Studio. Subsequently, a molecular dynamics simulation of 200ns for each protein-ligand complex was performed in GROMACS. The identified ligands are found to impart higher rigidity and stability to the protein-ligand complexes. Furthermore, the results were validated by performing the principal component analysis (PCA), and calculation of binding free energy using the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) approach. The identified ligands occupy smaller phase space, form stable clusters and exhibit stronger non-bonded interactions. Thus, our findings can be useful for the development of promising anti-fibrinolytic agents. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/512028v2_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@b762fforg.highwire.dtl.DTLVardef@1c47591org.highwire.dtl.DTLVardef@1028dc1org.highwire.dtl.DTLVardef@a884ad_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2022

Evidence for a Spoken Word Lexicon in the Auditory Ventral Stream

The existence of a neural representation for whole words (i.e., a lexicon) is a common feature of many models of speech processing. Prior studies have provided evidence for a visual lexicon containing representations of whole written words in an area of the ventral visual stream known as the "Visual Word Form Area" (VWFA). Similar experimental support for an auditory lexicon containing representations of spoken words has yet to be shown. Using fMRI rapid adaptation techniques, we provide evidence for an auditory lexicon in the "Auditory Word Form Area" (AWFA) in the human left anterior superior temporal gyrus that contains representations highly selective for individual spoken words. Furthermore, we show that familiarization with novel auditory words sharpens the selectivity of their representations in the AWFA. These findings reveal strong parallels in how the brain represents written and spoken words, showing convergent processing strategies across modalities in the visual and auditory ventral streams. HighlightsO_LIIndividual auditory word form areas (AWFA) were defined via an auditory localizer C_LIO_LIThe AWFA shows tuning for individual real words but not untrained pseudowords C_LIO_LIThe AWFA develops tuning for individual pseudowords after training C_LI

neuroscience↗

bioRxiv2022

A Synthetic ERRα Agonist Induces an Acute AerobicExercise Response and Enhances Exercise Capacity

Repetitive physical exercise induces physiological adaptations in skeletal muscle that improves exercise performance and is effective for the prevention and treatment of several diseases. Here we report the identification of a synthetic agonist for the orphan nuclear receptor ERR (estrogen receptor-related receptor ), SLU-PP-332, that activates an acute aerobic exercise genetic program in skeletal muscle in an ERR-dependent manner. SLU-PP-332 increases mitochondrial function and cellular respiration consistent with induction of this genetic program. When administered to mice, SLU-PP-332 increased the type IIa oxidative skeletal muscle fibers and enhanced exercise endurance. These data indicate the feasibility of targeting ERR for development of compounds that act as exercise mimetics that may be effective in treatment of numerous metabolic disorders and to improve muscle function in the aging.

pharmacology and toxicology↗

bioRxiv2022

Dynamic trade-offs between biomass accumulation and division determine bacterial cell size and proteome in fluctuating nutrient environments

Bacteria dynamically regulate cell size and growth rate to thrive in changing environments. While much work has been done to characterize bacterial growth physiology and cell size control during steady-state exponential growth, a quantitative understanding of how bacteria dynamically regulate cell size and growth in time-varying nutrient environments is lacking. Here we develop a dynamic coarse-grained proteome sector model which connects growth rate and division control to proteome allocation in time-varying environments in both exponential and stationary phase. In such environments, growth rate and size control is governed by trade-offs between prioritization of biomass accumulation or division, and results in the uncoupling of single-cell growth rate from population growth rate out of steady-state. Specifically, our model predicts that cells transiently prioritize ribosome production, and thus biomass accumulation, over production of division machinery during nutrient upshift, explaining experimentally-observed size control behaviors. Strikingly, our model predicts the opposite behavior during downshift, namely that bacteria temporarily prioritize division over growth, despite needing to upregulate costly division machinery and increasing population size when nutrients are scarce. Importantly, when bacteria are subjected to pulsatile nutrient concentration, we find that cells exhibit a transient memory of the previous metabolic state due to the slow dynamics of proteome reallocation. This phenotypic memory allows for faster adaptation back to previously-seen environments when nutrient fluctuations are short-lived.

systems biology↗

bioRxiv2022

Nanoscale infrared spectroscopy identifies parallel to antiparallel beta sheet transformation of Aβ fibrils

