Glycobiology

Here we present the GLYCAM Bacterial Carbohydrate Builder (https://glycam.org/cb), an enhanced version of the GLYCAM-Web Carbohydrate Builder1 structure modeller that integrates support for modelling bacterial glycans, enabling the straightforward generation of three-dimensional structural models. The tool integrates bacterial monosaccharide parametrisations into a curated, user-friendly web-based resource. It provides an intuitive interface for the generation of carbohydrate sequences and generates 3D structural models in PDB file format, as well as the input files required for performing molecular dynamics simulations with the AMBER software package. The current implementation includes a library of 18 bacterial monosaccharides, which can be used in combination with the already parametrised eukaryotic sugars to construct complex bacterial glycans. Common derivatives, including acetylation, methylation, and sulfation are also supported. By validating and integrating bacterial sugar parameters into the GLYCAM-Web Carbohydrate Builder, this work reduces the technical barriers associated with bacterial glycan modelling and facilitates computational studies of complex bacterial glycoconjugates.

{beta}-mannans are plant structural and storage polysaccharides prevalent in the human diet. Their degradation in the gastrointestinal tract is mediated by the human gut microbiota (HGM) through expression of a plethora of carbohydrate-active enzymes (CAZymes), although our understanding of the details of mannan breakdown is lacking. In this study, a prominent HGM member, Bacteroides cellulosilyticus, was found to be exceptionally efficient at utilising {beta}-mannans, mediated by the expression of a single polysaccharide utilisation locus (PUL). Amongst the predicted surface CAZymes encoded in the PUL, we identified a family 26 glycoside hydrolase of an unusual molecular architecture. BcWH2_GH26 contains a putative carbohydrate-binding module (CBM) directly intercalated into its catalytic domain, unlike classical CBMs which are located at the N- or C-termini of the catalytic domain. Phylogenetic and functional analyses of this internal CBM, and a homologue from another mannan user Bacteroides uniformis, revealed a narrow specificity for {beta}-mannan and support their classification as a novel CBM family. To investigate the evolutionary basis for the unusual enzyme architecture, the effect of the CBM on the catalytic activity of the enzyme was assessed. No significant differences in the kinetic parameters were found between the full-length and CBM deletion constructs against both soluble and insoluble mannans. The potential role of the internal CBM in enzyme function is discussed in the context of the likely localisation of the BcWH2_GH26 in the outer membrane utilisome encoded by the Bc mannan PUL.

GD3 synthase (GD3S) is a key enzyme in the production of gangliosides, sialylated membrane glycosphingolipids with essential physiological roles in mammalian brains. To elucidate the molecular bases of neuropathological findings associated with GD3S deficiency, we performed a multilayered analysis focused on the functionality of ion transporters Na +/K+-ATPase (NKA) and plasma membrane Ca2+-ATPase (PMCA) in the cortex and cerebellum of GD3S-deficient mice (GD3S-/-). We examined global transcriptomes, NKA and PMCA gene and protein expression, the influence of membrane lipid composition on lipid raft integrity, and the activity of both ATPases, pairing them with an exploratory principal component analysis. Transcriptomic data reveal that sets of genes involved in ion transport and membrane dynamics are differentially expressed in the absence of GD3S, whereas qRT-PCR data confirm changes in gene expression of specific NKA and PMCA subunits or isoforms. Altered protein expression and significantly lower activity of both NKA and PMCA were found in the cerebral cortex of GD3S-/- mice. Detailed lipidomic analysis revealed segregation of cholesterol into lipid rafts, which may lead to disordered membrane lipid architecture in GD3S deficiency. Additionally, altered ganglioside composition was found to affect the activities of NKA and PMCA in the brain tissue of GD3S-/- mice. Our results confirm that an imbalance in membrane ganglioside composition leads to significant alterations in ion transporter function. Experimental restoration of ATPase activity in cortical homogenates by administering exogenous b-series gangliosides may aid in developing therapeutic strategies targeting deficits in GD3S and other enzymes of ganglioside biosynthesis.

