Biology subjects

Rao, C.

Publications and source records attributed to Rao, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Early Prenatal Cell-Free DNA Methylation Landscape Informs Epigenetic Purification of the Circulating Fetal Genome

Circulating fetal cfDNA is the analyte of a screening test used by millions of pregnant women annually. However, low fetal fraction limits current testing. Fetal cfDNA can be enriched via hypomethylation, but the informative sites require a first-trimester fetal methylome reference. Here we sequenced fetal cfDNA (median purity 94.7%) derived from discarded media from cultured chorionic villus biopsies (n=30) and assembled a first-trimester methylome across 26M CpG methylation sites at a median depth of 207x. We then built an analytical enrichment model that identified millions of CpG markers and assigned predicted enrichment scores. Guided by this model, we adapted epiSIFT as a genome-wide assay selective for unmethylated DNA molecules. In 28 prenatal plasma cases, epiSIFT raised the median fetal fraction from 5.1% to 28.4% (5.6-fold gain) using the top eighth of markers. These findings establish a first-trimester fetal cfDNA methylome reference and demonstrate epigenetic enrichment of the fetal genome.

genomics↗

bioRxiv2026

A Novel PD-L1 Splice Isoform Modulates β Cell Communication in Response to Interferon Signaling

{beta} cell expression of the immune checkpoint ligand PD-L1 (encoded by CD274) limits autoimmune {beta} cell destruction in type 1 diabetes (T1D). {beta} cell display PD-L1 not only at the cell surface but also secreted on extracellular vesicles (EVs), which bind PD-1 and restrain CD8+ T cell activation. {beta} cell IFN signaling is an early driver of T1D pathogenesis, yet the mechanisms linking IFN signaling to the fate of PD-L1 protein remain undefined. Here, we identify an IFN- and coxsackievirus-inducible alternatively spliced isoform of PD-L1, PD-L1{Delta}3, lacking cassette exon 3 and generated in human {beta} cells and islets in response to IFN signaling or viral infection. PD-L1{Delta}3 transcripts are elevated in islets from donors with single autoantibody positivity (AAB+) and T1D. Unlike full-length PD-L1, which localizes to the plasma membrane, PD-L1{Delta}3 is retained intracellularly and loses the capacity to bind PD-1. Functionally, PD-L1{Delta}3 fails to suppress CD8+ and CD4+ T cell proliferation, activation, and cytotoxic cytokine release, and is not efficiently sorted into EVs. Convergently, a germline CD274 splice-site variant (c.682+1G>A) found in siblings with neonatal T1D yields a protein with the same intracellular retention, reduced {beta} cell PD-1 binding, and reduced {beta} cell and circulating EV PD-L1. Together these findings define PD-L1{Delta}3 as an IFN-induced splice variant that diverts PD-L1 away from its immunoregulatory, EV-competent form, revealing a post-transcriptional axis that shapes {beta} cell immune communication.

cell biology↗

bioRxiv2026

Carbamylome analysis reveals regulatory roles for lysine carbamylation in β-cell glycolytic and insulin-processing enzymes

In type 1 diabetes (T1D), insulin-producing {beta} cells are destroyed by an autoimmune response driven by pro-inflammatory cytokines, including interferons. {beta}-cell cytokine signaling is mediated in part by post-translational modifications, such as phosphorylation and acetylation. However, the role of other post-translational modifications in {beta}-cell cytokine signaling represents an important knowledge gap. In the context of autoimmune diseases, lysine carbamylation has gained attention for its role in pathogenesis. Here, we investigate the role of carbamylation in T1D. We found that pancreatic islet cells from the T1D model, non-obese diabetic (NOD) mice, exhibit 11% carbamylation-positive cells, whereas non-diabetic CD1 mice have only 5%. Proteomics analysis of the MIN6 insulin-producing cell line treated with a cocktail of three pro-inflammatory cytokines IFN{gamma} + IL-1{beta} + TNF identified 284 carbamylated peptides from 222 proteins impacted by the cytokine treatment. Integration of carbamylation and acetylation provided a deep view of the cytokine-regulated PTMs and potential points of interplay. A functional-enrichment analysis revealed that carbamylation was enriched in pathways related to autoimmune diseases, metabolism, DNA replication, and protein translation. Moreover, functional testing demonstrated that carbamylation inhibits the glycolytic enzyme aldolase A and the insulin-processing enzyme carboxypeptidase E, identifying a possible role for cytokine-induced {beta}-cell dysfunction. In summary, protein carbamylation is elevated in islets from NOD mice, and pro-inflammatory cytokine treatment regulates protein carbamylation in MIN6 cells. These data identify carbamylation as a potential regulatory mechanism for {beta}-cell metabolism and insulin production in the context of islet inflammation.

