Biology subjects

Basu, B.

Publications and source records attributed to Basu, B..

At least 19 recordsLinked to original sources

bioRxiv2026

JEV helicase targets host MTOC and participates in the organization of pericentriolar viroplasm

Flaviviruses contribute significantly to the global disease burden and are known to remodel host organelles to their advantage. Centrosomal microtubule-organizing centres (MTOCs) having established role in cell division and signalling are also targeted by viruses. However, it remains uncertain whether they act as bystanders or actively engage in viral processes. Here, we elucidate the centrosome and cytoskeletal involvement during Japanese Encephalitis Virus (JEV) infection. Our virus-free expression studies revealed a centrosome-targeting region within the C-terminal helicase domain that mediates the association of JEV-NS3 with host MTOCs. When expressed exogenously, JEV-NS3 formed pericentriolar aggresomes resembling the helicase-containing viroplasm distribution pattern of infected cells. These pericentriolar viroplasm appears in the non-neuronal cell types with centriolar consequences while the neuronal cell types fail to show such phenotypes. Centriole depletion assays revealed the proviral role of centrosome, where the viroplasm organization depends on centrosomal MTOCs, and vimentin cages. Additionally, microtubule disruption and dynarrestin blockade assays emphasized the roles of microtubules and dynein in concentrating NS3-containing vesicular packets towards centrosomes, highlighting the centrosome-cytoskeleton axis as a potential target for intervention.

cell biology↗

bioRxiv2026

Prpf8N1531S homozygous mutant mouse embryos have multiple defects in cardiac development and show aberrant splicing of the cardiac transcription factor Tead1

Mutations in the spliceosomal gene PRPF8 are associated with a range of human diseases. Studies in mouse and zebrafish suggest that Prpf8 also has a developmental function. Here, using a Prpf8 mutant mouse line isolated from a chemical induced mutagenesis screen, we uncover a previously unrecognised and essential role for Prpf8 in heart development, consistent with the embryonic lethality observed in Prpf8N1531S homozygous mutants. Prpf8N1531S mutant embryos display severe defects in ventricular trabeculation and compact zone formation, accompanied by increased cardiomyocyte proliferation specifically in the compact zone. Mutant embryonic hearts also exhibit disrupted cellular organisation, altered cytoskeletal architecture and changes in extracellular matrix protein expression. Notably, these cardiac abnormalities were exacerbated in embryos exhibiting cardiac looping defects. Transcriptomic analysis identified multiple aberrantly spliced transcripts in Prpf8N1531S mutant embryos, among which the cardiac transcription factor Tead1 was selected as a key functional candidate due to it known role in cardiac ventricle wall developemnt. Tead1 mis-splicing generated an in-frame, lower molecular weight protein isoform, associated with reduced overall TEAD1 expression. The Tead1 mis-spliced isoform showed altered nuclear localisation and dysregulation of TEAD1-dependent gene network important for heart development, including known cardiac sarcomeric genes. In addition, we observed reduced levels of the intracellular domain of the NOTCH1 receptor (NICD1), indicating impaired Notch signalling.. These findings suggest that impaired TEAD1-dependent transcription and Notch signalling contribute to abnormal cardiac trabeculation and compact zone development, highlighting a critical role for Prpf8 in maintaining proper heart development through the regulation of cardiac transcription factor expression and associated signalling networks. This study offers new mechanistic insights into congenital heart diseases linked to spliceosomal gene mutations.

developmental biology↗

bioRxiv2026

Factors shaping frugivory patterns of Asian mammals using a continental-scale dataset

