Biology subjects

Sarkar, S.

Publications and source records attributed to Sarkar, S..

At least 91 records · Page 5Linked to original sources

bioRxiv2024

Genomic and Phenomic Prediction for Soybean Seed Yield, Protein, and Oil

Developments in genomics and phenomics have provided valuable tools for use in cultivar development. Genomic prediction (GP) has been used in commercial soybean [Glycine max L. (Merr.)] breeding programs to predict grain yield and seed composition traits. Phenomic prediction (PP) is a rapidly developing field that holds the potential to be used for the selection of genotypes early in the growing season. The objectives of this study were to compare the use and performance of GP and PP for predicting soybean seed yield, protein content, and oil content. We additionally conducted Genome Wide Association Studies (GWAS) to identify significant SNPs associated with the traits of interest. These SNPs were also used to train the GP models. The GWAS panel of 292 diverse accessions was grown in six environments in replicated trials. Spectral data were collected at three timepoints during the growing season. A GBLUP model was trained on 268 accessions, while three separate machine learning (ML) models were trained on vegetation indices (VIs) and canopy traits. We observed that for PP, Random Forest (RF) algorithm had the highest rank correlation between the predicted and the actual phenotype rank. PP had a higher correlation coefficient than GP for seed yield, while GP had higher correlation coefficients for seed protein and oil contents. VIs with high feature importance were used as covariates in a new GBLUP model, and a new RF model was trained with the inclusion of selected SNPs from the GWAS results. These models did not outperform the original GP and PP models. These results show the capability of using ML for in-season predictions for specific traits in soybean breeding and provide insights on PP and GP inclusions in breeding programs. PLAIN LANGUAGE SUMMARYObtaining DNA information can often be costly for a public breeding program to obtain, which is a barrier of entry for using genomic prediction in these programs to make selections. Phenomic prediction provides an alternative opportunity that does not require the recurring costs of DNA sequencing. This research aimed to compare genomic and phenomic prediction in a diverse panel of soybeans. We found that phenomic prediction had the highest accuracy for seed yield, while genomic prediction was the best model for seed protein and oil. Combining genomic and phenomic data did not improve the predictive ability of the models. Core ideas O_LIIn-season phenomic prediction is able to outperform genomic prediction for seed yield. C_LIO_LIGenomic prediction outperformed in-season phenomic prediction models for seed protein and oil. C_LIO_LIOverlapping spectral indices were identified as the most predictive for seed yield, protein, and oil. C_LIO_LIFusion of genomic and phenomic prediction did not increase the predictive ability of the combined models. C_LI

plant biology↗

bioRxiv2024

Divergent Functions of Late ESCRT Components in Giardia lamblia: Insights from Subcellular Distributions and Protein Interactions

Giardia lamblia, a human gut pathogen, possesses a minimal ESCRT (Endosomal Sorting Complex Required for Transport) machinery. Paradoxically, there are multiple paralogs of some late-ESCRT components. There are three paralogs for Vps4, GlVps4a, GlVps4b, and GlVps4c, and two for Vps46, GlVps46a, and GlVps46b. This study addressed whether these paralogs discharge overlapping and/or distinct cellular functions by determining the sub-cellular distribution of the paralogs in trophozoites and during encysting. Consistent with the distribution of orthologs in model organisms, most of these components were found to be associated with various cellular membranes, particularly in regions of acute membrane bending. Some of these paralogs are also associated with microtubule structures, such as cytoplasmic axonemes and the median body. Considering their diverse sub-cellular distributions, it is likely that they perform non-overlapping functions within the cell. Furthermore, their redistribution during encystation indicates that they may play a role in the morphological and functional changes accompanying this transition. The study also characterized GlIst1, an ESCRT-III accessory protein that undergoes unique post-translational myristoylation at lysine 43, potentially aiding its membrane recruitment. GlIst1 selectively interacts with GlVps4b through non-canonical MIT-MIM interactions. GlIst1 also exhibits selective interaction with GlVps46b. Such selective interaction of GlIst1 with only specific paralogs of GlVps4 and GlVps46 further underscores the distinct cellular roles of these paralogs. Author SummaryGiardia lamblia, a unicellular protozoan parasite, is the causative agent of giardiasis, a water-transmitted disease affecting millions globally. This disease poses a substantial threat to public health, especially in less developed countries, where clean water and proper sanitation are scarce. The parasite manifests in two morphologically distinct forms, trophozoites and cysts. Transformation between these forms is essential for the organisms survival, spread, and infection processes. Trophozoites, the active and motile form of Giardia, inhabit the small intestine of the host and trigger infections. These trophozoites can transform into cysts through encystation, enabling the parasite to endure harsh external environments and spread between hosts through contaminated water or food sources. The transition between these states necessitates extensive membrane restructuring. Such changes are likely to involve the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, as it has been shown to participate in both prokaryotic and eukaryotic membrane remodeling events. Our research sheds light on the ESCRT machinery in G. lamblia, a crucial membrane-shaping system that may facilitate the transition between trophozoites and cysts. The ESCRT machinery in G. lamblia is distinct from that in yeast and humans, representing one of the most basic ESCRT systems. Our investigation provides valuable information about the intracellular distribution of various late-ESCRT components under different conditions, their potential functions, and their interactions with other late-ESCRT components. These findings may contribute significantly to our understanding of the basic operation of the ESCRT machinery in this parasite.

