Biology subjects

Bhattacharjee, S.

Publications and source records attributed to Bhattacharjee, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Post-GWAS functional analyses of CNTNAP5 suggests its role in glaucomatous neurodegeneration

Primary angle closure glaucoma (PACG) affects more than 20 million people worldwide, with an increased prevalence in south-east Asia. In a prior haplotype-based GWAS, we identified a novel CNTNAP5 genic region, significantly associated with PACG. In the current study, we have extended our perception of CNTNAP5 involvement in glaucomatous neurodegeneration in a zebrafish model, through investigating phenotypic consequences pertinent to retinal degeneration upon knockdown of cntnap5 by translation-blocking morpholinos. While cntnap5 knockdown was successfully validated using an antibody, immunofluorescence followed by western blot analyses in cntnap5-morphant (MO) zebrafish revealed increased expression of acetylated tubulin indicative of perturbed cytoarchitecture of retinal layers. Moreover, significant loss of Nissl substance is observed in the neuro-retinal layers of cntnap5-MO zebrafish eye, indicating neurodegeneration. Additionally, in spontaneous movement behavioural analysis, cntnap5-MO zebrafish have a significantly lower average distance traversed in light phase compared to mismatch-controls, whereas no significant difference was observed in the dark phase, corroborating with vision loss in the cntnap5-MO zebrafish. This study provides the first direct functional evidence of a putative role of CNTNAP5 in visual neurodegeneration.

genetics↗

bioRxiv2024

Methionine cycle in a pair of serotonergic neurons regulates diet-dependent behavior and longevity through a neuron-gut signaling

The folate-methionine cycle (Met-C) is a central metabolic pathway that is regulated by vitamin B12 (B12), a micronutrient obtained exclusively from diet and microbiota. This metabolic hub supports amino acid, nucleotide and lipid biosynthesis apart from its central role of providing one carbon (-CH3) moiety for methylation reactions. While deficiency of B12 as well as polymorphism in enzymes of the Met-C has been clinically attributed to neurological and metabolic disorders, how this pathway cell non-autonomously regulates systemic physiological processes is less understood. Using a B12-sensitive mutant of Caenorhabditis elegans, we show that the neuronal Met-C responds to differential B12 content in diet to regulate p38-MAPK activation in intestinal cells, thereby modulating cytoprotective gene expression, stress tolerance and longevity. Mechanistically, B12-driven changes in the metabolic flux through the Met-C in the serotonergic ADF neurons of the mutant lead to the release of serotonin (5-hydroxytryptamine, 5-HT). 5-HT activates its receptor, MOD-1, in the post-synaptic interneurons that then secretes the neuropeptide FLR-2. FLR-2 binds to FSHR-1, its cognate receptor in the intestine, and induces the phase transition of the SARM domain protein TIR-1, thereby activating the p38-MAPK pathway. Importantly, this cascade influences the foraging behaviour of the mutant worms such that they prefer a B12-rich diet. Together, our study reveals a dynamic neuron-gut signaling axis that helps an organism modulate behaviour and life history traits based on the neuronal Met-C metabolic flux determined by B12 availability in its diet. Understandably, disruption of the optimum functioning of this axis may have debilitating effects on the health of an organism and the survival of the species.

cell biology↗

bioRxiv2024

An arch worth revisiting: A study on the feline humeral supracondylar foramen and its evolutionary significance