biochemistry↗

bioRxiv2026

PTMOverlay: A Proteomic Tool to Visualize Post-Translational Modifications Across Evolution

Evolutionary conservation has been considered a hallmark of essential basic functions in cells. Therefore, the study of evolutionarily conserved post-translational modifications (PTMs) can provide insight into their role in protein function. In this context, mass spectrometry can identify and quantify thousands of PTM sites. However, a major bottleneck lies in analyzing the large amounts of data collected by the mass spectrometer. Here we address the need for a protein sequence alignment tool for multiple PTMs across several species. We developed a tool named PTMOverlay that takes peptide identification output files and overlays PTM sites onto multiple protein sequence alignments. Examining 31 bacteria isolates, we combined their protein sequences with select PTM types, including acetylation, phosphorylation, monomethylation, dimethylation, and trimethylation. The tool revealed a variety of conserved modification sites on the bacterial central carbon metabolism. Further structural analysis revealed possible interactions between methylated arginine and lysine residues with phosphothreonine/serine sites on the homodimer interface of enolase. Overall, this tool can parse large amounts of mass spectrometry data and allows for more informed and efficient selection of sites for future studies of protein function.

systems biology↗

bioRxiv2025

Engineering sialylation in pigs: A promising strategy for overcoming xenograft rejection and revolutionizing organ transplantation.

The organ shortage crisis leaves over 100,000 people waiting for transplants, causing 6,000 deaths annually. To address this, pigs are being explored as potential donors. Despite advances like the FDA-approved GalSafe pig, immunological challenges remain. Key issues include strong antibody, innate, and cellular immune responses, along with coagulation problems due to differences in glycosides and sialic acid linkages, which prevent long-term xenograft survival. Hyperacute rejection, caused by instant blood-mediated immune reaction (IBMIR), is a persistent problem, characterized by inflammatory and thrombotic responses when, for example, xenogeneic islets contact blood or reperfusion post anastomosis of transplanted organ. To overcome IBMIR, and other innate immune mediated rejection, we expressed human sialyltransferase (ST8Sia6) in otherwise wild type, porcine endothelium, creating human-like sialic linkages on porcine glycoproteins and lipids. This increased expression of human sialic acid 2,8- linkage on porcine endothelial cells, enhanced sialic-acid-binding immunoglobulin-like lectin (Siglec) binding reducing immune effector mechanisms such as complement activation and cell cytotoxicity. It also correlated with reduced CTL-targeted killing, lower levels of CD107a, perforin, and IFN-{gamma} production. This coincided with higher immunoreceptor tyrosine-based inhibition motif (ITIM) induction, mirroring immune tolerance seen in fetal development and tumor immune evasion. Moreover, the landscape of the expression induced transcriptome of ST8Sia6, overexpression in porcine kidney cells revealed differential expression of genes involved in immune downregulation, cell signaling, and metabolic alteration. Expression of 2,8-linked disialic acids on porcine cells protected against immune effector mechanisms, reducing complement activation, immune cell activation, and CTL killing. These findings suggest that enhanced 2,8- linked disialic acid expression can modulate the innate and adaptive immune response, reducing xenograft rejection. This approach may improve xenotransplantation success, mitigate primary non-function in xenografts, and be applied to human iPSC-derived islets and other cell products. Further research into the specific mechanisms of these immunomodulatory effects could guide the development of effective strategies for xenotransplantation.

immunology↗

bioRxiv2024

Beta cell extracellular vesicle PD-L1 as a novel regulator of CD8+ T cell activity and biomarker during the evolution of Type 1 Diabetes