cell biology↗

bioRxiv2024

Cigarette smoke impairs the endocytotic process in Saccharomyces cerevisiae

The accumulation of misfolded proteins inside the cells has been considered to be an important contributor to the development of cigarette smoke-mediated diseases. Since endocytosis plays a crucial role in protein trafficking and clearance, impaired endocytosis may contribute to cigarette smoke-mediated protein accumulation. Therefore, the current study investigated the effects of cigarette smoke extract (CSE) on the endocytosis process in yeast Saccharomyces cerevisiae. The current study showed that treatment of cells with CSE caused reduced uptake of FM4-64 stain, indicating impaired endocytosis. Further analysis revealed that CSE treatment resulted in a defect in the recruitment of proteins involved in endocytosis. Also, aberrant actin morphology was found upon CSE treatment, which might interfere with vesicle budding from the membrane. Moreover, the current study showed that the PI4,5P2 level in the plasma membrane in CSE treated cells is reduced due to the failed translocation of MSS4 kinase to the membrane. This reduced PI4,5P2 results in aberrant actin morphology. Thus, the current study demonstrates that CSE treatment causes endocytosis defects and provides insight into this defective endocytosis.

cell biology↗

bioRxiv2024

Evolutionary history with chronic malnutrition enhances pathogen susceptibility at older ages

Juvenile malnutrition is a global public health concern that negatively impacts the development and maturation of the immune system, leading to increased susceptibility to infectious diseases. Such adverse effects on immunity might increase with ageing, worsening disease conditions later in life. Furthermore, malnutrition may persist across generations, imposing strong natural selection to survive the nutrient shortage. However, it is unclear how the evolutionary history of ancestral generations with chronic malnutrition could influence pathogen resistance and infection susceptibility, as well as their age-specific changes in extant generations. To address this, we used Drosophila melanogaster populations adapted against chronic juvenile malnutrition and exposed them to a bacterial pathogen, Providencia rettgeri, during their early and late adulthood. Surprisingly, we observed that in populations adapted to chronic malnutrition, young flies survived infection better by tolerating the infection, while control flies displayed higher infection susceptibility despite carrying a similar pathogen load. However, this pattern in post-infection survival is reversed with ageing. There was no change in pathogen resistance, but evolved flies succumbed more to infection than control flies regardless of the input infection doses. Our study thus revealed new evolutionary insights into the development of contrasting early vs late-life immune strategies and age-specific vulnerabilities to infection as a function of early-life malnutrition.

evolutionary biology↗

bioRxiv2024

The binary protein interactome mapping of the Giardia lamblia proteasome lid reveals extra proteasomal functions of GlRpn11

