Biochimie

This study presents a phytochemical and pharmacological investigation of Aruncus dioicus, a medicinal plant collected from the northeastern coastal region of Vietnam. In light of the growing global prevalence of type 2 diabetes mellitus (T2DM), the search for natural compounds capable of modulating key enzymes involved in glucose metabolism, particularly Protein Tyrosine Phosphatase 1B (PTP1B) and -glucosidase, remains an important research objective. The experimental methods employed included: botanical identification, extraction, chromatographic separation, and biological activity evaluation. As a result, eleven pure compounds were isolated. Structural determination via 1H- and 13C-NMR spectroscopy revealed these constituents as phenylpropanoids, phenolic acids, nucleosides, and ester derivatives, thereby establishing a distinctive chemical profile for the Vietnamese population of A. dioicus. In vitro enzyme inhibition tests demonstrated significant biological activity. p-coumaric acid (Compound 3) and cinnamic acid (Compound 4) exhibited effects on PTP1B, with IC{square}{square} values of 0.25 {micro}M and 1.16 {micro}M, respectively, higher than the activity of the reference compound ursolic acid (IC{square}{square} = 3.5 {micro}M). Furthermore, ethylparaben (Compound 7) and cinnamic acid exhibited -glucosidase inhibition, with potencies approximately five- to six-fold greater than that of acarbose. These findings suggest that A. dioicus is a potentially valuable source of antidiabetic agents and emphasize the significance of phenylpropanoid derivatives in enzyme inhibition associated with glucose metabolism, thereby providing a scientific foundation for subsequent pharmacological investigations.

The KRAS oncogene, central to cellular signaling via MAPK and PI3K-AKT pathways, is a notorious cancer driver frequently activated in pancreatic, colorectal, and lung carcinomas. Regulation of human KRAS oncogene expression is important due to its capital role in cell growth, proliferation, and survival. Misregulation of its expression contributes directly to the development and progression of multiple types of cancer. In previous studies, the role of G-quadruplexes elements in both the promoter and 5 UTR regions have shown to play important roles in KRAS expression, particularly when these G4s elements interact with regulatory protein hnRNPA1. In this study, we reveal that KRAS expression is also modulated at the post-transcriptional level through the formation of RNA G-quadruplexes (rG4s) situated at the 5 untranslated region (5UTR) of the mRNA. Biophysical and binding studies were carried out to probe the interaction. Through isothermal titration calorimetry (ITC), we quantified a strong binding affinity between the UP1 domain of hnRNPA1 and short-nucleotide RNA segments capable of adopting different G-quadruplex fold. The binding interaction is characterized by a favorable Gibbs free energy change in the range of {Delta}G {approx} -32 to -34 kJ/mol, suggesting a specific and energetically favorable association. One-dimensional and two-dimensional 1H-15N HSQC NMR spectroscopy revealed pronounced chemical shift changes in residues of both RNA recognition motifs (RRMs) of UP1, signifying direct contact with the rG4 structure.

Translation is a crucial regulatory mechanism involved in several diseases, including cancer, where pro-inflammatory conditions within the microenvironment have been shown to modulate the translation of specific mRNAs. In the present study, we focused on the regulation of insulin growth factor-like family member 1 (IGFL1) in MCF7 breast cancer cells in response to pro-inflammatory IL-1{beta} and observed an induction of both transcription and translation. We characterized the 3 untranslated region as regulatory hub for the post-transcriptional regulation and identified a distinct G-rich region to confer the IL-1{beta}-dependent translational increase. Our study therefore provides new insights into the translation regulation of IGFL1 in the context of an inflammatory tumor microenvironment.