Over the past two decades, Higher Education Institutions have increasingly prioritised transferrable skills to enhance graduate employability. Graduate Attributes (GAs) now act as key indicators of student competencies for both learners and employers. Final-year research projects, typically high in credit value, represent capstone experiences that promote subject expertise and GA development through research, written work, and oral presentations. This study analyses pre- and post-project survey data from RQF Level 6 biomedical and biomolecular science students at a Russell Group University over four years (2019-2023). Most projects were laboratory-based, though the 2020-2021 cohort completed theirs remotely due to COVID-19. Students reflected on expectations and experiences of GA development, subject knowledge, and employability. Initial responses revealed anxiety and uncertainty, particularly among the 2020-2021 cohort, but most anticipated gains in skills and employability. Post-project feedback confirmed this, identifying critical thinking, confidence, resilience, collaboration, and future focus as key outcomes. Digital capability was notably strengthened, especially during remote delivery. The findings emphasise the importance of a shared understanding of GAs in bioscience education and the value of embedding structured reflection and preparatory support to help students recognise and articulate their evolving skills.

Our current understanding of carbohydrate-binding module (CBM) function is limited by the fact that most CBM research has focused on single-binding-site modules. CBM family 92 (CBM92) is a recently characterized family of predominantly trivalent proteins that bind {beta}-1,3- and {beta}-1,6-glucans with high specificity. CpCBM92A from Chitinophaga pinensis stands out as the first trivalent member of the family to be structurally determined. Multivalent CBM families are rare, and the way in which the three binding sites cooperate in ligand recognition remains unclear. Here, we use NMR spectroscopy to demonstrate how each of the proteins binding sites plays distinct roles in ligand binding. One binding site, referred to as the {beta} site, can be identified as the primary attachment point because of its higher affinity for all tested ligands, consistent with previous biochemical data suggesting it is the strongest binding site on CpCBM92A. The other two binding sites, referred to as and {gamma}, preferentially bind longer segments of {beta}-1,3- and {beta}-1,6-glucan chains, respectively. We further show that the glycosidic bond position and anomeric configuration of the binding glucosyl unit strongly affects protein affinity due to a preferred ligand pose in the binding sites. Our results provide insight into how the trivalent architecture of CBM92 might enable cross-linking of scleroglucan chains, which may guide the development of new applications for CBMs in biotechnology.

Heterotrophic marine microorganisms have the capability to degrade and metabolize laminarin, which is the most abundant source of energy and nutrients in the marine environment, via enzymes encoded by genes clustered in polysaccharide utilization loci (PULs). In this study, a PUL potentially responsible for laminarin utilization was identified in the genome of the marine bacterium Muricauda lutaonensis strain ISCAR-4703, with conserved synteny in the genus Muricauda. A GH17 laminarinase (MlGH17A) encoded in the newly identified PUL was cloned, produced, and characterized as an endo-acting laminarinase, exhibiting the ability to degrade laminarin and laminari-oligosaccharides with a degree of polymerization (DP) greater than four into laminaribiose, laminaritriose, and laminaritetraose, with laminaritriose as the main product making up >50% of the produced oligosaccharide products. The three-dimensional model of the enzyme revealed the presence of seven putative subsites, including four glycone subsites (-4 to -1) and three aglycone subsites (+1 to +3), with a wide cleft to accommodate branches at the -2 subsite, enabling it to act on {beta}-1,3 linked backbones in polysaccharides with {beta}-1,6 linked branches. This enzyme is, along with the recently characterized {beta}-1,3 glucanosyltransglycosylase (MlGH17B), conserved in several Muricauda species and is suggested to play a crucial role in the utilization of laminarin by these bacteria. ImportanceLaminarin, a {beta}-1,3-glucan with occasional {beta}-1,6 branching, is the most abundant source of energy and nutrients in the marine environment. In this study, polysaccharide utilization loci (PULs) for laminarin degradation were identified in various marine Muricauda species, encoding a range of glycoside hydrolases and transglycosylases. In Muricauda lutaonensis ISCAR-4703, the PUL included two GH17 enzymes, separated by a GH30 enzyme and a major facilitator superfamily (MFS) transporter, a feature observed in all corresponding Muricauda PULs. A novel endoacting laminarinase from the PUL, MlGH17A, was characterized and shown to hydrolyze laminarin into laminaribiose, laminaritriose, and laminaritetraose, with laminaritriose as main product. Bioinformatic analysis showed that the enzyme lacked the typical subdomain found in GH17 plant {beta}-glucanases, leading to a lower number of aglycone subsites (+1 to +3). Instead, MlGH17A possessed more glycone subsites (-1 to -4), attributed to the {beta}3-3 loop, which was longer than in GH17 plant {beta}-glucanases.