O_LIFrugivores vary in their selection of fruit traits and their fruit handling methods, leading to differences in the plant species they consume for fruits. While fruit consumption patterns of birds are relatively well understood, much less is known about those of mammals. C_LIO_LIGiven the wide morphological and physiological diversity of mammals, fruit consumption patterns and fruit traits selected by different mammal groups may vary substantially. C_LIO_LIWe investigated differences in fruit consumption among three mammal groups - primates, herbivores, and carnivores - in Asia based on peer-reviewed and secondary literature. We assessed both morphological traits and taxonomic composition and compared patterns across vegetation types and for figs and non-figs. We found that primates (29%) and carnivores (21%) consumed more unique fruit genera than herbivores (6%). Carnivores and primates shared more fruit genera with each other (17%) than with herbivores. These patterns were consistent across vegetation types and for figs and non-figs. C_LIO_LIMorphological traits such as fruit size, colour, type, habit, seed number, and seed arillation showed no major differences among mammal groups. C_LIO_LIThere was no significant relationship between mammal body size and the mean or maximum fruit diameter consumed. However, among mammals that handle fruits exclusively with their mouthparts, body size was positively related to the maximum fruit diameter consumed. In contrast, for mammals that handle fruits using opposable thumbs (primates), body size showed a negative relationship with the mean fruit size consumed. There was no significant relationship between mammal activity patterns and the colour of the fruits they consumed. C_LIO_LIOur results suggest that fruit consumption patterns among mammal groups are not strongly differentiated by the morphological traits investigated; however, carnivores and primates are more similar in their preferred fruit genera. Moreover, morphological trait selection may be influenced by fruit handling methods. C_LIO_LIHerbivores consumed larger fruits and, like primates, preferred dull-coloured fruits, whereas carnivores more often fed on liana and shrub fruits across a wider colour range C_LIO_LIFuture research should focus on chemical and quantitative visual traits, such as volatile profiles and nutritional composition, to better understand the drivers of mammal fruit consumption. C_LI

ecology↗

bioRxiv2025

Spatial transcriptomic profiling uncovers the molecular effects of the neurotoxicant polychlorinated biphenyls (PCBs) in the brains of adult mice

Environmental toxicants, such as polychlorinated biphenyls (PCBs), are highly stable synthetic organic compounds that are present in air, water, and soil. PCBs have been identified in post-mortem human brains of individuals with neurodegenerative diseases, indicating a possible link between environmental factors and disease risk. Research has revealed an association between PCB exposure and cognitive decline. Therefore, it is crucial to evaluate how PCB mixtures relevant to humans affect brain function and cognition. To investigate the effects of PCBs on memory and transcriptomic profiles, we exposed adult male C57BL/6J mice orally to a synthetic PCB mixture daily. After seven weeks of exposure, the mice were assessed in a spatial object recognition task (SOR) to evaluate long-term spatial memory. Our findings showed that mice exposed to PCBs exhibited deficits in long-term spatial memory. To examine the molecular effects of PCB on the brain, we used a spatial transcriptomics technique to analyze gene expression changes in five brain regions: the hippocampus, neocortex, thalamus, caudal putamen, and fiber tracts. Our analysis of spatial gene expression revealed the molecular signatures influenced by PCB in these susceptible brain regions of mice. Network analysis suggests that these changes are associated with higher chlorinated PCBs present in the brain. Additionally, we show that PCB exposure disrupts the expression of tight junction proteins, which are crucial for maintaining the integrity of the blood-brain barrier (BBB). Thus, our results offer mechanistic insights into how PCB exposure affects brain function and cognition.

neuroscience↗

bioRxiv2025

Emetine dihydrochloride inhibits Chikungunya virus nsP2 helicase and shows antiviral activity in the cell culture and mouse model of virus infection

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes frequent epidemics of chikungunya fever across the world and has become a significant medical challenge, warranting the development of novel antivirals. A cell imaging-based high-content screening of the Spectrum collection of small molecules, containing all the US and international drug molecules, identified Emetine dihydrochloride (ED) as a potent antiviral against CHIKV. ED inhibited CHIKV replication in the C57BL/6 mouse model of chikungunya disease, resulting in significantly lower viremia. Importantly, clinical symptoms of joint swelling were altogether absent in the CHIKV-infected mice treated with ED. In the ERMS cells, ED inhibited the CHIKV uptake, and the virus replication early during the virus life cycle by inhibiting the viral RNA synthesis, resulting in the inhibition of the viral protein synthesis. ED was predicted to bind the helicase domain of the CHIKV non-structural protein nsP2 by in silico methods. The ED binding to the nsP2 protein and its helicase domain was validated in vitro by microscale thermophoresis and isothermal titration calorimetry. Importantly, ED inhibited the CHIKV nsP2 helicase activity in vitro in a dose-dependent manner. These data demonstrate the potential of ED to be repurposed as a novel CHIKV antiviral, warranting clinical development. AUTHOR SUMMARYChikungunya virus (CHIKV) spreads through the bites of the virus-infected Aedes mosquitoes. The virus causes frequent epidemics of chikungunya fever involving severe joint edema and muscle pain. The virus activity has been recorded in more than 100 countries in Asia, Africa, South and Central America, and Europe, and it has become endemic in several regions of the world. In 2024, around 480,000 CHIKV cases and over 200 deaths were reported worldwide. While a CHIKV vaccine was recently approved, no virus-specific antiviral therapy is available. Considering the global medical significance of the virus, the development of effective CHIKV antivirals is a priority. With a view to repurposing an existing drug, we screened a collection of all the approved US and international drug molecules and identified Emetine dihydrochloride (ED) as a potent inhibitor of CHIKV replication in the cell culture model. The CHIKV-infected mice treated with ED showed reduced virus replication and no joint swelling, which is a typical clinical symptom of CHIKV disease. Our studies show that ED binds the CHIKV nsP2, inhibiting its helicase action, which is required for replicating the viral genomic RNA and producing the viral proteins. In the past, emetine has been used in humans to treat amoebiasis. Our work thus shows the potential of the drug emetine to be repurposed for treating CHIKV patients.