Aims/hypothesisSurviving beta cells in type 1 diabetes respond to inflammation by upregulating programmed death-ligand 1 (PD-L1) to engage immune cell programmed death-1 (PD-1) and limit destruction by self-reactive immune cells. Extracellular vesicles (EVs) and their cargo can serve as biomarkers of beta cell health and contribute to islet intercellular communication. We hypothesized that the inflammatory milieu of type 1 diabetes increases PD-L1 in beta cell EV cargo and that EV PD-L1 may protect beta cells against immune-mediated cell death. MethodsBeta cell lines and human islets were treated with proinflammatory cytokines to model the proinflammatory type 1 diabetes microenvironment. EVs were isolated using ultracentrifugation or size exclusion chromatography and analysed via immunoblot, flow cytometry, and ELISA. EV PD-L1: PD-1 binding was assessed using a competitive binding assay and in vitro functional assays testing the ability of EV PD-L1 to inhibit NOD CD8 T cells. Plasma EV and soluble PD-L1 were assayed in plasma of individuals with islet autoantibody positivity (Ab+) or recent-onset type 1 diabetes and compared to non-diabetic controls. ResultsPD-L1 protein colocalized with tetraspanin-associated proteins intracellularly and was detected on the surface of beta cell EVs. 24-h IFN- or IFN-{square} treatment induced a two-fold increase in EV PD-L1 cargo without a corresponding increase in number of EVs. IFN exposure predominantly increased PD-L1 expression on the surface of beta cell EVs and beta cell EV PD-L1 showed a dose-dependent capacity to bind PD-1. Functional experiments demonstrated specific effects of beta cell EV PD-L1 to suppress proliferation and cytotoxicity of murine CD8 T cells. Plasma EV PD-L1 levels were increased in islet Ab+ individuals, particularly in those with single Ab+, Additionally, in from individuals with either Ab+ or type 1 diabetes, but not in controls, plasma EV PD-L1 positively correlated with circulating C-peptide, suggesting that higher EV-PD-L1 could be protective for residual beta cell function. Conclusions/interpretationIFN exposure increases PD-L1 on the beta cell EV surface. Beta cell EV PD-L1 binds PD1 and inhibits CD8 T cell proliferation and cytotoxicity. Circulating EV PD-L1 is higher in islet autoantibody positive patients compared to controls. Circulating EV PD-L1 levels correlate with residual C-peptide at different stages in type 1 diabetes progression. These findings suggest that EV PD-L1 could contribute to heterogeneity in type 1 diabetes progression and residual beta cell function and raise the possibility that EV PD-L1 could be exploited as a means to inhibit immune-mediated beta cell death. Research in contextO_ST_ABSWhat is already known about this subject? (maximum of 3 bullet points)C_ST_ABSO_LIExtracellular vesicles (EVs) serve as paracrine effectors in the islet microenvironment in health and disease. C_LIO_LIInterferon-alpha (IFN-) and IFN-gamma (IFN-{square}) are key cytokines contributing to type 1 diabetes pathophysiology and islet IFN signalling increases beta cell programmed death-ligand 1 (PD-L1) expression. C_LIO_LIUp-regulation of beta cell PD-L1 in the non-obese diabetic (NOD) mouse model delays progression of type 1 diabetes. C_LI What is the key question? (one bullet point only; formatted as a question)O_LIDo beta cells exposed to IFNs upregulate EV PD-L1 and can these changes be detected in circulation? C_LI What are the new findings? (maximum of 3 bullet points)O_LIIFN- or IFN-{square} exposure increases beta cell EV PD-L1 cargo in beta cell lines and human islets. C_LIO_LIPD-L1 is present on the surface of beta cell EVs, binds PD-1 and EV PD-L1 inhibits proliferation, activation and cytotoxicity of murine CD8 T cells. C_LIO_LIEV PD-L1 levels are higher in islet autoantibody positive individuals compared to nondiabetic controls and levels of circulating EV PD-L1 positively correlate with residual beta cell function in islet autoantibody positive individuals as well as in individuals with recent-onset type 1 diabetes. C_LI How might this impact on clinical practice in the foreseeable future? (one bullet point only)O_LIA beneficial effect of PD-L1+ EVs could ultimately be harnessed as an intervention to prevent autoimmune beta cell destruction. Circulating EV PD-L1 cargo has potential as a minimally invasive and informative biomarker to offer insights into the pathogenesis and progression of type 1 diabetes. C_LI