Human RNase 2 (eosinophil-derived neurotoxin, EDN) is a major eosinophil granule protein of the vertebrate-specific RNase A superfamily and is involved in antiviral response and inflammation. Identifying ligand-binding pockets in EDN is thus relevant to structure-based drug design. In our laboratory we identified by protein crystallography a conserved site at the protein surface binding to carboxylic anion molecules (malonate, tartrate and citrate). Searching for potential biomolecules rich in anion groups and considering previous report of EDN binding to glycosaminoglycans, we explored the protein binding to saccharides. Next, EDN crystals were soaked with mono- and disaccharides, and the 3D structures of ten complexes were solved by X-ray crystallography at atomic resolution. We identified protein binding pockets to glucose, fucose, mannose, sucrose, galactose, trehalose, N-acetyl-D-glucosamine, N-acetylmuramic acid, and the sialic acid N-acetylneuraminic acid. A main site for glucose, fucose, and galactose was located adjacent to the spotted carboxylic anion site. Secondarily, N-acetylneuraminic acid, N-acetylmuramic acid, sucrose, galactose, and mannose shared another protein surface region. Overall, the saccharides clustered into seven defined sites, outlining a conserved recognition pattern, which was further analysed by molecular modelling. Interestingly, within the RNase A family, we find amphibian RNases that were initially isolated as carbohydrate binding proteins and named as leczymes, combining enzymatic and lectin properties. The present data is the first systematic structural characterization of a mammalian sugar-binding RNase within the family. The results highlight unique EDN residues that mediate its sugar specific interactions, of particular interest for a better understanding of the protein physiological role. HighlightsO_LIstructure of RNase 2 in complex with mono and disaccharides at atomic resolution C_LIO_LIidentification of RNase 2 unique sugar binding sites C_LIO_LIcharacterization of a mammalian RNase A family enzyme with lectin properties C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/713198v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1d805f7org.highwire.dtl.DTLVardef@16fcc49org.highwire.dtl.DTLVardef@ccfd92org.highwire.dtl.DTLVardef@1b8f1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Biodesign is an interdisciplinary research domain that incorporates principles from design and the life sciences to develop new systems, processes, and objects. Collegiate biodesign educators face unique pedagogical challenges, including an absence of relevant scholarship on curriculum design and instructional best practices for cultivating student scientific literacy. These difficulties may be overcome with newly available technologies, like generative AI systems, that enable personalized learning through domain-specific semantic spaces. This article examines the instructional value of one such domain-specific LLM, Biodesign Buddy, through a mixed-methods analysis of an eight-week study involving 64 students participating in an international biodesign competition. Results indicate strong support for integrating AI into biodesign coursework. Surveys captured attitudes toward AI, scientific literature, and learning experiences to assess AIs impact on learning outcomes. Findings suggest that integrating AI into biodesign pedagogy can meaningfully redress conceptual issues in biodesign while informing broader debates on AIs role in higher education. Impact StatementThis article introduces Biodesign Buddy, a domain-specific generative AI system for collegiate biodesign education, and reports on its exploratory deployment, offering design principles and preliminary findings to inform the development of AI-supported pedagogies for interdisciplinary biodesign instruction.