microbiology↗

bioRxiv2025

The RNA splicing factor PRPF8 is required for left-right organiser cilia function and determination of cardiac left-right asymmetry via regulation of Arl13b splicing

Cilia function in the left-right organizer (LRO) is critical for determining internal organ asymmetry in vertebrates. To further understand the genetics of left-right asymmetry, we isolated a mouse mutant with laterality defects, l11Jus27, from a random mutagenesis screen. l11Jus27 mutants carry a missense mutation in the pre-mRNA processing factor, Prpf8. cephaloph[o]nus (cph) mutant zebrafish, carrying a protein truncating mutation in prpf8, phenocopy the laterality defects of l11Jus27 mutants. Prpf8 mutant mouse and fish embryos have increased expression of an alternative transcript encoding the cilium-associated protein, ARL13B, that lacks exon 9. In zebrafish, over-expression of the arl13b transcript lacking exon 9 perturbed cilium formation and caused laterality defects. The shorter ARL13B protein isoform lacked interactions with intraflagellar transport proteins. Our data suggest that PRPF8 plays a prominent role in LRO cilia by through the regulation of alternative splicing of ARL13B, thus uncovering a new mechanism for cilia-linked developmental defects.

developmental biology↗

bioRxiv2025

AGPAT1 is a novel Chikungunya virus receptor on human cells

Chikungunya virus (CHIKV) is a medically important alphavirus whose host receptor is not fully understood. We identified AGPAT1 from the human Huh7 cell plasma membrane binding with CHIKV particles in vitro. The CHIKV binding with AGPAT1 was demonstrated on Huh7, HAP1, and ERMS plasma membrane by confocal microscopy. The AGPAT1 antibody inhibited CHIKV binding to cells, reducing the virus uptake in Huh7 and ERMS cells. CHIKV binding, uptake, and replication were significantly reduced in the AGPAT1 knockout HAP1 cells, and the ectopic expression of AGPAT1 rescued the reduced virus binding, uptake, and replication. AGPAT1 interacted with the E1 surface on the E1-E2 dimer of CHIKV envelope proteins in silico. The E1-AGPAT1 interacting amino acid residues identified in the computational study were experimentally verified. AGPAT1 was also shown to have a role in the binding and uptake of another alphavirus, Ross River virus. These data demonstrate an important role for AGPAT1 as a novel host receptor involved in CHIKV binding and uptake in human cells. AUTHOR SUMMARYChikungunya virus (CHIKV) is a medically important alphavirus. Identifying its receptor on human cells will aid the development of novel antivirals. We have identified AGPAT1 as a novel CHIKV receptor on several human cells. We provide data showing the interaction of AGPAT1 with the E1 protein of CHIKV E1-E2 dimer present on the virion surface and identify the amino acid residues involved in the interaction. AGPAT1 was also shown to have a role in the binding and uptake of Ross River virus, another alphavirus. AGPAT1 is a protein involved in lipid metabolism and has not been reported to have a direct role in any virus infection, or as a virus receptor. Thus, this work identifies AGPAT1 as a novel receptor for CHIKV on human cells, and has implications for understanding the virus pathogenesis and designing the receptor-blocking CHIKV antivirals.