cell biology↗

bioRxiv2024

KSR1 regulates small-cell lung carcinoma tumor initiation and therapy resistance

Small-cell lung cancer (SCLC) has a dismal five-year survival rate of less than 7%, with limited advances in first line treatment over the past four decades. Tumor-initiating cells (TICs) contribute to resistance and relapse, a major impediment to SCLC treatment. Here, we identify Kinase Suppressor of Ras 1 (KSR1), a molecular scaffold for the Raf/MEK/ERK signaling cascade, as a critical regulator of SCLC TIC formation and tumor initiation in vivo. We further show that KSR1 mediates cisplatin resistance in SCLC. While 50-70% of control cells show resistance after 6-week exposure to cisplatin, CRISPR/Cas9-mediated KSR1 knockout prevents resistance in >90% of SCLC cells in ASCL1, NeuroD1, and POU2F3 subtypes. KSR1 KO significantly enhances the ability of cisplatin to decrease SCLC TICs via in vitro extreme limiting dilution analysis (ELDA), indicating that KSR1 disruption enhances the cisplatin toxicity of cells responsible for therapeutic resistance and tumor initiation. The ability of KSR1 disruption to prevent cisplatin resistant in H82 tumor xenograft formation supports this conclusion. Previous studies indicate that ERK activation inhibits SCLC tumor growth and development. We observe a minimal effect of pharmacological ERK inhibition on cisplatin resistance and no impact on TIC formation via in vitro ELDA. However, mutational analysis of the KSR1 DEF domain, which mediates interaction with ERK, suggests that ERK interaction with KSR1 is essential for KSR1-driven cisplatin resistance. These findings reveal KSR1 as a key regulatory protein in SCLC biology and a potential therapeutic target across multiple SCLC subtypes. Statement of ImplicationGenetic manipulation of the molecular scaffold KSR1 in small-cell lung cancer cells reveals its contribution to cisplatin resistance and tumor initiation.

cancer biology↗

bioRxiv2023

Regulation of bacterial stringent response by an evolutionary conserved ribosomal protein L11 methylation

Lysine and arginine methylation is an important regulator of enzyme activity and transcription in eukaryotes. However, little is known about this covalent modification in bacteria. In this work, we investigated the role of methylation in bacteria. By reanalyzing a large phyloproteomics dataset from 48 bacterial strains representing 6 phyla, we found that almost a quarter of the bacterial proteome is methylated. Many of these methylated proteins are conserved across diverse bacterial lineages, including those involved in central carbon metabolism and translation. Among the proteins with the most conserved methylation sites is ribosomal protein L11 (bL11). bL11 methylation has been a mystery for five decades, as the deletion of its methyltransferase PrmA causes no cell growth defects. A comparative proteomics analysis combined with a guanosine polyphosphate assay of the {Delta}prmA mutant in Escherichia coli revealed that bL11 methylation is important for stringent response signaling. Moreover, we show that the {Delta}prmA mutant has an abnormal polysome profile, suggesting a role in ribosomal homeostasis during stationary growth phase. Overall, our investigation demonstrates that the evolutionary conserved bL11 methylation is important for stringent response signaling and ribosomal homeostasis. ImportanceProtein methylation in bacteria was first identified over sixty years ago. Since then, its functional role has been identified for only a few proteins. To better understand the functional role of methylation in bacteria, we analyzed a large phyloproteomics dataset encompassing 48 diverse bacteria. Our analysis revealed that ribosomal proteins are often methylated at conserved residues, suggesting that methylation of these sites may have a functional role in translation. Further analysis revealed that methylation of ribosomal protein L11 is important for stringent response signaling and ribosomal homeostasis.