Background & AimsClinically relevant postoperative pancreatic fistula (CR-POPF) remains a major cause of morbidity following pancreatic surgery, potentially due to fatty acid release by lipase activity. This study investigated how the biochemical composition of CR-POPF effluents drives cellular injury and transcriptional stress responses. MethodsDrain effluents from 14 patients undergoing pancreatoduodenectomy (7 with CR-POPF and 7 controls) were analyzed using gas chromatography-mass spectrometry. Candidate lipids were tested on human foreskin fibroblasts, mesothelial cells, and pancreatic epithelial cells using viability and cytotoxicity assays. Effluents were applied directly to cultures, and RNA sequencing was performed on cells exposed to the two most cytotoxic CR-POPF samples. ResultsMetabolomic profiling revealed lipolytic traits characterized by long-chain saturated fatty acids, including palmitic and stearic acid, and the palmitic acid monoacylglycerol monopalmitin, in drain effluents. These fatty acids accounted for over 70% of the variance in multivariate metabolomic analyses between CR-POPF and control groups. Dose-response assays confirmed concentration-dependent cytotoxicity (p < 0.0001), with a subtoxic threshold of 0.2 mM. Two effluents (AES1448 and GR1479) consistently reduced cell viability across models (F > 19, p < 0.0001). Transcriptomic profiling showed enrichment of inflammatory, unfolded-protein, and stress-response pathways, along with suppression of proliferation modules. GR1479 induced metabolic adaptation, whereas AES1448 and monopalmitin triggered overt lipotoxic stress. ConclusionsLipolysis-derived lipids may mediate stromal and mesothelial injury in CR-POPF. Integrating metabolomic, functional, and transcriptomic data uncovers a spectrum of cellular responses, spanning from adaptive remodeling to lipolysis-driven proteotoxic stress. These findings support lipid toxicity as a biochemical property of CR-POPF and a potential target for prevention. SynopsisThis study identifies long-chain saturated fatty acids in postoperative pancreatic effluents as key mediators of cytotoxic and inflammatory stress. Integrating metabolomic and transcriptomic analyses link effluent composition directly to cellular injury and impaired healing after pancreatic surgery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/705517v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@2ba175org.highwire.dtl.DTLVardef@752522org.highwire.dtl.DTLVardef@d8f823org.highwire.dtl.DTLVardef@8acb7f_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Xanthan gum is a widely used industrial polysaccharide employed as a thickening and stabilizing agent in food, pharmaceutical, and technological applications. Its biosynthesis involves membrane-associated glycosyltransferases that assemble the repeating unit at the cytoplasmic side of the inner membrane. Among them, GumH and GumI catalyze consecutive reactions using the same donor substrate, guanosine 5-diphospho-alpha-D-mannose, but with opposite stereoselectivity. Despite their biochemical characterization, structural insights into their catalytic mechanisms and membrane interactions remain limited, hindering a detailed understanding of their function and future engineering efforts. In this work, we combined artificial intelligence-based structure prediction with atomistic molecular dynamics simulations to investigate the structural organization and substrate-binding modes of GumH (family GT4) and GumI (family GT94). The predicted apo structures exhibit a conserved GT-B fold but differ in interdomain flexibility and membrane-anchoring strategies. GumH displays a more structured interdomain linker and a defined clamp-like region in the acceptor-binding domain, consistent with stable membrane interaction, whereas GumI shows a more flexible linker and an open groove architecture. Modeling of the donor-bound complexes reveals distinct substrate-binding modes. In GumH, it adopts a geometry consistent with its retaining stereochemical outcome, positioning the sugar close to the conserved catalytic residue. In contrast, GumI exhibits a different donor orientation, lacking a clearly positioned catalytic base near the reactive center, suggesting a substrate-assisted catalytic mechanism. Although the predicted ternary complexes show limited stability in our simulations, they provide chemically reasonable conformations and offer structural insights into substrate recognition, membrane association, and stereochemical control in these two glycosyltransferase families. Significance statementXanthan gum is an industrially important polysaccharide widely used in food and other technological products. Although several enzymes in its biosynthetic pathway have been studied, structural information remains limited. Using AI-based structure predictions and molecular simulations, we revealed how these enzymes sit in the membrane and bind sugar substrates. These structural insights clarify xanthan biosynthesis and could help improve or engineer its production.