microbiology↗

bioRxiv2025

Single-cell resolution spatial transcriptomic signature of the retrosplenial cortex during memory consolidation

The retrosplenial cortex (RSC) is a critical brain region activated during spatial memory tasks and plays an underlying role in long-term memory consolidation. The RSC comprises multiple cell types, including different classes of excitatory neurons across laminar layers. These layer-specific cells form the hub of neuronal connection between the RSC and other brain regions, including the hippocampus. Despite the established role of the RSC in spatial memory, the transcriptomic signature of the neuronal sub-types in the RSC during spatial memory consolidation remained elusive. Here we used both unbiased and targeted spatial transcriptomic approaches to illuminate the transcriptional signature of the RSC following a spatial memory task. We found that genes related to transcription regulation, protein folding, and mitogen-activated protein kinase pathways were upregulated in the RSC after spatial learning during an early time window of memory consolidation. Further, cell type and excitatory neuronal layer-specific changes in gene expression were resolved using Xenium spatial transcriptomics. The distinct signatures of memory-responsive genes were observed in excitatory neurons across the laminar layers of the RSC following learning. Finally, we observed that blocking RSC excitatory neurons during the early temporal window after learning using a chemogenetic approach impaired long-term spatial memory. Overall, our results uncover a molecular signature of the RSC after learning and demonstrate the role of RSC excitatory neurons during the early time points of memory consolidation. This study underscores the importance of the learning-induced transcriptional signature of the RSC in long-term spatial memory consolidation and reveals a cell-type specific signature of memory-responsive gene expression.

neuroscience↗

bioRxiv2025

Histone Lysine Crotonylation Regulates Long-Term Memory Storage

Histone post-translational modifications (PTMs), particularly lysine acetylation (Kac), are critical epigenetic regulators of transcriptional programs underlying long-term memory consolidation. Beyond Kac, several other non-acetyl acylations have been identified with the ability to regulate transcription; however, their role in memory consolidation remains unknown. Here, we identify histone lysine crotonylation (Kcr) as a molecular switch for long-term memory and glutamatergic neurotransmission. We find that spatial learning induces distinct spatiotemporal patterns of Kcr across hippocampal subregions, and that Kcr stimulates learning-induced gene expression. Through genetic and pharmacological manipulations, we show that reducing hippocampal Kcr levels impairs memory, while increasing Kcr enhances long-term memory. Single-nuclei transcriptome and chromatin accessibility analyses reveals that Kcr facilitates activation of genes regulating glutamate signaling. Cell-cell communication analysis further infers that Kcr enhancement strengthens glutamatergic signaling within principal hippocampal neurons. Real-time fluorescence imaging with genetically encoded sensors functionally validates our multiomics and computational findings--demonstrating the role of Kcr in regulating presynaptic glutamate release and neuronal activity. In summary, our findings elucidate a novel mechanism underlying long-term memory consolidation and excitatory neurotransmission, linking epigenetic events to synaptic function and behavior.

neuroscience↗

bioRxiv2024

Histone H2B Monoubiquitylation Regulates Elongation-to-Termination Transition in RNA Polymerase II Transcription

RNA Polymerase II (Pol II)-dependent transcription is meticulously regulated during initiation, elongation, termination, and two pivotal transitions: from initiation to elongation and from elongation to termination 1. The successful transition from elongation to termination is vital for accurate transcription termination 2,3. While elongation and termination have been extensively studied, the regulatory mechanisms governing the transition between these stages are not fully understood. Here we report that, in fission yeast Schizosaccharomyces pombe (S. pombe) and human cells, Cdk9 and its cyclin partner (Cyclin T in humans, and Pch1 in S. pombe) disengage from the elongation machinery near gene 3-ends, as Pol II crosses the cleavage and polyadenylation signal (CPS). Concurrently, chromatin immunoprecipitation (ChIP) data shows an increased association of Dis2 (the PP1 ortholog in S. pombe)4 beyond the CPS. Additionally, ChIP-seq indicates that histone H2B monoubiquitylation (H2Bub1) is pivotal for Cdk9 occupancy but serves differential roles along the gene. H2Bub1 facilitates Cdk9 chromatin recruitment during elongation but prompts Cdk9 dissociation from the elongation complex as Pol II traverses the CPS. Furthermore, in S. pombe, disruptions of H2Bub1 in htb1-K119R 5 and sustained levels in ubp8D 6 inversely affect the chromatin occupancy of Pol II, Pol II CTD phosphorylation within the heptad repeats, Spt5, phosphorylated Spt5 (pSpt5), Dis2, components of the mRNA 3-end processing complex (Pfs2 and Pla1), and termination factors Rhn1 (Rtt103) and Pcf1 around the CPS-- diminishing with H2Bub1 loss and increasing with H2Bub1 stabilization. In essence, our data indicates that the H2Bub1-mediated eviction of Cdk9 as Pol II navigates the CPS precipitates a rise in PP1 binding, which, in turn, dephosphorylates pSpt5, slowing Pol II to facilitate efficient termination. The unique modulation of Cdk9 occupancy by H2Bub1 during transcription progression presents a compelling, novel concept requiring further exploration.