The importance of human aldehyde oxidase (hAOX1) has increased over the last decades due to its involvement in drug metabolism. Inhibition studies concerning hAOX1 are extensive and a common reducing agent, dithiothreitol (DTT), was recently found to inactivate the enzyme. However, in previous crystallographic studies of hAOX1, DTT was found to be essential for crystallization. To surpass this concern another reducing agent used in crystallization trials. Using tris(2-carboxyethyl)phosphine (TCEP), a sulphur-free reducing agent, it was possible to obtain well-ordered crystals from hAOX1 wild type and variant, hAOX1_6A, which diffracted beyond 2.3 [A]. Instead of the typical star-shaped crystals of hAOX1, at pH 4.7, plates are obtained in the orthorhombic space group (P22121) with two molecules in the asymmetric unit. Activity assays with the enzyme incubated with both reducing agents show that contrary to DTT, TCEP does not lead to irreversible inactivation of the enzyme. The replacement of DTT with TCEP in crystallization of hAOX1 provides a strategy to circumvent enzyme inactivation during crystallographic studies, allowing future applications of new assays, such as time-resolved crystallography.

Metabolic dysfunction-associated steatohepatitis (MASH) is associated with increased expression of peroxisome proliferator-activated receptor gamma (PPAR{gamma}, Pparg) and reduced expression of genes involved in methionine metabolism in the liver. The nuclear receptor PPAR{gamma} is activated by fatty acids, and the knockout of Pparg in hepatocytes (Pparg{Delta}Hep) reduced the negative effects of MASH on methionine metabolism. Here, we sought to determine whether hepatocyte Pparg is required for the transcriptional regulation of genes involved in hepatic methionine metabolism in conditions with altered fatty acid flux to the liver: fasting, refeeding, and high-fat diet (HFD)-induced obesity/steatosis. Fasting induced liver steatosis and increased the expression of key genes involved in the methionine metabolism in the liver, while 6h-refeeding reversed these effects and reduced the expression of phosphatidylethanolamine N-methyltransferase (Pemt) and cystathionine beta synthase (Cbs). Overall, fasting and refeeding did not alter hepatocyte Pparg expression nor Pparg{Delta}Hep affected fasting and refeeding-mediated regulation of methionine metabolism gene expression. Diet-induced steatosis reduced hepatic Pemt expression in control (Pparg-intact) mice, and the thiazolidinedione (TZD)-mediated activation of PPAR{gamma} in diet-induced obese control (Pparg-intact) mice reduced the expression of betaine homocysteine S-methyltransferase (Bhmt) and Cbs. However, diet-induced steatosis increased hepatocyte Pparg expression, and Pparg{Delta}Hep blocked the negative effects of HFD and TZD on hepatic methionine metabolism. The PPAR{gamma}-dependent reduction of hepatic Bhmt and Cbs expression was confirmed in mouse primary hepatocytes. Taken together, hepatocyte Pparg may serve as a negative regulator of hepatic methionine metabolism in diet-induced obese mice and these actions could contribute to promoting the onset of MASH.

Obesity affects millions of people worldwide and has serious complications such as cardiovascular disease and diabetes. Current treatments for obesity target proteins such as the receptors for glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP) and/or glucagon (GCG). These interventions have revolutionized the treatment of obesity and represent first-line pharmacotherapeutic strategies. One major weakness to these strategies is that once drug treatment stops, most patients are unable to maintain the new body weight setpoint, often gaining weight back rapidly. Thus, the identification of new therapies that focus on the ability to maintain homeostatic setpoint are necessary. The glucocorticoid receptor (GR) has been implicated in several pathways including reward-seeking, inflammation, stress and energy balance. Here, we investigated the effects of 30 days treatment with PT150 (40 mg/kg), a novel GR antagonist, alone and in combination with semaglutide (30 nmol/kg) on food intake, glucose homeostasis, body weight and setpoint maintenance using a C57Bl/6 diet-induced obesity (DIO) mouse model. We monitored food intake and body weight throughout treatment and after drug washout for 20 days to evaluate defended body weight maintenance (body weight setpoint). Our results indicate that treatment with PT150 alone does not significantly alter body weight but in combination with semaglutide it shows the most promising effects in body weight reduction and homeostatic setpoint maintenance. Together, these data suggest that PT150, a GR modulator, may be effective as a homeostatic setpoint modulator when combined with semaglutide.