Using its 9 sequentially-placed cohesin (Coh) domains as bait, each CipA scaffoldin chain in a Clostridium thermocellum cellulosome binds to (and displays) some combination of 9 out of over [~]70 available dockerin (Doc) domain-bearing lignocellulose-degrading enzymes, at any time. Domains numbered Coh3 through Coh8 show over 94 % pairwise sequence identity with each other, but only [~] 61-76 % identity with Coh1, Coh2, and Coh9. Such identities are much lower ([~] 40-60 %) amongst Doc domains; however, amongst both Coh and Doc domains, polypeptide backbone folds are highly conserved, suggesting that loading preferences of enzyme-bearing Doc domains upon Coh domains must depend upon their relative abundances, and pairwise affinities. To explore this further, we used microscale thermophoresis (MST), size exclusion chromatography (SEC), native polyacrylamide gel electrophoresis (NPGE), mass spectrometry (MS) and bioinformatics-based approaches (BIBA), to examine 28 Coh-Doc pairwise interactions involving recombinant Coh [Coh1, Coh2, Coh3, Coh9] and enzyme-bearing Doc [Cel8A, Cel9F, Man26/5H, Cel9R, Xyn10C, Xyn11D, Xyn10Z] domains. Interactions were found to occur with varying affinities, suggesting that Coh1 prefers Xyn11D; Coh2 prefers Xyn10C; Coh3 prefers Cel9R; Coh9 prefers Cel9R; Coh1/Coh2 prefer Xyn partners; Coh3/Coh9 prefer Cel partners. Dual modes of binding are shown by Coh1 with Xyn10C and Xyn11D; Coh2 with Xyn10Z, Cel8A, and Cel9R; Coh3 with Cel8A, and Cel9R; and Coh9 with Xyn10C, suggesting that Doc domains use either of their two homologous helices (1 and 3) to bind to Coh domains, as earlier proposed. ImportanceBacteria such as Clostridium thermocellum use extracellular enzyme complexes called cellulosomes to degrade and use cellulose. Each complex uses a linear chain of nine cohesin (Coh) domains called a scaffoldin to bind to (and display) any nine of over seventy available xylan or cellulose-degrading enzymes that bear dockerin (Doc) domains. Understanding interactions between Coh and Doc domains facilitates an appreciation of how cellulosomes are assembled and supports the building of protein-engineered constructs that utilize such interactions for many conceivable enzymatic and other applications. The significance of the presented research lies in its demonstration of the differential modes and pairwise affinities of different Coh-Doc interactions, using recombinant protein constructs and a combination of quantitative, semi-quantitative and qualitative analytical methods.

Glycosylation of bacterial surface proteins, such as flagellin (FliC), is important for their function and is often involved in virulence of pathogens. Glycans can be further modified by so-called post-glycosylation modifications (PGMs) often resulting in exclusive molecular structures. In Clostridioides difficile a unique glycan structure (Type A) decorates FliC (which forms the flagellar filament) that consists of an O-linked N-acetyl-{beta}-D-glucosamine (GlcNAc) modified with an N-methyl-L-threonine via a phosphodiester linkage. This PGM is synthesized by a set of four enzymes encoded in one operon (ftaABCD), but the exact biosynthesis pathway and biosynthetic intermediates remain unknown. In this study, we chemically synthesized two hitherto undescribed biosynthetic intermediates that we predicted based on bioinformatic analyses, CDP-threonine and CDP-N-methylthreonine. We showed that they are involved in the Type A PGM biosynthesis, as evidenced by mass spectrometric analyses of extracts of a set of C. difficile mutant strains. Furthermore, we characterized FtaC to be a SAM-dependent CDP-threonine N-methyltransferase, that installs the methyl group on CDP-threonine prior to transfer of the PGM to GlcNAc-FliC, and we revealed FtaD as the CDP-N-methylthreonine:GlcNAc N-methylthreoninephosphotransferase. Finally, using recombinantly expressed FtaC and FtaD in combination with synthetic CDP-threonine, we reconstituted the biosynthesis pathway of the Type A PGM in vitro. Overall, our results open avenues to explore these unique biosynthesis enzymes in molecular detail to provide new points of entry for the development of biosynthesis inhibitors and tools to study the role of this PGM in virulence and flagellar assembly.