systems biology↗

bioRxiv2023

Nitrogen starvation causes lipid remodeling in Rhodotorula toruloides

The oleaginous yeast Rhodotorula toruloides is a promising chassis organism for the biomanufacturing of value-added bioproducts. It can accumulate lipids at a high fraction of biomass. However, metabolic engineering efforts in this organism have progressed at a slower pace than those in more extensively studied yeasts. Few studies have investigated the lipid accumulation phenotype exhibited by R. toruloides under nitrogen limitation conditions. Consequently, there have been only a few studies exploiting the lipid metabolism for higher product titers. Here, we present a multi-omic investigation of the lipid accumulation phenotype under nitrogen limitation. Through an integrative lens of transcriptomic and lipidomic analysis, we identify that R. toruloides undergoes lipid remodeling during nitrogen limitation, wherein the pool of phospholipids gets remodeled to mostly storage lipids. This insight into the mechanisms of lipid accumulation can lead to the success of future metabolic engineering strategies for overproduction of oleochemicals. HighlightsO_LIThe oleaginous yeast R. toruloides displays enhanced lipid accumulation during nitrogen starvation. C_LIO_LIA multi-omic investigation of the lipid accumulation phenotype was carried out. C_LIO_LILipid remodeling was observed during the accumulation phase, wherein carbon was transferred from phospholipids to storage lipids. C_LIO_LIMulti-omic analysis suggested that selective regulation within lipid biosynthesis controls for the specific increase of storage lipids. C_LI

systems biology↗

bioRxiv2022

SOS1 and KSR1 modulate MEK inhibitor responsiveness to target resistant cell populations based on PI3K and KRAS mutation status

KRAS is the most commonly mutated oncogene. Targeted therapies have been developed against mediators of key downstream signaling pathways, predominantly components of the RAF/MEK/ERK kinase cascade. Unfortunately, single-agent efficacy of these agents is limited both by intrinsic and acquired resistance. Survival of drug-tolerant persister cells (DTPs) within the heterogeneous tumor population and/or acquired mutations that reactivate receptor tyrosine kinase (RTK)/RAS signaling can lead to outgrowth of tumor initiating cells (TICs) and drive therapeutic resistance. Here, we show that targeting the key RTK/RAS pathway signaling intermediates SOS1 or KSR1 both enhances the efficacy of, and prevents resistance to, the MEK inhibitor trametinib in KRAS-mutated lung (LUAD) and colorectal (COAD) adenocarcinoma cell lines depending on the specific mutational landscape. The SOS1 inhibitor BI-3406 enhanced the efficacy of trametinib and prevented trametinib resistance by targeting spheroid initiating cells (SICs) in KRASG12/G13-mutated LUAD and COAD cell lines that lacked PIK3CA co-mutations. Cell lines with KRASQ61 and/or PIK3CA mutations were insensitive to trametinib and BI-3406 combination therapy. In contrast, deletion of the RAF/MEK/ERK scaffold protein KSR1 prevented drug-induced SIC upregulation and restored trametinib sensitivity across all tested KRAS mutant cell lines in both PIK3CA- mutated and PIK3CA wildtype cancers. Our findings demonstrate that vertical inhibition of RTK/RAS signaling is an effective strategy to prevent therapeutic resistance in KRAS- mutated cancers, but therapeutic efficacy is dependent on both the specific KRAS mutant and underlying co-mutations. Thus, selection of optimal therapeutic combinations in KRAS-mutated cancers will require a detailed understanding of functional dependencies imposed by allele-specific KRAS mutations. Significance StatementWe provide an experimental framework for evaluating both adaptive and acquired resistance to RAS pathway-targeted therapies and demonstrate how targeting specific RAS pathway signaling intermediates SOS1 or KSR1 enhanced effectiveness of and prevented resistance to MEK inhibitors in KRAS-mutated cancer cells with genotypic precision. The contribution of either effector was dependent upon the mutational landscape: SOS1 inhibition synergized with trametinib in KRASG12/G13-mutated cells expressing WT PI3K but not in KRASQ61-mutated cells or if PIK3CA is mutated. KSR1 deletion inhibited MEK/ERK complex stability and was effective in cells that are unresponsive to SOS1 inhibition. These data demonstrate how a detailed understanding of functional dependencies imposed both by allele specific KRAS mutations and specific co-mutations facilitates the optimization of therapeutic combinations.