The newly identified signaling system comprising C-X-C motif chemokine ligand 17 (CXCL17) and G protein-coupled receptor 25 (GPR25) is involved in immune regulation and tumor development. However, the evolutionary origin of this pair has remained unclear because CXCL17 orthologs in lower vertebrates exhibit extreme sequence variation and cannot be identified through conventional homology-based searches. In this study, we identified seven possible CXCL17 orthologs in primitive cartilaginous fishes, including sharks and rays, using an integrated approach based on key amino acid sequence features as well as gene synteny, architecture, and RNA sequencing data in the NCBI gene database. To validate these candidates, a representative ortholog from the cloudy catshark (Scyliorhinus torazame), termed St-CXCL17, was prepared via bacterial overexpression and in vitro refolding. In cell-based functional assays, St-CXCL17 demonstrated high binding affinity and activation potency toward its corresponding receptor, St-GPR25. Further analysis revealed that removing three conserved C-terminal residues almost completely abolished this activity. While these cartilaginous fish CXCL17s share considerable homology with one another, they lack significant overall similarity to orthologs in mammals, amphibians, or bony fishes. These findings identify functional CXCL17 orthologs in cartilaginous fishes for the first time, implying that the CXCL17-GPR25 signaling pair likely originated in ancient cartilaginous fish ancestors or earlier and has been conserved throughout the evolution of jawed vertebrates.

IntroductionDrug repurposing offers a cost-effective and time-efficient strategy for cancer therapy by leveraging existing drugs with established safety profiles, thus functioning as an alternative therapeutic strategy in demanding diseases such as cancer. Antidiabetic agents, in particular, have demonstrated encouraging anticancer potential. Among them, the non-sulfonylurea insulin secretagogue repaglinide (RPG) has shown emerging anticancer potential, yet its effects on breast and lung cancers remain largely unexplored. Thus, this study investigates the anticancer activity of repaglinide in human breast (MCF-7) and lung (A549) cancer cell lines, focusing on its cytotoxic, pro-apoptotic, anti-proliferative, and anti-migratory effects and the underlying possible molecular mechanisms. Methodology and ResultsMTT cytotoxic assay revealed that RPG reduced cell viability in a dose-/time-dependent manner, with an IC (48h) of 100.8 {+/-} 3.98 {micro}M for MCF-7 and 104 {+/-} 3 {micro}M for A549. Further, the apoptotic effect of RPG on both cell lines was evidenced by double staining assays, comet assay, and western blotting analysis, suggesting that RPG explicitly caused DNA damage and activated intrinsic and extrinsic apoptosis pathways. Additionally, RPG suppressed clonogenicity and enforced G1 arrest in MCF7 and A549 cells by modulating cell cycle regulations as well as cell proliferation pathways. Moreover, RPG markedly suppressed cell motility, as demonstrated by scratch and Transwell migration/invasion assays, which is correlated with reduced MMP-2 and MMP-9 expression, confirmed by gelatin zymography and western blotting. ConclusionConclusively, Repaglinide exerts potent anticancer effects in breast and lung cancer cells by modulating key oncogenic signaling pathways, and thus can be considered a promising candidate for repurposing in cancer therapy.