molecular biology↗

bioRxiv2023

A novel role of USP7 in imparting partial EMT state in colorectal cancer through the DDX3X-β-catenin axis

Epithelial mesenchymal transition (EMT) is a fundamental and highly regulated process that is normally observed during embryonic development and tissue repair but is deregulated during advanced cancer. Classically, through the process of EMT, cancer cells gradually transition from a predominantly epithelial phenotype to a more invasive mesenchymal phenotype. Increasing studies have, however, brought into light the existence of unique intermediary states in EMT, often referred to as partial EMT states. Through our studies we have found the deubiquitinase USP7 to be strongly associated with the development of such a partial EMT state in colon cancer cells, characterized by the acquisition of mesenchymal characteristics but without the reduction in epithelial markers. We found USP7 to be overexpressed in colon adenocarcinomas and to be closely associated with advancing tumor stage. We found that functional inhibition or knockdown of USP7 is associated with a marked reduction in mesenchymal markers and in overall migration potential of cancer cells. Starting off with a proteomics-based approach we were able to identify and later on verify the DEAD box RNA helicase DDX3X to be an interacting partner of USP7. We then went on to show that USP7, through the stabilization of DDX3X, augments Wnt/{beta}-catenin signaling, which has previously been shown to be greatly associated with colorectal cancer cell invasiveness. Our results strongly suggest a positive role of USP7 in the development of a partial mesenchymal phenotype in colorectal cancer.

cancer biology↗

bioRxiv2021

Computational study on Strontium ion modified Fibronectin-Hydroxyapatite interaction

Protein adsorption is the first key step in cell-material interaction. The initial phase of such adsorption process can only be probed using modelling approaches like molecular dynamics (MD) simulation. Despite a large number of studies on the adsorption behaviour of proteins on different biomaterials including hydroxyapatite (HA); little attention has been paid towards quantitative assessment of the effects of various physicochemical influencers like surface modification, pH, and ionic strength. Among these factors, surface modification through isomorphic substitution of foreign ions inside the apatite structure is of particular interest in the context of protein-HA interaction as it is widely used to tailor the biological response of HA. Given this background, we present here the molecular-level understanding of fibronectin (FN) adsorption mechanism and kinetics on Sr2+-doped HA (001) surface, at 300K by means of all-atom molecular dynamics simulation. Electrostatic interaction involved in adsorption of FN on HA was found to be significantly modified in presence of Sr2+ doping in apatite lattice. In harmony with the published experimental observations, the Sr-doped surface was found to better support FN adhesion compared to pure HA, with 10 mol% Sr-doped HA exhibiting best FN adsorption. Sr2+ ions also influence the stability of the secondary structure of FN, as observed from the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) analysis. The presence of Sr2+ enhances the flexibility of specific residues (residue no. 20-44, 74-88) of the FN module. Rupture forces to disentangle FN from the biomaterials surface, obtained from steered molecular dynamics (SMD) simulations, were found to corroborate well with the results of equilibrium MD simulations. One particular observation is that, the availability of RGD motif for the interaction with cell surface receptor integrin is not significantly influenced by Sr2+ substitution. Summarizing, the present work establishes a quantitative foundation towards the molecular basis of the earlier experimentally validated better cytocompatibility of Sr-doped HA. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/461618v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@19614a6org.highwire.dtl.DTLVardef@25b39eorg.highwire.dtl.DTLVardef@13368eborg.highwire.dtl.DTLVardef@2d44ec_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Identification of Novel Long non-coding RNAs and Expression Analyses in Rectal Neuroendocrine Tumors