cancer biology↗

bioRxiv2022

PGC1β and ERRα promote glutamine metabolism and colorectal cancer survival via transcriptional regulation of PCK2

Previous studies have shown that Peroxisome Proliferator-Activated Receptor Gamma, Coactivator 1 Beta (PGC-1{beta}) and Estrogen-Related Receptor Alpha (ERR) are over-expressed in colorectal cancer and promote tumor survival. In this study, we show that amino acid motif LRELL on PGC-1{beta} is responsible for the physical interaction with ERR and promotes ERR mRNA and protein expression. We used RNAsequencing to determine the genes regulated by both PGC-1{beta} & ERR and found that mitochondrial Phosphoenolpyruvate Carboxykinase 2 (PCK2) was the gene that decreased most significantly after depletion of both genes. Depletion of PCK2 in colorectal cancer cells was sufficient to reduce anchorage-independent growth and inhibit glutamine utilization by the TCA cycle. Lastly, shRNA-mediated depletion of ERR decreased anchorage-independent growth and glutamine metabolism, which could not be rescued by plasmid derived expression of PCK2. These findings suggest that transcriptional control of PCK2 is one mechanism used by PGC-1{beta} and ERR to promote glutamine metabolism and colorectal cancer cell survival.

cancer biology↗

bioRxiv2022

Post-transcriptional control of SRSF9 promotes the epithelial-to-mesenchymal transition (EMT) in colorectal cancer cells

In human colorectal cancer (CRC) cells the Raf/MEK/ERK scaffold Kinase Suppressor of Ras 1 (KSR1)-dependent signaling is required for the epithelial-to-mesenchymal transition (EMT)-like phenotype. Here we show that KSR1 promotes the association of differentially spliced mRNA bearing recognition sites for the Serine/Arginine-Rich (SR) splicing factor SRSF9. CRISPR/Cas9 disruption of KSR1 destabilizes SRSF9 protein, which interacts preferentially with mRNA encoding Epithelial Stromal Interaction 1 (EPSTI1). EPSTI1 protein mediates Ras and KSR1-dependent induction of EMT. Analysis of EPSTI1 splice variants reveals that inclusion of exon 8 is critical to the ability of EPSTI1 to promote the E-to N-cadherin switch and CRC cell motile and invasive behavior. These data reveal a mechanism in CRC cells in which Ras-induced and KSR1-dependent signaling affects pre-mRNA splicing to control behaviors critical to cancer cell dissemination and metastasis.

cell biology↗

bioRxiv2022

Regulation of translation by lysine acetylation in Escherichia coli

N{varepsilon}-lysine acetylation is a common post-translational modification observed in diverse species of bacteria. Aside from a few central metabolic enzymes and transcription factors, little is known about how this post-translational modification regulates protein activity. In this work, we investigated how lysine acetylation affects translation in Escherichia coli. In multiple species of bacteria, ribosomal proteins are highly acetylated at conserved lysine residues, suggesting that this modification may regulate translation. In support of this hypothesis, we found that the addition of the acetyl donors, acetyl phosphate or acetyl-Coenzyme A, inhibits translation but not transcription using an E. coli cell-free system. Further investigations using in vivo assays revealed that acetylation does not appear to alter the rate of translation elongation but rather increases the proportion of dissociated 30S and 50S ribosomes, based on polysome profiles of mutants or growth conditions known to promote lysine acetylation. Furthermore, ribosomal proteins are more acetylated in the disassociated 30S and 50S ribosomal subunit than in the fully assembled 70S complex. The effect of acetylation is also growth rate dependent, with disassociation of the subunits most pronounced during late exponential and early stationary phase growth - the same growth phase where protein acetylation is greatest. Collectively, our data demonstrate that lysine acetylation inhibits translation, most likely by interfering with subunit association. These results have also uncovered a new mechanism for coupling translation to the metabolic state of the cell. IMPORTANCENumerous cellular processes are regulated in response to the metabolic state of the cell. One such regulatory mechanism involves lysine acetylation, a covalent modification involving the transfer of an acetyl group from the central metabolites acetyl coenzyme A or acetyl phosphate to a lysine residue in a protein. This post-translational modification is known to regulate some central metabolic enzymes and transcription factors in bacteria, though a comprehensive understanding of its effect on cellular physiology is still lacking. In the present study, lysine acetylation was also found to inhibit translation in Escherichia coli by impeding ribosome association, most likely by disrupting salt-bridges along the binding interface of the 30S and 50S ribosomal subunits. These results further our understanding of lysine acetylation by uncovering a new target of regulation, protein synthesis, and aid in the design of bacteria for biotechnology applications where the growth conditions are known to promote lysine acetylation.