Giardia lamblia does not encode Rpn12 and Sem1, two proteins crucial for assembling the proteasome lid. To understand how the interactions between the giardial proteasome lid subunits may have changed to compensate for their absence, we used the yeast two-hybrid assay to generate a binary protein interaction map of the Giardia lid subunits. Most interactions within the Giardia proteasome lid are stronger than those within the Saccharomyces cerevisiae lid. These may compensate for the absence of Rpn12 and Sem1. A notable exception was the weaker interaction between GlRpn11 and GlRpn8, compared to the strong interaction between Rpn11-Rpn8 of yeast. The Rpn11-Rpn8 dimer provides a platform for lid assembly and their interaction involves the insertion of a methionine residue of Rpn11 into a hydrophobic pocket of Rpn8. Molecular modeling indicates that GlRpn8s pocket is wider, reconciling the experimental observation of its weak interaction with GlRpn11. This weaker interaction may have evolved to support extra proteasomal functions of GlRpn11, which localizes to multiple subcellular regions where other proteasome subunits have not been detected. One such location is the mitosome. Functional complementation in yeast shows that GlRpn11 can influence mitochondrial function and distribution. This, together with its mitosomal localization, indicates that GlRpn11 functions at the mitosome. Thus, this parasites proteasome lid has a simpler subunit architecture and structural attributes that may support dual functionalities for GlRpn11. Such parasite-specific proteasome features could provide new avenues for controlling the transmission of Giardia. 1 Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/613619v1_ufigs1.gif" ALT="Figure 0"> View larger version (33K): org.highwire.dtl.DTLVardef@989e69org.highwire.dtl.DTLVardef@1c8134aorg.highwire.dtl.DTLVardef@6a9794org.highwire.dtl.DTLVardef@5ac20_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGiardia genome does not encode two proteasomal lid subunits: Rpn12 and Sem1 C_LIO_LIUnique interactions within the lid may compensate for the absence of these two C_LIO_LIGlRpn8:GlRpn11 weakly interacts to support GlRpn11s extra-proteasomal distribution C_LIO_LIGlRpn11 localizes at mitosomes, OZ of VD, and to the VFP C_LIO_LIThe 182-218 fragment of GlRpn11 may regulate mitosomal function C_LI

cell biology↗

bioRxiv2024

Chaperone-mediated heterotypic phase separation prevents the amyloid formation of the pathological Y145Stop variant of the prion protein

Biomolecular condensates formed via phase separation of proteins and nucleic acids are crucial for the spatiotemporal regulation of a diverse array of essential cellular functions and the maintenance of cellular homeostasis. However, aberrant liquid-to-solid phase transitions of such condensates are associated with several fatal human diseases. Such dynamic membraneless compartments can contain a range of molecular chaperones that can regulate the phase behavior of proteins involved in the formation of these biological condensates. Here, we show that a heat shock protein 40 (Hsp40), Ydj1, exhibits a holdase activity by potentiating the phase separation of a disease-associated stop codon mutant of the prion protein (Y145Stop) either by recruitment into Y145Stop condensates or via Y145Stop-Ydj1 two-component heterotypic phase separation that prevents the conformational conversion of Y145Stop into amyloid fibrils. Utilizing site-directed mutagenesis, multicolor fluorescence imaging, single-droplet steady-state and picosecond time-resolved fluorescence anisotropy, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy, we delineate the complex network of interactions that govern the heterotypic phase separation of Y145Stop and Ydj1. We also show that the properties of such heterotypic condensates can further be tuned by RNA that promotes the formation of multicomponent multiphasic protein-RNA condensates. Our vibrational Raman spectroscopy results in conjunction with atomic force microscopy imaging reveal that Ydj1 effectively redirects the self-assembly of Y145Stop towards a dynamically-arrested non-amyloidogenic pathway, preventing the formation of typical amyloid fibrils. Our findings underscore the importance of chaperone-mediated heterotypic phase separation in regulating aberrant phase transitions and amyloid formation associated with a wide range of deadly neurodegenerative diseases.

biophysics↗

bioRxiv2024

Two paralogues of N-ethylmaleimide sensitive factor: An exception to the minimal vesicular trafficking machinery of Giardia

Vesicular trafficking plays a critical role in the survival of the human gut pathogen Giardia lamblia as it drives nutrient uptake and morphological stage transition. Unlike most eukaryotes, Giardia has a minimal vesicular trafficking machinery. Herein, we report a rare exception to this minimalism wherein two paralogues of NSF, a crucial factor driving vesicular trafficking by uncoupling the cis-SNARE bundle, are present in this unicellular parasite. While GlNSF114776 and GlNSF112681 share very high sequence homology, they are likely to have distinct cellular roles as they exhibit differences in their affinities towards the Gl-SNAPs and display non-overlapping distribution in encysting trophozoites. Under multiple stress conditions (nutritional, oxidative and nitrosative), while GlNSF112681 remains at peripheral vesicles, GlNSF114776 relocalizes to the anterior flagella-associated striated fibres, indicating a possible role in regulating flagellar motility. At this location, GlNSF114776 is likely to perform a 20S complex independent function as neither Gl-SNAPs nor GlSNAREs are present there. The two paralogues are likely needed for stress adaptation as both copies have also been retained in Giardia genomes isolated from clinical samples. This non-canonical function of the GlNSF114776 may have evolved to support the unique architecture and motility of the anterior flagella.