Spontaneous aggregation of amyloid beta (A{beta}) proteins leading to the formation of oligomers and eventually into fibrils has been identified as a key pathological signature of Alzheimers disease. Structure of late stage aggregates have been studied in depth by conventional structural biology techniques including Nuclear Magnetic Resonance, X-ray crystallography and Infrared Spectroscopy; however the structure of early-stage aggregates is less known due to their transient nature. As a result, the structural evolution of amyloid aggregates from its early oligomers to mature fibril is still not fully understood. Here we have applied AFM-IR nanospectroscopy to investigate the aggregation of A{beta} 16-22, which spans the amyloidogenic core of the amyloid beta peptide. Our results demonstrate that A{beta} 16-22 involves a structural transition from oligomers with parallel beta sheets to antiparallel fibrils through disordered and possibly helical intermediate fibril structures, contrary to the known aggregation pathway of full-length A{beta}.

biophysics↗

bioRxiv2022

AlphaFold2-based fusion design deciphers crucial role of the E3 UFL1 N-terminal helix in E2 UFC1 binding and ufmylation

Ufmylation, a protein modification by Ubiquitin-like (UBL) protein UFM1, plays a crucial role in several cellular processes including DNA damage response, protein translation and ER homeostasis. To date, little is known how the enzymes responsible for this modification coordinate their action. Here we have studied the details of UFL1 (E3) activity, its binding to UFC1 (E2), and its relation to UBA5 (E1), using a combination of structural modeling with Alphafold2, X-ray crystallography, NMR, and in vitro biochemical activity assays. Guided by an Alphafold2 model, we generated an active UFL1 fusion construct that includes its cofactor DDRGK1, and solved the first crystal structure of this critical interaction. This fusion construct also unveiled the importance of the N-terminal helix of UFL1 for its binding to UFC1, which was validated by ITC and NMR experiments. Importantly, the binding site suggested by our structural model of the UFL1-UFC1 interaction reveals a conserved interface, and suggests a competition for binding to UFC1 between UFL1 and UBA5, which we reconfirmed by NMR. Altogether, our study reveals a novel, terminal helix-mediated regulatory mechanism which coordinates the cascade of E1-E2-E3 mediated transfer of UFM1 to its substrate, and provides new leads to target this important modification. Significance statementUfmylation is an important post-translational modification, but little is known about the mechanistic details of its machinery, and in particular how the UFM1 E3 ligase (UFL1) binds and functions together with the E2 conjugating enzyme (UFC1). We combined AlphaFold2 modeling, X-ray crystallography, NMR and biochemical experiments to reveal crucial elements that govern UFL1 activity and ufmylation. We discover a crucial role for the UFL1 N-terminal helix in binding to UFC1 and productive ufmylation. This helix competes with the E1 (UBA5) C-terminal helix for binding to UFC1. Altogether, our findings uncover a new, helix-mediated regulatory mechanism in ufmylation.

biochemistry↗

bioRxiv2022

Musleblind-1 regulates microtubule cytoskeleton in C. elegans mechanosensory neuron through tubulin mRNAs

Regulation of microtubule cytoskeleton is fundamental for the development and maintenance of neuronal architecture. Recent studies have shown that regulated RNA processing is also critical for the establishment and maintenance of neural circuits. In a genetic screen using mechanosensory neurons of C. elegans, we identified a mutation in muscleblind-1 as a suppressor of loss of kinesin-13 family microtubule destabilizing factor klp-7. Muscleblind-1(MBL-1) is an RNA-binding protein that regulates the splicing, localization, and stability of RNA. We found that mbl-1 is required cell-autonomously for axon growth and synapse formation in the posterior lateral microtubule (PLM) neuron. Loss of mbl-1 affects stability and plus-end-out organization of microtubules in the anterior process of PLM. These defects are also accompanied by abnormal axonal transport of the synaptic protein RAB-3 and loss of gentle touch sensation in mbl-1 mutant. Our data showed that mbl-1 is genetically epistatic to mec-7 ({beta} tubulin) and mec-12 (a tubulin) for axon growth. The immunoprecipitation of MBL-1 pulls down the mec-7, mec-12, and sad-1 mRNAs. Additionally, the mbl-1 mutants show a reduction in the level and stability of mec-7 and mec-12 transcripts. Independently, mbl-1 is epistatic to sad-1 for synapse formation. Our work elucidated a previously unknown link between RNA binding protein and cytoskeletal machinery for the development and maintenance of the nervous system.