Altered expression of long-non-coding RNAs (lncRNAs) is associated with various pathological conditions, including cancers. LncRNAs associated with rectal neuroendocrine tumors (rNETs) remain unexplored. Here, we report the identification of 418 high confidence novel lncRNAs by analyzing publicly available RNA-Seq datasets of primary rNETs and rNETs with lymph node or liver metastases. Novel lncRNAs had basic characteristics similar to the known lncRNAs and showed individual-specific expressions. Expression analysis in the normal rectal mucosa and pancreas NETs (pNETs) and small intestine NETs (siNETs) revealed high expression of two lncRNAs, namely MSTRG30689.1 and MSTRG341161.1. Functional characterization revealed that 9 lncRNAs could work as the ce-RNA and one as miRNAs precursor. Cis- and trans- analysis of the 9 PCG-associated lncRNAs revealed that these lncRNAs could regulate various important cancer-related genes. Further, an integrated regulatory mechanism for selected cancer hallmark genes such as SMAD3, ZEB1, TCF4 and ATM through lncRNAs is predicted. Taken together, our analyses reveal common and unique lncRNAs associated with rNETs and their metastatic variants, their PCG targets, and predict mechanisms of lncRNA mediated regulation of target PCGs.

genomics↗

bioRxiv2020

Drug and siRNA screens identify ROCK2 as a therapeutic target for ciliopathies.

Primary cilia are microtubule-based organelles that act as cellular antennae to mediate vertebrate development and growth factor signalling. Defects in primary cilia result in a group of inherited developmental conditions known as ciliopathies. Ciliopathies often present with cystic kidney disease, a major cause of early renal failure that requires renal replacement therapies. Currently, only one drug, Tolvaptan, is licensed to slow the decline of renal function for the ciliopathy polycystic kidney disease. Novel therapeutic interventions for these conditions remain a pressing clinical need. We screened clinical development compounds for positive effects on cilia formation and function and identified fasudil hydrochloride as the top hit. Fasudil is a generic, off-patent drug that is a potent but broadly selective Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. In a parallel whole genome siRNA-based reverse genetics phenotypic screen of positive modulators of cilia formation, we identified ROCK2 as the target molecule. We demonstrate that ROCK2 is a key mediator of cilium formation and function through effects on actin cytoskeleton remodelling. Our results indicate that specific ROCK2 inhibitors such as belumosudil (KD-025) could be repurposed for pharmacological intervention in cystic kidney disease. We propose that ROCK2 inhibition represents a novel, disease-modifying therapeutic approach for heterogeneous ciliopathies.

cell biology↗

bioRxiv2020

Somatic chromosomal number alterations affecting driver genes inform in-vitro and clinical drug response in high-grade serous ovarian cancer

The genomic complexity and heterogeneity of high-grade serous ovarian cancer (HGSOC) has hampered the realisation of successful therapies and effective personalised treatment is an unmet clinical need. Here we show that primary HGSOC spheroid models can be used to predict drug response and use them to demonstrate that somatic copy number alterations (SCNAs) in frequently amplified HGSOC cancer genes significantly correlate with gene expression and drug response. These genes are often located in areas of the genome with frequent clonal SCNAs. MYC chromosomal copy number is associated with ex-vivo and clinical response to paclitaxel and ex-vivo response to mTORC1/2 inhibition. Activation of the mTOR survival pathway in the context to MYC-amplified HGSOC is mostly due to increased prevalence of SCNAs in genes from the PI3K pathway. These results suggest that SCNAs encompassing driver genes could be used to inform therapeutic response in the context of clinical trials testing personalised medicines.

cancer biology↗

bioRxiv2020

Electric field mediated fibronectin hydroxyapatite interaction: A molecular insight