cell biology↗

bioRxiv2024

Single-cell analysis of human diversity in circulating immune cells

Lack of diversity and proportionate representation in genomics datasets and databases contributes to inequity in healthcare outcomes globally1,2. The relationships of human diversity with biological and biomedical phenotypes are pervasive3, yet remain understudied, particularly in a single-cell genomics context. Here we present the Asian Immune Diversity Atlas (AIDA), a multi-national single-cell RNA-sequencing (scRNA-seq) healthy reference atlas of human immune cells. AIDA comprises 1,265,624 circulating immune cells from 619 healthy donors and 6 controls, spanning 7 population groups across 5 countries. AIDA is one of the largest healthy blood datasets in terms of number of cells, and also the most diverse in terms of number of population groups. Though population groups are frequently compared at the continental level, we identified a pervasive impact of sub-continental diversity on cellular and molecular properties of immune cells. These included cell populations and genes implicated in disease risk and pathogenesis as well as those relevant for diagnostics. We detected single-cell signatures of human diversity not apparent at the level of cell types, as well as modulation of the effects of age and sex by self-reported ethnicity. We discovered functional genetic variants influencing cell type-specific gene expression, including context-dependent effects, which were under-represented in analyses of non-Asian population groups, and which helped contextualise disease-associated variants. We validated our findings using multiple independent datasets and cohorts. AIDA provides fundamental insights into the relationships of human diversity with immune cell phenotypes, enables analyses of multi-ancestry disease datasets, and facilitates the development of precision medicine efforts in Asia and beyond.

genomics↗

bioRxiv2024

Highly synchronized inhibition from Purkinje cells entrains cerebellar output in zebrafish

Cerebellar function, known to be important for motor learning and motor coordination, is mediated by efferent neurons that project to diverse motor areas. To understand cerebellar function, it is imperative to study how these efferent neurons integrate inputs from the principal neurons of the cerebellar cortex, the inhibitory Purkinje neurons (PNs). In zebrafish, PNs are bistable and we show here that bistability influences spike synchrony among PNs. Bistability also alters spike correlation with motor bouts. We asked how PN population synchrony influences Eurydendroid cells (ECs), which are postsynaptic targets of PNs and are the cerebellar efferent cells in zebrafish. Using optogenetics, we artificially modulated population synchrony of PNs over millisecond time scales and showed that under conditions of high synchrony, EC firing is briefly suppressed and entrained by PN spiking. However, the magnitude of such modulation is relatively small and indicates a strong combined influence of other synaptic inputs on EC spiking. Key PointsO_LICerebellar Purkinje neurons (PN) in larval zebrafish alter simple spike correlations with each other based on cellular state. C_LIO_LIThey also alter simple spike correlations with motor bouts as a function of state. C_LIO_LIWe altered PN population synchrony in a graded manner using optogenetics. C_LIO_LIPN targets are cerebellar efferent neurons, which in teleosts are called eurydendroid cells. C_LIO_LIWhen PN population is firing with high synchrony, eurydendroid cells are entrained better than when the PN input is asynchronous. C_LIO_LIThis can explain how PNs use bistability to modulate their influence on cerebellar output and ultimately, motor behavior. C_LI

neuroscience↗

bioRxiv2024

Conformational dynamics of electrostatically charged Intrinsically Disordered Proteins

We experimentally and computationally study the dynamics of homogeneously electrically charged intrinsically disordered proteins (IDPs) in ionic solutions (aqueous solutions containing salt). Computationally, this is achieved by extending a recently introduced discrete elastic rod (DER) model to include electrostatic interactions. The respective model includes only three free parameters: bending stiffness, bond length, and hydrodynamic radius. These parameters were obtained by fitting the measured conformational dynamics of uncharged polypeptides as reported in a previous publication [8]. We compare our computational results with photo-electron transfer fluorescence correlation spectroscopy (PET-FCS) measurements of the conformational dynamics of the highly charged (-44 e-) intrinsically disordered protein Prothymosin . We here report on an agreement between the loop-closing rates obtained from our computational model and the respective experimental measurements, with rate values significantly slower than those observed for uncharged IDPs.