Lipopolysaccharide (LPS), a ubiquitous bacterial component of food, is neutralized by a variety of mechanisms that help to establish a threshold, which when exceeded results in an inflammatory TLR4 mediated response. Notably both CEACAM1 and CD36 affect downstream signaling of TLR4 to LPS. Furthermore, CEACAM1 associates with CD36 in hepatocytes, regulating lipid storage and bile acid (BA) secretion that includes reverse transport of LPS to the intestine. Direct binding of LPS-Ra micelles to soluble CEACAM1 or soluble CD36 was analyzed by surface plasmon resonance (SPR), size exclusion chromatography (SEC) and transmission electron microscopy (TEM). Direct binding of CEACAM1 to CD36 was analyzed by SPR and proximity ligation assays. Molecular models were generated by Alpha Fold and Molecular Dynamics. LPS Binding: SPR binding constants of KD= 1.04 x 10-10 M and KD= 3.38 x 10-10 M were obtained for LPS-Ra micelle binding to sCEACAM1 and sCD36, respectively. On SEC, the molecular sizes of LPS-Ra micelles bound to sCEACAM1 and sCD36 were approximately 500 and 800 kDa, respectively. In addition, LPS binding to both was reduced by sodium cholate and sodium deoxycholate. Alpha Fold predicted a binding site of LPS-Ra to CD36, while Molecular Dynamic studies of an N-domain mutant of CEACAM1, that breaks a conserved salt bridge, revealed the presence of an open form that is predicted to bind LPS. sCEACAM1 to sCD36 Binding: A KD of 5.28 x 10-8 M was obtained for sCEACAM1 binding to immobilized sCD36 by SPR. Antibody-based-proximity ligation demonstrated the association of the ectodomains of CEACAM1 and CD36 on hepatic cells and when co-expressed in HEK cells. In addition, biotin-based proximity ligation demonstrated association of the cytoplasmic domains of CEACAM1 and a CD36-BioID2 fusion protein when co-expressed in HEK cells. Alpha Fold predicted both head-to-head (trans) and side-to-side (cis) binding of the N-domain of CEACAM1 to CD36, from which a membrane model of their cis-interaction could account for the proximity ligation results. Both CEACAM1 and CD36 share a common LPS micelle binding function, as well as binding to each other, and together, may regulate uptake and excretion of micellar LPS.

Hydroxycarboxylic acid receptors (HCARs) are class A G-protein-coupled receptors that function as metabolic sensors. This receptor family includes three members (HCAR1, HCAR2, and HCAR3) expressed in metabolically active tissues and immune cells, where they link cellular metabolic status to physiological responses. This study aims to elucidate the evolutionary history of the most recently originated members of the HCAR gene family, namely HCAR2 and HCAR3, in primates. According to our phylogenetic analyses, the duplicative history of these genes involved multiple independent duplication events during ape evolution. Thus, most ape lineages possess independently originated duplicated copies, while non-ape primates retain the ancestral condition of a single-copy gene (HCAR2/3). Our analyses further indicate that this single-copy gene in non-ape primates is functionally equivalent to HCAR2, suggesting that the primary functional innovation in apes is associated with the physiological roles of HCAR3. Finally, gene expression analyses reveal that major divergence in tissue expression occurred after the initial duplication event that generated HCAR1 and the HCAR2/3 lineage, whereas HCAR2 and HCAR3 exhibit substantial overlap in their expression profiles. Thus, the more refined and context-dependent regulation of lipid metabolism that provides the HCAR3 receptor seems to have originated multiple times during the evolutionary history of apes.