The supracondylar foramen with a seemingly osseous peripheral arch noticed on the medio-distal feline humeri had remained disputed among anatomists. Some scholars have argued in favor of homology between this foramen and the supracondyloid foramen formed in the presence of the ligament of Struthers in humans. Other theories include its presence as a retinaculum holding the median nerve and brachial artery to their anatomical position in a flexed elbow. Unfortunately, these theories lack investigative rigor. The emergence of non-invasive imaging modalities, such as micro-computed tomography, has enabled researchers to inspect the internal anatomy of bones without dismantling. Thus, a micro-computed tomographic investigation was conducted on three feline humeri specimens while the internal anatomy of the supracondylar foramina was examined. Unlike the humerus, the thin peripheral arch of the feline supracondylar foramen failed to elicit any osseous trabeculae or foci of calcification. While adhering to the humeral periosteum at its origin, the non-osseous arch, typical of a muscular tendon or a ligament, inserts into a bony spur attached to the medial humeral epicondyle suggestive of a ligament or aponeurotic extension of a (vestigial) brachial muscle, with the coracobrachialis longus emerging to be the most likely candidate.

evolutionary biology↗

bioRxiv2024

Targeted Dab2 Restoration: A Novel Approach for Enhanced Wound Healing in Diabetic Conditions

Diabetes mellitus can cause impaired and delayed wound healing, leading to lower extremity amputations; however, the mechanisms underlying the regulation of vascular endothelial growth factor (VEGF)-dependent angiogenesis remain uncertain and could reveal new therapeutic targets. In our study, the molecular underpinnings of endothelial dysfunction in diabetes were investigated, focusing on the roles of Disabled-2 (Dab2) and Forkhead Box M1 (FoxM1) in VEGF receptor 2 (VEGFR2) signaling and endothelial cell (EC) function. Bulk RNA-sequencing analysis identified significant downregulation of Dab2 in high concentrations glucose treated primary mouse skin ECs, simulating hyperglycemic conditions in diabetes mellitus. In diabetic mice with a genetic EC deficiency of Dab2 angiogenesis was reduced in vivo and in vitro when compared with wild-type mice. Restoration of Dab2 expression by injected mRNA-containing lipid nanoparticles rescued impaired angiogenesis and wound healing in diabetic mice. At the same time, FoxM1 was downregulated in skin ECs subjected to high glucose conditions as determined by RNA-sequencing analysis. FoxM1 was found to bind to the Dab2 promoter, regulating its expression and influencing VEGFR2 signaling. The FoxM1 inhibitor FDI-6 reduced Dab2 expression and phosphorylation of VEGFR2. These findings indicate that restoring Dab2 expression through targeted therapies can enhance angiogenesis and wound repair in diabetes. To explore this therapeutic potential, we tested LyP-1-conjugated lipid nanoparticles (LNPs) containing Dab2 or control mRNAs to target ECs and found the former significantly improved wound healing and angiogenesis in diabetic mice. This study provides evidence of the crucial roles of Dab2 and FoxM1 in diabetic endothelial dysfunction and establishes targeted delivery as a promising treatment for diabetic vascular complications.

cell biology↗

bioRxiv2024

Variability in HIV-1 Transmitted/Founder Virus Susceptibility to Combined APOBEC3F and APOBEC3G Host Restriction