biophysics↗

bioRxiv2024

A general approach to reduce off-target radioactivity in vivo via Tetrazine-Knock-Out (TKO)

Monoclonal antibodies have had a remarkable impact on cancer therapy due to their high target specificity. However, their large molecular weight results in slow blood clearance, which can take weeks to clear from circulation. As companion nuclear imaging and diagnostic tools, these characteristics force delayed imaging and the use of isotopes with long half-lives such as 89Zr. For optimal clinical application, it is desirable that radioimmunoconjugates remain in the blood for just enough time to accumulate adequately in target tissues, while non-targeted or circulating radioactivity is ideally rapidly excreted from the body to maximize imaging contrast and minimize radiation dose to healthy tissues. We addressed the current challenges of antibody-based imaging by developing rituximab radioimmunoconjugates that accumulate sufficient activity for tumor imaging within 24 h of administration, while clearing circulating radioactivity via administration of a small molecule clearing agent. Rituximab, an anti-CD20 monoclonal antibody, is used as standard first-line therapy for diffuse large B-cell lymphoma. CD20 is expressed by 95% of B-lymphocytes and their malignant counterparts, making it a therapeutic target for B-cell malignancies. We attached 125I, 68Ga, and 89Zr to rituximab using a "clickable" linker containing trans-cyclooctene and tested the ability of tetrazines to induce the inverse electron demand Diels-Alder reaction (iEDDA) after antibody administration. This "tetrazine-knock-out" (TKO) approach liberates the radioactivity from rituximab in the bloodstream, resulting in its rapid renal excretion which enhances target-to-background ratios, and minimizes off-target radiation exposure. Due to the internalization of the radioimmunoconjugate in CD20+ tumor cells, no substantial clearance was observed from Raji xenografts. We characterized different leaving groups, several cellular models and antibodies with distinct internalizaing properties. The TKO approach opens opportunities to use radiolabeled antibodies for low-abundance or heterogeneously expressed biologic targets and may allow radioimmunotherapy (RIT) for targets traditionally untenable due to dose-limiting toxicities. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/596510v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@11bc2f0org.highwire.dtl.DTLVardef@19b4f72org.highwire.dtl.DTLVardef@17cee32org.highwire.dtl.DTLVardef@fa4491_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Spatial cell graph analysis reveals skin tissue organization characteristic for cutaneous T cell lymphoma

Cutaneous T cell lymphomas (CTCLs) are non-Hodgkin lymphomas caused by malignant T cells which migrate to the skin. The cancerous T cells lead to rash-like lesions which can be difficult to distinguish from inflammatory skin conditions like atopic dermatitis (AD) and psoriasis (PSO). To characterize CTCL in comparison to these differential diagnoses, we carried out multi-antigen imaging on 69 skin tissue samples (21 CTCL, 23 AD, 25 PSO). The resulting spatially resolved protein abundance maps were then analyzed via scoring functions to quantify heterogeneity of the individual cells neighborhoods within spatial graphs inferred from the cells positions in the tissue samples (available as a Python package at https://github.com/bionetslab/SHouT). Our analyses reveal several characteristic patterns of skin tissue organization in CTCL, including a combination of increased local entropy and egophily as characteristic properties of spatial T cell neighborhoods in CTCL as compared to AD and PSO. These results could not only pave the way for high-precision diagnosis of CTCL, but may also facilitate further insights into cellular disease mechanisms.

bioinformatics↗

bioRxiv2024

G-Quadruplex Mediated c-myc Specific Downregulation: A Unique Pathway of the Anticancer Action of Immunomodulator Drugs