Aiming to develop a high-throughput fluorimetric assay for the activity CYP1A2, we introduced 6-Methoxy-2-naphthoic acid (MONA) as a new fluorogenic substrate for this important metabolizer of antidepressants and psychotropic drugs in human liver. We demonstrated that oxidative demethylation of MONA by liver microsomes results in a red shift and a substantial increase in fluorescence. This effect, which is exceptionally well pronounced at alkaline pH, allowed us to develop a sensitive and robust high-throughput assay of MONA metabolism. Probing the activity of 15 individual recombinant human P450 enzymes, we found that only two P450 species exhibited activity in MONA demethylation: CYP1A2 (kcat=11.9{+/-}2.2 min-1, KM=578{+/-}106 {micro}M) and CYP2A6 (kcat=0.48{+/-}0.07 min-1, KM=54{+/-}15 {micro}M). Since the KM values of the two enzymes are well resolved and the turnover rate observed with CYP2A6 is much lower than that of CYP1A2, this new fluorogenic substrate is useful as a specific probe for CYP1A2 activity in HLM. Importantly, MONA is not metabolized by CYP1A1 and CYP2C19, which distinguishes it from all known CYP1A2 fluorogenic substrates. We then used MONA to investigate the effects of chronic alcohol exposure on CYP1A2 activity using a series of 23 proteomically characterized individual HLM preparations from donors with various levels of alcohol consumption. The substrate saturation profiles (SSP) acquired with these preparations were subjected to global kinetic analysis by approximating them with combinations of two Michaelis-Menten equations with globally optimized KM values of 11 and 553 {micro}M. The amplitudes (Vmax values) of both components showed a pronounced increase with increasing alcohol exposure of the liver donors. The Vmax of the minor high-affinity component was best correlated with the abundance of alcohol-inducible CYP2E1 enzyme. The correlation was further improved by combining it with the abundances of CYP2A6 and CPR. This finding suggests that this minor component reflects the activity of CYP2A6 in the complex with alcohol-inducible CYP2E1 protein. In contrast, the Vmax of the predominant CYP1A2-catalyzed low-affinity component revealed a pronounced correlation with the abundances of CYP1A2 and NADPH cytochrome P450 reductase (CPR). These results suggest a considerable increase in the rate of metabolism of drug substrates of CYP1A2 by chronic alcohol exposure that takes place despite an alcohol-induced decrease in CYP1A2 expression.

Mouse models of metabolic dysfunction-associated steatotic liver disease (MASLD) are valuable tools for identifying novel molecular mechanisms that drive progression from MASLD to metabolic dysfunction-associated steatohepatitis (MASH). However, generating a clinically relevant MASLD/MASH mouse model with obesity and peripheral metabolic dysfunction remains a challenge. In this study, we fed two different MASH-inducing diets to male mice with pre-existing high-fat (HF) diet-induced obesity. While a HF diet containing 40% Kcal from fat (mostly corn-oil shortening), 2% cholesterol, and 22% fructose reduced adiposity in these mice, a high-fat diet with 60% Kcal from fat (mostly lard), containing 2% cholesterol and supplemented with 10% fructose in the drinking water (HFC+Fr diet) promoted body weight and fat mass gain. Of note, 24 weeks of the HFC+Fr diet induced obesity, metabolic dysfunction, and liver steatosis in male and female mice, and promoted MASH with fibrosis in male mice. Furthermore, the HFC+Fr diet increased the expression of hepatocyte peroxisome proliferator-activated receptor {gamma} (Pparg), but the knockout of Pparg in hepatocytes (Pparg{Delta}Hep) reduced the development of MASH and fibrosis in male mice. In addition, the expression of key hepatic genes involved in methionine metabolism was downregulated by the HFC+Fr diet and upregulated by Pparg{Delta}Hep only in male mice. Overall, the HFC+Fr diet is obesogenic and promotes MASLD in both male and female mice. However, the HFC+Fr diet promotes MASH in a sex- and hepatocyte Pparg-specific manner, which may be associated with downregulation of hepatic methionine metabolism. New & NoteworthyWe explored how a new dietary intervention with fructose in the drinking water and added cholesterol to a high-fat diet extensively used to induce obesity and insulin resistance, promotes the onset of MASLD with obesity and metabolic dysfunction in male and female mice. This clinically relevant model of MASLD shows increased expression of hepatocyte PPAR{gamma} in both male and female mice, but only male mice have PPAR{gamma}-dependent impaired methionine metabolism and develop MASH with fibrosis.