Several APOBEC3 enzymes restrict HIV-1 replication by deaminating cytosine to form uracil in single-stranded proviral (-)DNA. However, HIV-1 Vif binds to APOBEC3 enzymes and counteracts their activity by inducing their proteosomal degradation. This counteraction by Vif is not complete as evidenced by footprints of APOBEC3-mediated mutations within integrated proviral genomes of people living with HIV-1. The APOBEC3 enzymes are co-ordinately expressed in CD4+T cells and relative contributions of APOBEC3s in HIV-1 restriction is not fully understood. In this study, we investigated the activity of co-expressed APOBEC3F and APOBEC3G against HIV-1 Subtype B and Subtype C Transmitted/Founder viruses. APOBEC3F and APOBEC3G when co-expressed were previously determined to form a hetero-oligomer that enables partial resistance of APOBEC3F to Vif-mediated degradation. Here, we determined that that APOBEC3F interacts with APOBEC3G through its N-terminal domain. We provide evidence that this results in protection from Vif-mediated degradation because the APOBEC3F N-terminal domain contains residues required for recognition by Vif. We also found subtype specific differences in activity of Transmitted/Founder Vifs against APOBEC3G and the APOBEC3F/APOBEC3G hetero-oligomer. HIV-1 Subtype C Vifs were more active in counteracting APOBEC3G compared to HIV-1 Subtype B Vifs when APOBEC3G was expressed alone. However, HIV-1 Subtype C Vifs were less active against APOBEC3G when APOBEC3F and APOBEC3G were co-expressed. Consequently, when APOBEC3F and APOBEC3G were expressed together HIV-1 Subtype C viruses showed a decrease in relative infectivity compared to that when APOBEC3G was expressed alone. Inspection of Vif amino acid sequences revealed that that differences in amino acids adjacent to conserved sequences influenced the Vif-mediated APOBEC3 degradation ability. Altogether, the data provide a possible mechanism for how combined expression of APOBEC3F and APOBEC3G could contribute to mutagenesis of HIV-1 proviral genomes in the presence of Vif and provide evidence for variability in the Vif-mediated degradation ability of Transmitted/Founder viruses. Author SummaryAPOBEC3 enzymes act as barriers to HIV infection by inducing cytosine deamination in proviral DNA, but their effectiveness is hindered by their counteraction by HIV Vif, which leads to APOBEC3 proteasomal degradation. The APOBEC3-Vif interaction has largely been determined using lab adapted HIV-1 Subtype B viruses and with singular APOBEC3 enzymes. Here we examined how primary isolates of HIV-1 replicated in the presence of APOBEC3F and APOBEC3G. APOBEC3F and APOBEC3G interact and this imparts partial resistance to Vif-mediated degradation. We determined that APOBEC3F interacts with APOBEC3G through its N terminal domain, and that APOBEC3F, like APOBEC3G has Vif-mediated degradation determinants in the N-terminal domain, providing a rational for protection from Vif-mediated degradation. We also demonstrate subtype-specific differences in the activity of Transmitted/Founder Vifs against APOBEC3G and the APOBEC3F/APOBEC3G hetero-oligomer. Through an analysis of Vif amino acid sequences, we identified variations influencing the Vif-mediated APOBEC3 degradation ability. This research uncovers previously unidentified mechanisms by which combined expression of APOBEC3F and APOBEC3G may contribute to HIV-1 proviral genome mutagenesis in the presence of Vif and emphasizes the contribution of amino acid variation outside of previously identified conserved regions in Vif-mediating APOBEC3 degradation.

microbiology↗

bioRxiv2024

Artemisinin-resistant Plasmodium falciparum Kelch13 mutant proteins display reduced heme-binding affinity and decreased artemisinin activation

The rapid emergence of artemisinin resistance (ART-R) poses a challenge to global malaria control efforts. ART potency is triggered by ferrous iron- and/or heme-mediated cleavage of the endoperoxide bond to generate reactive heme-ART alkoxy radicals and covalent heme-ART adducts that alkylate parasite targets or inhibit the detoxification of heme into {beta}-hematin crystals; both of which lead to parasite death. Mutations in the P. falciparum Kelch-containing protein Kelch13 (PfKekch13) confer clinical ART-R, in which the resistant parasites exhibit impaired hemoglobin uptake, reduced heme yield, and thus decreased ART activation. However, a more direct involvement of PfKelch13 in heme-mediated ART activation has not been reported. Here, we show that recombinant, purified PfKelch13 wild-type (WT) protein displays measurable binding affinity for both iron and heme, the main effectors for ART activation. Comparative biochemical analyses further indicate weaker heme-binding affinities in the two Southeast Asian ART-R PfKelch13 mutants C580Y and R539T compared to the ART-sensitive WT and A578S mutant proteins, which ultimately translates into reduced yield of heme-ART derivatives. In conclusion, this study provides the first evidence for regulated ART activation via the heme-binding propensity of PfKelch13, which may contribute towards modulating the level of ART-R in malaria parasites with PfKelch13 mutations.