neuroscience↗

bioRxiv2022

Phylophenomic and Phylogenomic analysis for Ovis aries reveals distinct identity of newly reported breed

Domestication and phylogenetics for Ovis aries is an important species to study, since there exists enormous biodiversity in terms of habitat and utility of sheep. The present study aimed at identification of the biodiversity existing within sheep breeds reared in different agroclimatic zones of the state West Bengal (Garole, Birbhum, Bonpala and Chotanagpuri) through phylogenetic analysis of phenotypic traits as growth and biomorphometric traits through principal component analysis, factor analysis, genetic correlation, multivariate cluster analysis through Hierarchial classification and k-means cluster analysis. Confirmation of the phylophenomic studies were later on carried out with phylogenomic analysis with microsatellite markers for sheep. Birbhum sheep from dry arid region of West Bengal is reported as the newly reported breed of sheep with distinct genetic identity.

genetics↗

bioRxiv2022

Molecular phylogenetic analysis for Ovis aries with whole mitochondrial genome sequencing

Domestication of Ovis aries has taken place time immorial across the different geographical regions across the globe. Biodiversity among the sheep population has been interesting to study by a group of researchers based on mitochondrial genes like Cytochrome B, D loop. The current study is a novel attempt to understand the molecular phylogenomics among Ovis aries through all the 37 genes of mitochondria. We have analyzed complete mitochondrial genome sequencing for sheep breeds for West Bengal as Garole, Chotanagpuri, Bonpala and Birbhum sheep. Phylogenetic analysis reveals genetic similarity between Garole and Chotanagpuri brred of sheep, where as Birbhum and Bonpala were found to be genetically distinct. Phylogenomics in a global prospect reveal three lineages, Lineage A comprise of sheep from West Bengal grouped with Tibetan sheep. Lineage B consists of sheep population across the other parts including Europe (France, Denmark), Africa and Syro-Arabian desert, whereas Lineage C emerged as smaller outgroup.

genomics↗

bioRxiv2022

Molecular evolution and characterization of domestic duck (Anas platyrynchos) and Goose (Anser indicus) with reference to its wild relatives through whole mitochondrial genome sequencing

It is important to study the evolution and domestication of the domesticated duck (Anas platyrynchos) population from the wide range of wild relatives of Anas spp. Whole mitochondrial genome sequencing was attempted for Anas platyrynchos (Bengal duck) and Anser indicus (goose) from same geographical region. The study deals with the Molecular evolution of domestic duck based on mitochondrial gene due to its sequence variability, and to find out the phylogenetic relationships among Anas platyrynchos and its wild relatives. In this study we have used 45 wild species of Anas spp to study the mitochondrial genes and phylogenomics. Our result signifies that duck species were effectively discriminated with respect to mitochondrial genes, which could then be used for an appropriate genetic conservation program for the wild duck and domestic duck breeds. The DNA sequences from any unknown sample of the mitochondrial gene may be determined and can compare with those on a DNA database and can do blast for phylogenetic analysis of unknown wild duck, which gives its future scope. In silico analysis for 3D structure for Anas platyrynchos with the closest relative as Anas poecilorhyncha (Indian spot-billed duck) was attempted. Anas platyrynchos was also compared with Anser indicus. Therefore, this experiment was conducted to investigate the genetic diversity of West Bengal wild ducks with reference to its wild relatives based on mitochondrial gene.

genomics↗

bioRxiv2022

Gene amplification acts as a molecular foothold to facilitate cross-species adaptation and evasion of multiple antiviral pathways