The enzyme nitric oxide synthase (NOS) breaks down the semi-essential amino acid L-arginine (L-Arg) in the cell to produce citrulline and nitric oxide (NO). NO is a crucial signalling molecule in cells that controls the metabolism of fats and carbohydrates. The aim of this study was to investigate two important genes in the L-Arg-NOS-NO signalling pathway, AMPK and ACC-1, as markers of the molecular mechanisms that are triggered when liver cells sense elevated L-Arg. Mouse liver epithelial insulin-sensitive BNL CL2 cells were used as a model system and cultured with 0, 400 or 800 {micro}M L-Arg. Cell growth parameters were analysed alongside qRT-PCR based analysis of target transcripts involved in lipid and glucose metabolic pathways. In a further experiment, NOS inhibitor; L-NAME (40 mM) and external NO donor; SNAP (100 {micro}M) were added and the effect on target gene expression analysed. L-Arg addition impacted culture viability and cell growth. AMP-activated protein kinase (AMPK) was regulated in response to L-Arg addition with increasing extracellular concentrations elevating AMPK mRNA and protein expressions. L-NAME decreased target gene expression in an L-Arg addition dependent manner. SNAP (100 {micro}M) addition increased target gene expression after 6 and 24 h. NO, produced as a result of L-Arg addition and the factors L-NAME and SNAP, that regulate NO bioavailability, impacted BNL CL2 cell NO/AMPK/ACC-1 signalling pathways via regulating mRNA expression and subsequently protein expression.

A method to measure telomere length in S. cerevisiae was developed based on bioluminescence resonance energy transfer (BRET). The system uses energy transfer between a luciferase-Rif2 fusion protein and fluorescently tagged Rap1. The study demonstrates that the BRET ratio correlates with the Rap1/Rif2 complex at the telomeres and thus the availability of telomeric Rap1 binding sites. This enables the measurement of telomere length in living cells. The system was able to reproduce reported deviations in telomere length in mutants lacking telomere length regulators, cells treated with telomere length modifying compounds and strains expressing inducible telomerase. The BRET ratio linearly correlated with the average number of telomeric nucleotides derived from long-read sequencing data using a novel algorithm for telomere length calculation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/711003v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1850c4dorg.highwire.dtl.DTLVardef@1ead295org.highwire.dtl.DTLVardef@1a76358org.highwire.dtl.DTLVardef@6b3183_HPS_FORMAT_FIGEXP M_FIG C_FIG

A cassane diterpene, 6{beta}-cinnamoyl-7-hydroxyvouacapen-5-ol (6{beta}CHV), isolated from Caesalpinia pulcherrima, has emerged as a promising anticancer drug lead with reported Wnt/{beta}-catenin pathway inhibitory activity and in vivo safety. The present study reports the in vivo pharmacokinetics and tissue distribution of 6{beta}CHV in Wistar rats following a single oral dose of 200 mg/kg. A reproducible RP-HPLC-UV method was developed and validated for quantifying 6{beta}CHV in rat plasma and tissues. Chromatographic separation was achieved using a gradient elution of methanol and water. The method was subsequently applied to investigate the pharmacokinetics and tissue distribution of 6{beta}CHV. Plasma pharmacokinetic analysis revealed delayed and moderate absorption, with a Tmax of 4 h and a Cmax of 1314.12 ng/mL. Following absorption, 6{beta}CHV is distributed widely across peripheral tissues, including the liver, heart, lungs, spleen, and kidneys, as well as pharmacological sanctuary sites such as the brain and testes. The highest concentrations were observed in the stomach, small intestine, and liver, with detectable levels persisting up to 24 h, reflecting extensive tissue partitioning and retention. Overall, these findings demonstrate that oral administration of 6{beta}CHV is feasible. However, the delayed absorption suggests that further optimization of formulation or alternative administration routes may enhance systemic exposure. This study provides the first comprehensive pharmacokinetic and tissue distribution profile of 6{beta}CHV, supporting its continued preclinical development as a potential anticancer therapeutic. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/715187v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@4ae86forg.highwire.dtl.DTLVardef@1e1e51aorg.highwire.dtl.DTLVardef@1881c43org.highwire.dtl.DTLVardef@f7789f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Postprandial hyperglycemia is a major concern in type 2 diabetes, and inhibition of intestinal -glucosidases [maltase-glucoamylase (MGAM)] is an established method for controlling post-meal glucose excursions. In this study, we conducted an in-silico screening of phytochemicals from different well-known medicinal plants (Withania somnifera, Rauwolfia serpentina, Curcuma longa, and Camellia sinensis) against MGAM, using the clinically approved inhibitor miglitol as reference for docking protocol validation. Molecular docking revealed that miglitol binds to MGAM with a binding energy of -6.86kcal/mol and an RMSD of 1.04 (with co-crystal structure; PBD ID:3L4W); however, several phytochemicals exhibited binding affinities equal to or stronger than miglitol. Among these, Withanolide B (-9.25kcal/mol) and Withanone (-7.57kcal/mol) from Withania somnifera showed the highest predicted affinities, indicating robust engagement of the MGAM catalytic pocket. Rauwolfia serpentina alkaloids such as yohimbine (-8.50kcal/mol) and raubasine (-8.46kcal/mol) also displayed strong binding energies, whereas curcuminoids (curcumin -6.36kcal/mol; deoxycurcumin -6.35kcal/mol) and tea catechins (e.g., epicatechin gallate -6.85 kcal/mol) demonstrated moderate affinity. Interaction analysis showed that top-ranking compounds formed extensive hydrogen-bonding and hydrophobic interactions with key catalytic residues of MGAM, suggesting stable occupancy of the active site. In-silico ADME profiling predicted favorable gastrointestinal absorption for lead phytochemicals, supporting their potential for oral intestinal action. Collectively, these results identify plant-derived ligands with binding energies comparable to or exceeding that of miglitol, highlighting Withania somnifera withanolides as priority candidates for experimental validation in enzyme inhibition assays and glucose tolerance models, and providing a focused set of natural MGAM inhibitors for further translational investigation in postprandial glucose control.