biochemistry↗

bioRxiv2024

Epsins promote atherosclerosis through VSMC phenotypic modulation

Smooth muscle cells in major arteries play a crucial role in regulating coronary artery disease. Conversion of smooth muscle cells into other adverse cell types in the artery propels the pathogenesis of the disease. Curtailing artery plaque buildup by modulating smooth muscle cell reprograming presents us a new opportunity to thwart coronary artery disease. Here, our report how Epsins, a family of endocytic adaptor proteins oversee the smooth muscle cell reprograming by influencing master regulators OCT4 and KLF4. Using single-cell RNA sequencing, we characterized the phenotype of modulated smooth muscle cells in mouse atherosclerotic plaque and found that smooth muscle cells lacking epsins undergo profound reprogramming into not only beneficial myofibroblasts but also endothelial cells for injury repair of diseased endothelium. Our work lays concrete groundwork to explore an uncharted territory as we show that depleting Epsins bolsters smooth muscle cells reprograming to endothelial cells by augmenting OCT4 activity but restrain them from reprograming to harmful foam cells by destabilizing KLF4, a master regulator of adverse reprograming of smooth muscle cells. Moreover, the expression of Epsins in smooth muscle cells positively correlates with the severity of both human and mouse coronary artery disease. Integrating our scRNA- seq data with human Genome-Wide Association Studies (GWAS) identifies pivotal roles Epsins play in smooth muscle cells in the pathological process leading to coronary artery disease. Our findings reveal a previously unexplored direction for smooth muscle cell phenotypic modulation in the development and progression of coronary artery disease and unveil Epsins and their downstream new targets as promising novel therapeutic targets for mitigating metabolic disorders.

cell biology↗

bioRxiv2023

Targeting endothelial FOXO1 protects diabetic β-cells and improves wound healing

The forkhead box O1 (FOXO1) transcription factor plays critical roles in regulating not only metabolic activity but also angiogenesis in the vascular endothelium1-4. Our previous studies show that epsin endocytic adaptors can regulate both angiogenesis and lymphangiogenesis5-7. Endothelial cells (ECs) lining the inside of blood vessels are continuously exposed to circulating insulin and insulin-like growth factors (IGFs). Emerging evidences suggest that ECs can affect {beta}-cell function8-11. Excessive IGF2, especially elevated local IGF2 levels in islets, may represent a risk factor for developing diabetes12-15; however, the underlying molecular mechanisms by which aberrant angiogenesis and endothelium-derived factors regulate pancreatic {beta}-cell function in diabetes remain unclear. Here, we report that the pancreas of diabetic patients as well as the pancreas, skin, and plasma of streptozotocin/high fat diet (STZ/HFD)-induced diabetic mice and db/db mice contains excess IGF2, which can lead to {beta}-cell dysfunction and apoptosis. Single-cell transcriptomics combined with mass spectrometry analysis reveal that endothelial-specific knockout of FOXO1 increases circulating soluble and cell-membrane or intracellular expression levels of IGF type 2 receptor (IGF2R) and CCCTC-binding factor (CTCF), while decreasing IGF2 levels in diabetes. Both IGFR215-17 and CTCF18-21 can reduce IGF2 levels and may ameliorate {beta}-cell decline associated with excess IGF2 in diabetes. Furthermore, depletion of FOXO1, epsins, or knockdown of ULK1 inhibits autophagy formation in ECs, preventing degradation of vascular endothelial growth factor receptor 2 (VEGFR2) to promote angiogenesis and improve wound healing in diabetes. Our findings reveal that endothelial FOXO1 regulates epsin-dependent angiogenesis and affects {beta}-cell function and fate through CTCF and IGF2-IGF2R, providing a potential strategy for ameliorating diabetes and accelerating cutaneous wound healing.

cell biology↗

bioRxiv2023

RNA Binding of Syntaxin 5 Synergises Membrane Fusion to Accelerate miRNA Export and Clear Pathogens