Cross-species spillover events are responsible for many of the pandemics in human history including COVID-19; however, the evolutionary mechanisms that enable these events are poorly understood. We have previously modeled this process using a chimeric vaccinia virus expressing the rhesus cytomegalovirus-derived PKR antagonist RhTRS1 in place of its native PKR antagonists; E3L and K3L (VACV{Delta}E{Delta}K+RhTRS1). Using this virus, we demonstrated that gene amplification of rhtrs1 occurred early during experimental evolution and was sufficient to fully rescue virus replication in partially resistant African green monkey (AGM) fibroblasts. Notably, this rapid gene amplification also allowed limited virus replication in otherwise completely non-permissive human fibroblasts, suggesting that gene amplification may act as a "molecular foothold" to facilitate viral adaptation to multiple species. In this study, we demonstrate that there are multiple barriers to VACV{Delta}E{Delta}K+RhTRS1 replication in human cells, mediated by both PKR and RNase L. We experimentally evolved three AGM-adapted virus populations in human fibroblasts. Each population adapted to human cells bimodally, via an initial 10-fold increase in replication after only two passages followed by a second 10-fold increase in replication by passage nine. Using our Illumina-based pipeline, we found that some SNPs which had evolved during the prior AGM adaptation were rapidly lost, while 13 singlebase substitutions and short indels increased over time, including two SNPs unique to HFF adapted populations. Many of these changes were associated with components of the viral RNA polymerase, although no variant was shared between all three populations. Taken together, our results demonstrate that rhtrs1 amplification was sufficient to increase viral tropism after passage in an "intermediate species" and subsequently enabled the virus to adopt different, species-specific adaptive mechanisms to overcome distinct barriers to viral replication in AGM and human cells.

evolutionary biology↗

bioRxiv2022

Localized Leishmania major infection causes systemic iron deficiency that can be controlled by oral iron supplementation

Leishmania major (L. major) and its related parasitic species infection causes human cutaneous leishmaniasis that results into disfiguring skin lesion. Although L. major infection has been found to alter macrophage iron homeostasis we have limited understanding on whether it can also manipulate the same at systemic level. In fact, localized L. major infection found to promote iron deficiency anemia in children by an unknown mechanism. To address these unresolved issues, Balb/c mouse were infected with L. major and iron status in different organs were monitored systematically with the development of cutaneous lesion. At week 10 post infection when there was maximum lesion development in the parasite infected left hind footpad, the iron content increased significantly in this tissue with the concomitant increase in parasite burden. L. major infection mediated iron accumulation in infected mouse footpad was found to be due to transferrin receptor upregulation and natural resistance-associated macrophage protein 1 (Nramp1) downregulation. Surge in iron level was found to be associated with the reduced hepatic iron storage that resulted increased serum iron. Limited iron storage in liver and bone-marrow of infected mice caused reduced hemoglobin level and production of deformed erythrocytes. Interestingly, L. major infected mice developed splenomegaly with significant upregulation of erythroid related genes. Importantly, oral iron supplementation post infection rescued the development of cutaneous lesion in infected mice. Together, our study unravelled a comprehensive mechanism behind developing iron deficiency anemia during cutaneous leishmaniasis and a novel therapeutic route of treating this infection by delivering iron.

immunology↗

bioRxiv2022

Super-exponential growth and stochastic shape dynamics in rod-like bacteria

Proliferating bacterial cells exhibit stochastic growth and size dynamics but the regulation of noise in bacterial growth and morphogenesis remains poorly understood. A quantitative understanding of morphogenetic noise control, and how it changes under different growth conditions, would provide better insights into cell-to-cell variability and intergenerational fluctuations in cell physiology. Using multigenerational growth and width data of single Escherichia coli and Caulobacter crescentus cells, we deduce the equations governing growth and size dynamics of rod-like bacterial cells. Interestingly, we find that both E. coli and C. crescentus cells deviate from exponential growth within the cell cycle. In particular, the exponential growth rate increases during the cell cycle, irrespective of nutrient or temperature conditions. We propose a mechanistic model that explains the emergence of super-exponential growth from autocatalytic production of ribosomes, coupled to the rate of cell elongation and surface area synthesis. Using this new model and statistical inference on large datasets, we construct the Langevin equations governing cell size and size dynamics of E. coli cells in different growth conditions. The single-cell level model predicts how noise in intragenerational and intergenerational processes regulate variability in cell morphology and generation times, revealing quantitative strategies for cellular resource allocation and morphogenetic noise control in different growth conditions.

biophysics↗