GPI-anchored proteins are crucial cell surface proteins with diverse, organism-specific functions, in eukaryotes. They are produced when the GPI transamidase (GPIT), a five-subunit membrane-bound enzyme complex, attaches a pre-formed GPI anchor to the C-terminal end of nascent proteins on the lumenal face of the endoplasmic reticulum. This process requires the removal of a C-terminal signal sequence (SS) on the substrate protein by the action of an endopeptidase subunit of the GPIT, Gpi8/ PIG-K. Using an AMC-tagged peptide in a cell free (post-mitochondrial fraction) assay, this manuscript studies the steady state kinetics of enzymatic cleavage of the substrate by GPIT of the human pathogenic fungus, C. albicans. We show that Mn+2 enhances activity by improving substrate binding but plays no direct role in substrate cleavage per se. Molecular dynamics simulations suggest that the divalent cation binds at a site away from the active site but provides compactness and stability to Gpi8. It also enables a conformation in which a flexible loop (219-244 residues) in the vicinity of the catalytic pocket is able to interact with and position the scissile bond for cleavage by Cys202. Steady state kinetics also indicate that peptides of lengths 7-mer to 9-mer are better bound than 4-mer or 15-mer peptide substrates. A bulky residue at the site of cleavage reduces the catalytic activity of the GPIT. This is the first detailed steady state kinetics study on the endopeptidase activity of a GPIT from any organism.

BackgroundMore than 6.2 million people have died already from COVID-19. Drug resistance and relapse cases from first generation therapeutics calls for development of new drugs in alternative medicine. Complementary and Alternative Medicines (CAM) that include herbal remedies and phytochemicals are usually not fully integrated into mainstream healthcare systems. The study proposes a CAM remedy, a new polyherbal formulation NAOQ19 against the SARS-CoV-2. MethodsThe present study consists of invitro and invivo evaluation of NAOQ19 against SARS-CoV-2 infection. First, invitro testing of NAOQ19 anti-viral activity was carried out on three relevant cell lines: Vero E6, A549ACE2 and Huh 7.5.1 ACE2TMPRSS2. Next, animal model testing of NAOQ19 was performed in Syrian golden hamsters along with positive control Remdesivir and infection control for 3 days to determine the efficacy and safety of the formulation. Finally, a double blind randomized clinical trial with mild to moderate COVID-19 infected patients were evaluated to test the efficacy of NAOQ19 in human settings. ResultsThis study demonstrated a strong anti-viral (low EC50) activity in cell culture with live virus and exhibits reduced plaque forming units (high antiviral activity) in the Syrian golden hamster model. Moreover, in the clinical trials, NAOQ19 shows high efficacy demonstrating early recovery and reduced levels of inflammatory biomarkers among COVID-19 infected patients. ConclusionThis novel polyherbal formulation NAOQ19, demonstrates strong anti-viral activity in preclinical and clinical study; thereby proving its candidacy as a low-cost alternative medicine with minimal adverse effects.