AbstractHydroxychloroquine (HCQ), and chloroquine (CQ) are in the preclinical trial stage for cancer along with their active application in autoimmune diseases and malaria. One of the critical hallmarks of cancer cells is the elevated expression of various oncogenes which promote cancer progression and contribute to poor prognosis. The upstream of the promoter region of these oncogenes often exhibits a G-quadruplex (G4) DNA structure which regulates the gene expression. Hence, targeting G4 structure has emerged as a promising therapeutic strategy for cancer. In this study, the recognition of HCQ and CQ with the G4 structure of different oncogenes and its effect on gene regulation has been explored by a combination of various biophysical and in-vitro and in-vivo biological methods. This study depicts that HCQ and CQ downregulate the c-myc oncogene transcription significantly in a G4-dependent manner compared to other oncogenes. The different biophysical techniques and molecular dynamics simulation studies illustrate that these drug molecules stack predominately at the terminal of the c-myc G4 and the binding of these molecules stabilizes c-myc G4 significantly higher than the G4 structure of other oncogenes. The in-vitro cell data exhibit a notable reduction in both c-myc mRNA and protein levels in a triple-negative breast cancer cell line following HCQ treatment. The pre-clinical breast cancer mouse model in-vivo data also indicate that HCQ reduces tumor growth through the downregulation of the c-myc oncogene. Simultaneously, HCQ also enhances the therapeutic efficacy of standard chemotherapeutic agents to be a potential candidate for combination therapy. This work demonstrates the alternative strategy of anticancer action of widely used drugs by specifically downregulating the c-myc oncogene in a G4-dependent manner.

biochemistry↗

bioRxiv2024

Genetic screening and metabolomics identify glial adenosine metabolism as a therapeutic target in Parkinson's disease

Neuronal alpha-synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB), lack disease-modifying therapies. We developed a Drosophila model for identifying novel glial-based therapeutic targets for alpha-synucleinopathies. In this model, human alpha-synuclein is expressed in neurons and individual genes are independently knocked down in glia. We performed a forward genetic screen, knocking down the entire Drosophila kinome in glia in alpha-synuclein expressing flies. Among the top hits were five genes (Ak1, Ak6, Adk1, Adk2, and awd) involved in adenosine metabolism. Knockdown of each gene increased brain adenosine levels, improved locomotor dysfunction, reduced alpha-synuclein oligomer levels, and rescued neurodegeneration. We determined that the mechanism of neuroprotection requires adenosine signaling through the neuronal adenosine receptor (AdoR) in Drosophila. These experiments support glial adenosine as a novel therapeutic target in PD.

neuroscience↗

bioRxiv2024

Eubacterium rectale detoxification mechanism increases resilience of the gut environment

Lachnospiraceae members were highly detected in dysbiotic IL-10 KO mice that displayed similar physiological outcomes as control mice. Lachnospiraceae is a highly diverse family of microbes that have been shown to display both commensal and pathogenic characteristics in the colon environment. We investigated the impact of genetic variation in five Lachnospiraceae strains on lowering cellular inflammation and ROS levels. Cell free spent media (CFSM) from Eubacterium rectale resulted in lowered ROS, and nitric oxide levels in stressed colon cells. We demonstrated through an array of multi omics and molecular techniques that glutathione (GSH) biosynthesized by E. rectale was able to alleviate host ROS damage. We further showed downregulation of cell stress and immune response genes by host RNA sequencing, which is evidence that E. rectale microbial products promote recovery and alleviate ROS stress. HighlightsO_LILachnospiraceae detection correlated with lower host inflammation in vertically transmitted dysbiotic mice C_LIO_LICell free spent media from E. rectale lowered ROS and nitric oxide levels when introduced to stressed Caco-2 cells more effectively than 4 other strains of Lachnospiraceae C_LIO_LIEubacterium rectale spent media products include high levels of glutathione (GSH), carbohydrates, and amino acids C_LIO_LIEubacterium rectale potential gene function for glutathione biosynthesis and metabolism found in the genome C_LIO_LILower expression of repair and stress genes of intestinal cells treated with hydrogen peroxide was observed with E. rectale CFSM as well as treatment with 15 mM GSH C_LI