Intercellular miRNA exchange acts as a key mechanism to control gene expression post-transcriptionally in mammalian cells. Regulated export of repressive miRNAs allows the expression of inflammatory cytokines in activated macrophages. Intracellular trafficking of miRNAs from endoplasmic reticulum to endosomes is a rate determining step in miRNA export process and plays an important role in controlling cellular miRNA level and inflammatory processes in macrophages. We have identified the SNARE protein Syntaxin5 to show a synchronized expression pattern with miRNA activity loss in activated mammalian macrophage cells. Syntaxin 5 is both necessary and sufficient for macrophage activation and clearance of the intracellular pathogen Leishmania donovani from infected macrophages. Exploring the mechanism of how Syntaxin5 acts as an immunostimulant, we have identified the de novo RNA binding property of this SNARE protein that binds specific miRNAs and facilitates their accumulation in endosomes in a cooperative manner with human ELAV protein HuR to ensure export of miRNAs and allows the expression of miRNA-repressed cytokines. Conversely, in its dual role in miRNA export, this SNARE protein prevents lysosomal targeting of endosomes by enhancing the fusion of miRNA-loaded endosomes with plasma membrane to ensure accelerated release of extracellular vesicles and associated miRNAs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/556844v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@bfabf8org.highwire.dtl.DTLVardef@1b35346org.highwire.dtl.DTLVardef@203a5corg.highwire.dtl.DTLVardef@1d7a15e_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIHuR transfers miRNAs to STX5 for export C_LIO_LISTX5 utilizes its N-terminal disordered domain to bind and target miRNAs to endosomes. C_LIO_LIIn its dual role, STX5 uses the SNARE domain to export miRNA by promoting EV biogenesis and release. C_LIO_LISTX5-mediated miRNA export activates macrophage to clear internalized parasites. C_LI

immunology↗

bioRxiv2023

CCT and Cullin1 regulate the TORC1 pathway to promote dendritic arborization in health and disease.

The development of cell-type-specific dendritic arbors is integral to the proper functioning of neurons within their circuit networks. In this study, we examine the regulatory relationship between the cytosolic chaperonin CCT, key insulin pathway genes, and an E3 ubiquitin ligase (Cullin1) in homeostatic dendritic development. CCT loss of function (LOF) results in dendritic hypotrophy in Drosophila Class IV (CIV) multidendritic larval sensory neurons, and CCT has recently been shown to fold components of the TOR (Target of Rapamycin) complex 1 (TORC1), in vitro. Through targeted genetic manipulations, we have confirmed that LOF of CCT and the TORC1 pathway reduces dendritic complexity, while overexpression of key TORC1 pathway genes increases dendritic complexity in CIV neurons. Both CCT and TORC1 LOF significantly reduce microtubule (MT) stability. CCT has been previously implicated in regulating proteinopathic aggregation, thus we examined CIV dendritic development in disease conditions as well. Expression of mutant Huntingtin leads to dendritic hypotrophy in a repeat-length-dependent manner, which can be rescued by TORC1 disinhibition via Cullin1 LOF. Together, our data suggest that Cullin1 and CCT influence dendritic arborization through regulation of TORC1 in both health and disease. SIGNIFICANCEThe insulin pathway has become an increasingly attractive target for researchers interested in understanding the intersection of metabolism and brain health. We have found connections between the insulin pathway and cytosolic protein maintenance in the development of neuronal dendrites. These pathways converge on the dendritic cytoskeleton, particularly microtubules. Neurons expressing mutant Huntingtin also show defects in dendritic development and the underlying cytoskeleton, and we find that disinhibition of the insulin pathway can rescue dendritic hypotrophy in these neurons. This work advances our understanding of the molecular interactions between the insulin pathway and neuronal development in both health and Huntingtons Disease conditions.