microbiology↗

bioRxiv2021

Characterization of opposing responses to phenol by Bacillus subtilis chemoreceptors

Bacillus subtilis employs ten chemoreceptors to move in response to chemicals in its environment. While the sensing mechanisms have been determined for many attractants, little is known about the sensing mechanisms for repellents. In this work, we investigated phenol chemotaxis in B. subtilis. Phenol is an attractant at low, micromolar concentrations, and a repellent at high, millimolar concentrations. McpA was found to be the principal chemoreceptor governing the repellent response to phenol and other related aromatic compounds. In addition, the chemoreceptors McpC and HemAT were found to the govern the attractant response to phenol and related compounds. Using receptor chimeras, McpA was found to sense phenol using its signaling domain rather than its sensing domain. These observations were substantiated in vitro, where direct binding of phenol to the signaling domain of McpA was observed using saturation-transfer difference nuclear magnetic resonance. These results further advance our understanding of B. subtilis chemotaxis and demonstrate that the signaling domain of B. subtilis chemoreceptors can directly sense chemoeffectors. IMPORTANCEBacterial chemotaxis is commonly thought to employ a sensing mechanism involving the extracellular sensing domain of chemoreceptors. Some ligands, however, appear to be sensed by the signaling domain. Phenolic compounds, commonly found in soil and root exudates, provide environmental cues for soil microbes like Bacillus subtilis. We show that phenol is sensed both as an attractant and a repellent. While mechanism for sensing phenol as an attractant is still unknown, we found that phenol is sensed as a repellent by the signaling domain of the chemoreceptor McpA. This study furthers our understanding of the unconventional sensing mechanisms employed by the B. subtilis chemotaxis pathway.

microbiology↗

bioRxiv2021

KSR1-and ERK-dependent Translational Regulation of the Epithelial-to-Mesenchymal Transition

The epithelial-to-mesenchymal transition (EMT) is considered a transcriptional process that induces a switch in cells from a polarized state to a migratory phenotype. Here we show that KSR1 and ERK promote EMT through the preferential translation of Epithelial-Stromal Interaction 1 (EPSTI1), which is required to induce the switch from E-to N-cadherin and coordinate migratory and invasive behavior. EPSTI1 is overexpressed in human colorectal cancer (CRC) cells. Disruption of KSR1 or EPSTI1 significantly impairs cell migration and invasion in vitro, and reverses EMT, in part, by decreasing the expression of N-cadherin and the transcriptional repressors of E-cadherin expression, ZEB1 and Slug. In CRC cells lacking KSR1, ectopic EPSTI1 expression restored the E-to N-cadherin switch, migration, invasion, and anchorage-independent growth. KSR1-dependent induction of EMT via selective translation of mRNAs reveals its underappreciated role in remodeling the translational landscape of CRC cells to promote their migratory and invasive behavior.

cancer biology↗

bioRxiv2020

The unconventional cytoplasmic sensing mechanism for ethanol chemotaxis in Bacillus subtilis