microbiology↗

bioRxiv2024

Microenvironment Drives Reentrant Condensation of Aβ40

Within the framework of liquid-liquid phase separation (LLPS), biomolecular condensation orchestrates vital cellular processes and its dysregulation is implicated in severe pathological conditions. Recent studies highlight the role of intrinsically disordered proteins (IDPs) in LLPS, yet the influence of microenvironmental factors has remained a puzzling factor. Here, via computationally simulating the impact of solution conditions on LLPS behavior of neurologically pathogenic IDP A{beta}40, we chanced upon a salt-driven reentrant condensation phenomenon, wherein A{beta}40 aggregation increases with low salt concentrations (25-50 mM), followed by a decline with further salt increments.. An exploration into the thermodynamic and kinetic signatures of reentrant condensation unveils a nuanced interplay between protein electrostatics and ionic strength as potential drivers. Notably, the charged residues of the N-terminus exhibit a non-monotonic response to salt screening, intricately linked to the recurrence of reentrant behavior in hydrophobic core-induced condensation. Intriguingly, our findings also unveil the reappearance of similar reentrant condensation phenomena under varying temperature conditions. Collectively, our study illuminates the profoundly context-dependent nature of A{beta}40s liquid-liquid phase separation behavior, extending beyond its intrinsic molecular framework, where microenvironmental cues wield significant influence over its aberrant functionality.

biophysics↗

bioRxiv2024

Potent neutralization by a receptor binding domain monoclonal antibody with broad specificity for SARS-CoV-2 JN.1 and other variants

SARS-CoV-2 continues to be a public health burden, driven in-part by its continued antigenic diversification and resulting emergence of new variants. While increasing herd immunity, current vaccines, and therapeutics have improved outcomes for some; prophylactic and treatment interventions that are not compromised by viral evolution of the Spike protein are still needed. Using a rationally designed SARS-CoV-2 Receptor Binding Domain (RBD) - ACE2 fusion protein and differential selection process with native Omicron RBD protein, we developed a recombinant human monoclonal antibody (hmAb) from a convalescent individual following SARS-CoV-2 Omicron infection. The resulting hmAb, 1301B7 potently neutralized a wide range of SARS-CoV-2 variants including the original Wuhan and more recent Omicron JN.1 strain, as well as SARS-CoV. Structure determination of the SARS-CoV-2 EG5.1 Spike/1301B7 Fab complex by cryo-electron microscopy at 3.1[A] resolution demonstrates 1301B7 contacts the ACE2 binding site of RBD exclusively through its VH1-69 heavy chain, making contacts using CDRs1-3, as well as framework region 3 (FR3). Broad specificity is achieved through 1301B7 binding to many conserved residues of Omicron variants including Y501 and H505. Consistent with its extensive binding epitope, 1301B7 is able to potently diminish viral burden in the upper and lower respiratory tract and protect mice from challenge with Omicron XBB1.5 and Omicron JN.1 viruses. These results suggest 1301B7 has broad potential to prevent or treat clinical SARS-CoV-2 infections and to guide development of RBD-based universal SARS-CoV-2 prophylactic vaccines and therapeutic approaches.

immunology↗

bioRxiv2024

Autoaggregation in Streptococcus intermedius is driven by the Pel polysaccharide

The Streptococcus Milleri Group (SMG) comprising of Streptococcus intermedius, anginosus and constellatus are commensal bacteria commonly found in healthy individuals. These bacteria are increasingly being recognized as opportunistic pathogens that can cause purulent infections at sterile body sites and have also been identified in the sputum of individuals with cystic fibrosis. Although the mechanisms of conversion to opportunistic pathogens are not well understood, auto-aggregation is a key driver of biofilm adhesion and cohesion in many Streptococci and Staphylococci. Here, we identify a gene cluster in the S. intermedius genome with significant homology to the pel operons in Bacillus cereus and Pseudomonas aeruginosa, which are required for Pel exopolysaccharide production and biofilm formation in these species. Characterization of a panel of clinical S. intermedius strains identified a range of aggregating phenotypes. Analysis of the pel operon in the hyper-aggregating C1365 strain revealed that each of the canonical pelDEADAFG genes, but not the four additional genes are required for aggregation. Further, we demonstrate that C1365 produces a GalNAc-rich exopolysaccharide and that aggregates can be disrupted by the 1,4 N-acetylgalactosaminidases, PelA and Sph3, but not other glycoside hydrolases, proteinase K or DNase I. Using an abscess model of mouse infection, we show that Pel driven aggregation leads to longer lasting infections, and that lack of Pel allows for the bacteria to be cleared more effectively. The polymer also affects how the bacteria interacts with the host immune system. Collectively, our data suggest that the pel operon has relevancy to S. intermedius pathogenicity.

microbiology↗