neuroscience↗

bioRxiv2023

Beyond Traditional Methods: Innovative Integration of LISS IV and Sentinel 2A Imagery for Unparalleled Insight into Himalayan Ibex Habitat Suitability

Despite advancements in remote sensing, satellite imagery is underutilized in conservation research. Multispectral data from various sensors have great potential for mapping landscapes, but distinct spectral and spatial resolution capabilities are crucial for accurately classifying wildlife habitats. Our study aimed to develop a technique for precisely discerning habitat categories for the Himalayan Ibex (Capra sibirica) using different satellite imagery. To address both spectral and spatial challenges, we utilized LISS IV and Sentinel 2A data and integrated the LISS IV data with Sentinel 2A data along with their corresponding geometric information. Employing multiple supervised classification algorithms, we found the Random Forest (RF) algorithm to outperform others. The integrated (LISS IV-Sentinel 2A) classified image achieved the highest accuracy, with an overall accuracy of 86.17% and a Kappa coefficient of 0.84. To map the suitable habitat of the Ibex, we conducted ensemble modeling using the Land Cover Land Use (LCLU) of all three image types (LISS IV, Sentinel 2A, Integrated) and other predictors such as topographical, soil type, vegetation, and water radiometric indices. The integrated model provided the most accurate prediction of suitable habitat for the Ibex, surpassing the other two LCLU classes derived from individual images. The Soil Adjusted Vegetation Index (SAVI) and elevation were identified as crucial factors in identifying suitable habitats These findings hold valuable implications for the development of effective conservation strategies, as accurate classification schemes enable the identification of vital landscape elements. By precisely classifying LULC satellite images and identifying crucial habitats for the Ibex, this pilot study provides a new and valuable strategy for conservation planning. It enhances our ability to preserve and protect the habitat of wildlife species in the mountain ecosystem of the Himalayas.

ecology↗

bioRxiv2023

Chromatin remodeling of histone H3 variants underlies epigenetic inheritance of DNA methylation

Epigenetic inheritance refers to the faithful replication of DNA methylation and histone modification independent of DNA sequence. Nucleosomes block access to DNA methyltransferases, unless they are remodeled by DECREASE IN DNA METHYLATION1 (DDM1Lsh/HELLS), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 activity results in replacement of the transcriptional histone variant H3.3 for the replicative variant H3.1 during the cell cycle. In ddm1 mutants, DNA methylation can be restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 [A] of DDM1 with a variant nucleosome reveals direct engagement at SHL2 with histone H3.3 at or near variant residues required for assembly, as well as with the deacetylated H4 tail. An N-terminal autoinhibitory domain binds H2A variants to allow remodeling, while a disulfide bond in the helicase domain is essential for activity in vivo and in vitro. We show that differential remodeling of H3 and H2A variants in vitro reflects preferential deposition in vivo. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1Dnmt1. DDM1 localization to the chromosome is blocked by H4K16 acetylation, which accumulates at DDM1 targets in ddm1 mutants, as does the sperm cell specific H3.3 variant MGH3 in pollen, which acts as a placeholder nucleosome in the germline and contributes to epigenetic inheritance.

molecular biology↗

bioRxiv2023

Local microtubule and F-actin distributions fully determine the spatial geometry of Drosophila sensory dendritic arbors

Dendritic morphology underlies the source and processing of neuronal signal inputs. Morphology can be broadly described by two types of geometric characteristics. The first is dendrogram topology, defined by the length and frequency of the arbor branches; the second is spatial embedding, mainly determined by branch angles and tortuosity. We have previously demonstrated that microtubules and actin filaments are associated with arbor elongation and branching, fully constraining dendrogram topology. Here we relate the local distribution of these two primary cytoskeletal components with dendritic spatial embedding. We first reconstruct and analyze 167 sensory neurons from the Drosophila larva encompassing multiple cell classes and genotypes. We observe that branches with higher microtubule concentration are overall straighter and tend to deviate less from the direction of their parent branch. F-actin displays a similar effect on the angular deviation from the parent branch direction, but its influence on branch tortuosity varies by class and genotype. We then create a computational model of dendritic morphology purely constrained by the cytoskeletal composition imaged from real neurons. The model quantitatively captures both spatial embedding and dendrogram topology across all tested neuron groups. These results suggest a common developmental mechanism regulating diverse morphologies, where the local cytoskeletal distribution can fully specify the overall emergent geometry of dendritic arbors.