In experimental research driven biomaterials science, the influence of different material properties (elastic stiffness, surface energy, etc.), and to a relatively lesser extent, the biophysical stimulation (electric/magnetic) on the cell-material interaction has been extensively investigated. Considering the central importance of the protein adsorption on cell-material interaction, the role of physiochemical factors on the protein adsorption is also probed. Despite its significance, the quantitative analysis of many such aspects remains largely unexplored in biomaterials science. In recent studies, the critical role of electric field stimulation towards modulation of cell functionality on implantable biomaterials has been experimentally demonstrated. Given this background, we investigated the influence of external electric field stimulation (upto 1.00 V/nm) on fibronectin (FN) adsorption on hydroxyapatite, HA (100) surface at 300K using all-atom MD simulation method. Fibronectin adsorption was found to be governed by the attractive electrostatic interaction, which changed with the electric field strength. Non-monotonous changes in structural integrity of fibronectin were recorded with the change in field strength and direction. This can be attributed to the spatial rearrangement of local charges and global structural changes of the protein. The dipole moment vectors of fibronectin, water and HA quantitatively exhibited similar pattern of orienting themselves parallel to the field direction, with field strength dependent increase in their magnitudes. No significant change has been recorded for radial distribution function of water surrounding fibronectin. Field dependent variation in the salt bridge nets and number of hydrogen bonds between fibronectin and hydroxyapatite were also examined. One of the important results in the context of the cell-material interaction is that the RGD sequence of FN was exposed to solvent side, when the field was applied along a direction outward perpendicular to HA (001) surface. Summarizing, the present study provides quantitative insights into the influence of electric field stimulation on biomolecular interactions involved in fibronectin adsorption on hydroxyapatite surface.

biophysics↗

bioRxiv2018

Higher order assembling of the mycobacterial essential polar growth factor DivIVA/Wag31

How proteins localize at the pole remains an enigma. DivIVA/Wag31, which is an essential pole organizing protein in mycobacteria, can assemble at the negatively curved side of the membrane at the growing pole to form a higher order structural scaffold for maintaining cellular morphology and localizing various target proteins for cell-wall biogenesis. A single-site phosphorylation in Wag31 is linked to the regulation of peptidoglycan biosynthesis for optimal mycobacterial growth. The structural organization of polar scaffold formed by coiled-coil rich Wag31, which is a target for anti-tubercular agent amino-pyrimidine sulfonamide, remains to be determined. Here, we report biophysical characterizations of a phospho-mimetic (T73E) and a phospho-ablative (T73A) form of mycobacterial Wag31 using circular dichroism, small angle solution X-ray scattering and atomic force microscopy. While data obtained from both variants of Wag31 in solution states suggested formation of alpha-helical, large, elongated particles, their structural organizations were different. Atomic force microscopic images of Wag31 indicate polymer formation, with occasional curving, sharp bending and branching. Observed structural features in this first view of the polymeric forms of Wag31 suggest a basis for higher order network scaffold formation for polar protein localization.

biophysics↗

bioRxiv2017

Human iPSC-derived RPE and retinal organoids reveal impaired alternative splicing of genes involved in pre-mRNA splicing in PRPF31 autosomal dominant retinitis pigmentosa

Mutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31+/- mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.\n\neTOCPRPF31 is a ubiquitously expressed pre-mRNA processing factor that when mutated causes autosomal dominant RP. Using a patient-specific iPSC approach, Buskin and Zhu et al. show that retinal-specific defects result from altered splicing of genes involved in the splicing process itself, leading to impaired splicing, loss of RPE polarity and diminished phagocytic ability as well as reduced cilia incidence and length in both photoreceptors and RPE.\n\nHighlightsO_LISuccessful generation of iPSC-derived RPE and photoreceptors from four RP type 11 patients\nC_LIO_LIRPE cells express the mutant PRPF31 protein and show the lowest expression of wildtype protein\nC_LIO_LIPRPF31 mutations result in altered splicing of genes involved in pre-mRNA splicing in RPE and retinal organoids\nC_LIO_LIPrpf31 haploinsufficiency results in altered splicing of genes involved in pre-mRNA splicing in mouse retina\nC_LIO_LIRPE cells display loss of polarity, reduced barrier function and phagocytosis\nC_LIO_LIPhotoreceptors display shorter and fewer cilia and degenerative features\nC_LIO_LIRPE cells display most abnormalities suggesting they might be the primary site of pathogenesis\nC_LIO_LIIn situ gene editing corrects the mutation and rescues key phenotypes\nC_LI

genetics↗