Motile bacteria sense chemical gradients using chemoreceptors, which consist of distinct sensing and signaling domains. The general model is that the sensing domain binds the chemical and the signaling domain induces the tactic response. Here, we investigated the unconventional sensing mechanism for ethanol taxis in Bacillus subtilis. Ethanol and other short-chain alcohols are attractants for B. subtilis. Two chemoreceptors, McpB and HemAT, sense these alcohols. In the case of McpB, the signaling domain directly binds ethanol. We were further able to identify a single amino-acid residue Ala431 on the cytoplasmic signaling domain of McpB, that when mutated to a serine, reduces taxis to ethanol. Molecular dynamics simulations suggest ethanol binds McpB near residue Ala431 and mutation of this residue to serine increases coiled-coil packing within the signaling domain, thereby reducing the ability of ethanol to bind between the helices of the signaling domain. In the case of HemAT, the myoglobin-like sensing domain binds ethanol, likely between the helices encapsulating the heme group. Aside from being sensed by an unconventional mechanism, ethanol also differs from many other chemoattractants because it is not metabolized by B. subtilis and is toxic. We propose that B. subtilis uses ethanol and other short-chain alcohols to locate prey, namely alcohol-producing microorganisms. ImportanceEthanol is a chemoattractant for Bacillus subtilis even though it is not metabolized and inhibits growth. B. subtilis likely uses ethanol to find ethanol-fermenting microorganisms for prey. Two chemoreceptors sense ethanol: HemAT and McpB. HemATs myoglobin-like sensing domain directly binds ethanol, but the heme group is not involved. McpB is a transmembrane receptor consisting of an extracellular sensing domain and a cytoplasmic signaling domain. While most attractants bind the extracellular sensing domain, we found that ethanol directly binds between inter-monomer helices of the cytoplasmic signaling domain of McpB, using a mechanism akin to those identified in many mammalian ethanol-binding proteins. Our results indicate that the sensory repertoire of chemoreceptors extends beyond the sensing domain and can directly involve the signaling domain.

microbiology↗

bioRxiv2020

A pathway of NADPH diaphorase positivity between central canal and pial surface at anterior fissure in spinal cord: Supra fissure area with hypothesis configuring from dog, rat, monkey and pigeon

The spinal cord is a cylinder structure in the vertebra and thought a simplified with the gray matter and white matter. Rexed lamination for the gray matter and regional sub-division for whiter matter are completely termed to date. Anterior commissure locates between the central canal and the anterior median fissure. However, some experimental data may still confront with new confined anatomical interpretation. By using NADPH diaphorase [N-d] enzyme histology, we found a vertical oriented neuronal pathway between the central canal and the anterior median fissure in the sacral spinal cord of young adult and aged dog. We used a term "supra fissure area" [SFA] to illustrate the region which consisted of the gray commissure and anterior white commissure. The N-d pathway was notably observable in aged animals. The vertical neurites revealed the cerebrospinal fluid [CSF] contacting neurites between the anterior median fissure and the central canal. We further examined the monkey, rat and pigeon in the region for better understanding of the structure and potential function. The neurodegeneration of N-d dystrophy was detected in the [SFA] in the thoracic spinal cord of the aged monkey. N-d positive fibers were detected in anterior fissure of the rat spinal cord. N-d fibrous structures were also detected in the pigeon spinal cord. These results suggested a new pathway of CSF contacting neurons and the neuronal communications about the central canal.

neuroscience↗

bioRxiv2020

Multi-kingdom quantitation reveals distinct ecological drivers of predictable early-life microbiome assembly

The infant gut microbiota develops remarkably predictably1-7, with pioneer species colonizing after birth, followed by an ordered succession of other microbes. This predictable assembly is vital to health8,9, yet the forces underlying it remain unknown. The environment, host and microbe-microbe interactions are all likely to shape microbiota dynamics, but in such a complex ecosystem identifying the specific role of any individual factor has remained a major challenge10-14. Here we use multi-kingdom absolute abundance quantitation, ecological modelling, and experimental validation to overcome this challenge. We quantify the absolute bacterial, fungal, and archaeal dynamics in a longitudinal cohort of 178 preterm infants. We uncover with exquisite precision microbial blooms and extinctions, and reveal an inverse correlation between bacterial and fungal loads the infant gut. We infer computationally then demonstrate experimentally in vitro and in vivo that predictable assembly dynamics are driven by directed, context-dependent interactions between microbes. Mirroring the dynamics of macroscopic ecosystems15-17, a late-arriving member,Klebsiella, exploits the pioneer,Staphylococcus, to gain a foothold within the gut. Remarkably, we find that interactions between kingdoms drive assembly, with a single fungal species,Candida albicans, inhibiting multiple dominant gut bacteria. Our work unveils the centrality of simple microbe-microbe interactions in shaping host-associated microbiota, a critical step towards targeted microbiota engineering.

microbiology↗