neuroscience↗

bioRxiv2023

The Zinc-BED transcription factor Bedwarfed promotes proportional dendritic growth and branching through transcriptional and translational regulation in Drosophila

Dendrites are the primary points of sensory or synaptic inputs to a neuron and play an essential role in synaptic integration and neural function. Despite the functional importance of dendrites, relatively less is known about the underlying mechanisms regulating cell-type specific dendritic patterning. Herein, we have dissected functional roles of a previously uncharacterized gene, CG3995, in cell-type specific dendritic development in Drosophila melanogaster. CG3995, which we have named bedwarfed (bdwf), encodes a zinc-finger BED-type protein which is required for proportional growth and branching of dendritic arbors, exhibits nucleocytoplasmic expression, and functions in both transcriptional and translational cellular pathways. At the transcriptional level, we demonstrate a reciprocal regulatory relationship between Bdwf and the homeodomain transcription factor (TF) Cut. We show that Cut positively regulates Bdwf expression and that Bdwf acts as a downstream effector of Cut-mediated dendritic development, whereas overexpression of Bdwf negatively regulates Cut expression in multidendritic sensory neurons. Proteomic analyses revealed that Bdwf interacts with ribosomal proteins and disruption of these proteins produced phenotypically similar dendritic hypotrophy defects as observed in bdwf mutant neurons. We further demonstrate that Bdwf and its ribosomal protein interactors are required for normal microtubule and F-actin cytoskeletal architecture. Finally, our findings reveal that Bdwf is required to promote protein translation and ribosome trafficking along the dendritic arbor. Taken together, these results provide new insights into the complex, combinatorial and multi-functional roles of transcription factors (TFs) in directing diversification of cell-type specific dendritic development.

neuroscience↗

bioRxiv2023

Mechanism of HflX-mediated recycling of E. coli ribosome studied by time-resolved cryo-EM

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here we introduce a novel time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 [A]. By employing this method, we show for the first time the mechanism of progressive HlfX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP, via capture of three high-resolution reaction intermediates within 140 ms.

biophysics↗

bioRxiv2022

Overexpression of Interleukin-8 and Interleukin-13 as potent immune markers associated with survival and dietary habits in esophageal squamous cell carcinoma.

BackgroundInterleukin-8 (IL8), Interleukin-12 (IL12) and Interleukin-13 (IL13) are cytokines that play regulatory role in cancer pathogenesis. We analysed their expression profile to evaluate as molecular biomarkers of esophageal squamous cell carcinoma (ESCC) and their association with different parameters. MethodsExpression analysis of IL8, IL12 and IL13 were performed by Real time qPCR in blood and tumor tissue of 120 ESCC patients. The expression profiles were associated with different clinicopathological and dietary factors. Survival and hazard analysis were also performed. ResultsWhen compared to normal controls, IL8 expression showed upregulation in 83% tissue samples (p=0.000) and 62% blood samples (p=0.388), IL12 expression showed upregulation in 62% tissue samples (p=0.435) and 57% blood samples (p=0.222) and IL13 expression showed upregulation in 83% tissue samples (p=0.001) and 68% blood samples (p=0.312). Significant positive correlation (p<0.05) was observed between tissue and blood level expression of IL8, IL12 and IL13. Different clinicopathological factors and dietary habits showed significant association (p<0.05) with IL8, IL12 and IL13 expression.. Statistically significant positive correlation were observed for IL8 and IL13 expression in tissue as well as IL13 and IL12 expression in both tissue and blood. Also significant negative correlation of IL8 and IL12 expression in blood and tissue were also observed. Tumor stage, node stage, metastasis, consumption of betel nut, tobacco, alcohol, hot food, smoked food, spices, IL8 expression in blood, IL13 expression in tissue and IL12 expression in blood and tissue showed significant association (p<0.05) with survival of ESCC patients. ConclusionsAltered expression of IL8, IL12 and IL13 may be associated with ESCC progression. Overexpression of IL8 and IL13 in tissue samples may be potential biomarkers for ECSS screening. Additionally, results from both survival and hazard analysis data indicate the effects of various parameters on the survival and mortality rate of ESCC patients.