In siRNA-based applications, cellular delivery remains one of the main hurdles. Many formulations are tested for the same and peptides came up as one of the optimal options. The latter have various advantages like natural biological presence, high specificity, and natural metabolism etc. siRNA in conjugation with peptides have exhibited enhanced mRNA silencing. Peptides aid siRNAs in condensation to smaller volumes, enhance nuclease protection, increase half-life, promote cell specific binding as well as endosomal escape and release in cytosol. Despite its prime importance, no resource is available for the peptide-based delivery of siRNAs, therefore to fill the gap we developed PEPsRNA web server. It includes 2266 entries of 270 different kinds of peptides, 106 different types of siRNAs and shRNAs along with more than 80 conjugate molecules targeting 55 different genes, experimentally tested for the delivery of the siRNAs. To provide the detailed insights of the procedure, we have incorporated analysis of the peptides (e.g. secondary structure, amino acid composition, polarity, hydrophobicity etc.), siRNAs (e.g. secondary structures with minimum free energies etc.) and associated conjugate molecules (e.g. structure, SMILES, Inchl). We have derived these values using various other tools and resources to make the web server comprehensive. We further compared various physicochemical properties with the efficacy of the peptide based on the target gene silencing, but these properties do not shown any distinct conclusive relationship. The data is available for browsing, searching and downloading freely on the web server with URL: http://bioinfo.imtech.res.in/manojk/pepsirna. Highlights PEPsRNA is the first database of experimentally tested peptides for siRNA delivery It comprised of 2266 entries with 270 peptides and about 80 conjugate molecules Analysis of peptides, siRNAs and details of conjugate molecules are provided Browse, search and various tools are incorporated for data retrieval and usage

C-X-C motif chemokine ligand 17 (CXCL17) has recently been identified as an agonist of the poorly characterized G protein-coupled receptor 25 (GPR25). Although GPR25 orthologs are widely distributed across vertebrates, non-mammalian CXCL17 orthologs have only been identified in some fish species in our recent studies. In this study, we systematically searched public databases for amphibian CXCL17 orthologs based on conserved C-terminal motif, gene synteny, and genomic architecture. Using this approach, we identified up to eighteen CXCL17 orthologs from diverse amphibian species. These amphibian CXCL17s exhibit no significant overall sequence similarity to known mammalian or fish CXCL17s, thus they were previously classified as uncharacterized proteins or even unannotated. Compared with known mammalian or fish CXCL17s, most amphibian CXCL17s display distinctive features, including four cysteine residues in their mature peptide and an additional residue following the conserved C-terminal Xaa-Pro-Yaa motif. A representative ortholog from the tropical clawed frog (Xenopus tropicalis) was recombinantly expressed and functionally characterized using cell-based assays, inducing ligand-receptor binding, {beta}-arrestin recruitment, and chemotactic cell migration. The recombinant amphibian CXCL17 directly bound to and efficiently activated its cognate GPR25 receptor and induced chemotactic migration of the transfected human embryonic kidney (HEK) 293T cells, but deletion of four C-terminal residues largely abolished its activity, indicating that all CXCL17 orthologs employ a conserved mechanism for receptor binding and activation. These findings establish the presence of a functional CXCL17-GPR25 signaling system in amphibians and provide new insights into the phylogenetic distribution and sequence diversity of CXCL17 orthologs across vertebrate lineages.