immunology↗

bioRxiv2022

Targeting Epsins to Inhibit FGF Signaling while Potentiating TGF-β Signaling Constrains Endothelial-to-Mesenchymal-Transition in Atherosclerosis

BACKGROUNDEpsin endocytic adaptor proteins are implicated in the progression of atherosclerosis; however, the underlying molecular mechanisms have not yet been fully defined. In this study, we determined how epsins enhance endothelial-to-mesenchymal transition (EndoMT) in atherosclerosis and assessed the efficacy of a therapeutic peptide in a preclinical model of this disease. METHODSUsing single cell RNA sequencing (scRNA-seq), combined with molecular, cellular, and biochemical analyses, we investigated the role of epsins in stimulating EndoMT using knock-out mouse models. The therapeutic efficacy of a synthetic peptide targeting atherosclerotic plaques was then assessed in Apoe-/- mice. RESULTSScRNA-seq and lineage tracing revealed that epsins 1 and 2 promote EndoMT, and the loss of endothelial epsins inhibits EndoMT marker expression as well as transforming growth factor-beta signaling in vitro and in atherosclerotic mice, which is associated with smaller lesions in Apoe-/- mouse model. Mechanistically, the loss of endothelial cell epsins results in increased fibroblast growth factor receptor-1 (FGFR1) expression that inhibits TGF-{beta} signaling and EndoMT. Epsins directly bind ubiquitinated FGFR1 through their ubiquitin-interacting motif (UIM), which results in endocytosis and degradation of this receptor complex. Consequently, administration of a synthetic UIM-containing peptide API significantly attenuates EndoMT and progression of atherosclerosis. CONCLUSIONSWe conclude that epsins potentiate EndoMT during atherogenesis by increasing TGF-{beta} signaling through FGFR1 internalization and degradation. Inhibition of EndoMT by reducing epsin-FGFR1 interaction with a therapeutic peptide may represent a novel treatment strategy for atherosclerosis.

cell biology↗

bioRxiv2022

Genome-Wide Large-Scale Multi-Trait Analysis Characterizes Global Patterns of Pleiotropy and Unique Trait-Specific Variants

Genome-wide association studies (GWAS) have found widespread evidence of pleiotropy, but characterization of global patterns of pleiotropy remain highly incomplete due to insufficient power of current approaches. We develop fastASSET, an extension of the method ASSET, to allow computationally efficient detection of variant-level pleiotropic association across a large number of traits. We analyze GWAS summary statistics of 116 complex traits of diverse types collected from the NIH GRASP repository and a number of other large GWAS consortia. We identify a total of 2,293 independent loci at the genome-wide significance level and found that the lead variants in nearly all of these loci ([~]99%) to be associated with to two or more (median = 6) traits. Further, the estimated degree of pleiotropy for the detected variants strongly predicted their degree of pleiotropy across a much larger number of traits (K=4,114) in the UK Biobank Study. Follow-up analyses of 21 unique trait-specific variants suggest that they are often linked to the expression in trait-related tissues for a small number of genes, some of which are well known to be involved in relevant biological processes. Our findings provide deeper insight into the nature of complex trait pleiotropy and leads to, for the first time, identification of highly unique trait-specific susceptibility variants.

genetics↗