The extracellular matrix (ECM) is a meshwork of proteins that orchestrates a broad range of cellular phenotypes, including proliferation, adhesion, migration, and differentiation. SNED1 is a newly characterized ECM glycoprotein that promotes cell adhesion and is essential for embryonic development. Its upregulation is also associated with breast cancer metastasis and poor prognosis for breast cancer patients. We recently showed that SNED1 assembles into fibrillar structures, but the mechanisms guiding its incorporation into the ECM scaffold remain unknown. Combining biochemical assays and confocal immunofluorescence imaging, we found that SNED1 assembly in the ECM occurs early in the process of ECM building and is concomitant and overlaps with the deposition of fibronectin and collagen I, two major ECM proteins. By knocking down fibronectin or destabilizing collagen I fibers, we further demonstrate that SNED1 requires the presence of these proteins for its assembly. Last, using biolayer interferometry, we identify collagen I as the first direct binding partner of SNED1. Altogether, our results lay the foundation for future studies aimed at determining the mechanisms by which SNED1 fibers contribute to SNED1 pathophysiological functions. SUMMARY STATEMENTThe novel protein SNED1 requires the presence of fibronectin and collagen I to assemble into fibrillar structures in the extracellular matrix scaffold.

Black rice oligosaccharides (BO) were extracted with 80% aqueous ethanol (v/v) and purified by charcoal-celite chromatography followed by dialysis using a 500 Da molecular weight cut-off (MWCO) membrane, yielding an oligosaccharide fraction with a degree of polymerisation (DP) between three and eight (DP3-DP8; 2.87 {+/-} 0.29% w/w). MALDI-TOF MS showed sodium adduct ions from m/z 527 to 1330, and GC-MS analysis of hydrolysed samples identified glucose and galactose as the major monomers, while ketosyl residues were detected in the intact fraction by selective staining and are most plausibly attributed to fructosyl units based on cereal origin and DP distribution. BO showed high resistance to simulated salivary, gastric, and pancreatic digestion (only 2.08 {+/-} 0.51%, 0.34 {+/-} 0.03%, and 4.29 {+/-} 0.73% hydrolysis, respectively) with approximately 93% remaining carbohydrate available for fermentation. All Lactobacillus strains showed positive prebiotic activity scores, with the highest response observed for Lactobacillus rhamnosus (1.165 {+/-} 0.255) and Lactobacillus plantarum (0.980 {+/-} 0.163). Fermentation produced metabolically relevant short-chain fatty acids (SCFA), mainly acetate (34.82 {+/-} 2.08 mM), as well as strain-dependent propionate and butyrate levels. BO greatly promoted probiotic biofilm formation, with biomass reaching 391.33 {+/-} 26.08% and viable cell counts of 9.01 {+/-} 0.70 log CFU/mL relative to the control. Collectively, the results indicate that BO represents a digestion-resistant, hexose-based oligosaccharide series that is selectively utilised by probiotic lactobacilli, promotes SCFA production and enhances biofilm development. To our knowledge, this work is the first to combine structural profiling with in vitro functional evaluation of a purified, low-DP oligosaccharide fraction obtained from black rice. HighlightsO_LIPurified oligosaccharides (DP3-DP8) were obtained from black rice using charcoal-celite chromatography followed by dialysis. C_LIO_LIStructural analysis confirmed that the oligosaccharides were hexose-based and composed mainly of glucose and galactose. C_LIO_LIBlack rice oligosaccharides exhibited higher resistance to simulated gastric and intestinal digestion compared with starch. C_LIO_LIPositive prebiotic activity scores were observed due to selective utilisation by probiotic Lactobacillus strains. C_LIO_LIFermentation of black rice oligosaccharides significantly increased short-chain fatty acid production. C_LIO_LIPurified oligosaccharides enhanced probiotic biofilm formation, indicating improved colonisation potential. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/705216v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@9a5915org.highwire.dtl.DTLVardef@14eac7aorg.highwire.dtl.DTLVardef@1db8baorg.highwire.dtl.DTLVardef@14ad50a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Microvascular inflammation and endothelial injury, triggered by interferon-gamma (IFN{gamma}), are hallmarks of antibody-mediated rejection (ABMR), the leading cause of premature kidney allograft loss. Glomerular extracellular matrix (ECM) remodeling and endothelial caveolae formation are important aspects of chronic ABMR. We found galectin-1, an immunomodulatory protein that interacts with the ECM, to be increased in the glomeruli of patients with ABMR, while its gene (LGALS1) expression was decreased by IFN{gamma} stimulation in glomerular endothelial cells. Mechanisms underlying endothelial dysfunction in ABMR, its links to ECM remodeling, and the role of immunomodulatory proteins such as galectin-1 remain incompletely understood. Here we studied the effects of galectin-1 modulation in glomerular microvascular endothelial cells (GMECs) in vitro. We demonstrated that galectin-1 was mainly expressed by glomerular endothelial cells in ABMR kidneys. To model key aspects of endothelial injury in ABMR, we knocked down LGALS1 in GMECs, followed by stimulation with IFN{gamma} and performed label-free quantitative proteomic and phosphoproteomic profiling of GMECs. Proteomic analysis identified 5446 proteins (FDR<0.01), of which 236, 827, and 267 were differentially expressed in response to LGALS1 knockdown, IFN{gamma} treatment, and their interaction, respectively (FDR<0.05). Both LGALS1 knockdown and the interaction between treatments significantly altered expression of adhesion proteins (FDR<0.01), particularly integrin subunit {beta}5, which was validated. Phosphoproteomic profiling identified 2727 phosphopeptides (FDR<0.01), with 28 that were differentially expressed across LGALS1 knockdown, IFN{gamma} treatment, and their interaction (P<0.01). Phosphorylation of CAVN1 and co-localization with its partner CAV1, critical for caveolar formation, were decreased in GMECs upon LGALS1 knockdown, IFN{gamma} stimulation, or both. In a microfluidic model of the glomerular microvasculature, addition of recombinant galectin-1 increased both endothelial permeability and secretion of proinflammatory cytokines, in LGALS1-silenced GMECs. Thus, endothelial signaling pathways regulated by inflammatory cues and galectin-1 contribute to endothelial injury and caveolae formation, highlighting galectin-1 as a potential therapeutic target in ABMR. SynopsisGalectin-1 is expressed by kidney glomerular endothelium. This study reveals that modifying galectin-1 in endothelial cells, in the presence of IFN{gamma} perturbs cytoskeletal, adhesion and caveolar proteins resulting in altered endothelial permeability. O_LILGALS1 knockdown increased ECM proteins and decreased interferon-induced proteins. C_LIO_LILGALS1 knockdown and IFN{gamma} treatment perturbed cell adhesion proteins such as ITGB5. C_LIO_LICAVN1 phosphorylation and colocalization with CAV1 decreased upon LGALS1 knockdown. C_LIO_LIExtracellular galectin-1 increased microvascular permeability in response to IFN{gamma}. C_LI

A human infection with clade 2.3.4.4b H5N1 influenza A virus in Canada revealed minority variants E190D and Q226H in the hemagglutinin (HA) receptor-binding site (RBS). Because mutations at positions 190 and 226 have been associated with altered receptor specificity in other influenza subtypes, we investigated their impact on receptor binding in H5 HA. Using a recombinant protein approach and an ELISA-based glycan-binding assay, we assessed binding to representative avian- and human-type sialylated glycans. Both single mutations and their combination resulted in a complete loss of detectable binding to the tested glycans. To evaluate whether this phenotype was background-dependent, Q226H was additionally introduced into two other H5 HA proteins, each representing a distinct clade. In both cases, the mutation similarly abolished receptor binding. These findings independently validate recent glycan microarray observations and demonstrate that the patient-derived E190D and Q226H substitutions severely impair receptor-binding capacity across multiple H5 backgrounds. Single mutations at key RBS residues in H5 often disrupt receptor binding rather than confer human-type receptor specificity, confirming complex mutational pathways required for adaptation to human-type receptors.

Developing scientific reading skills is critical for undergraduate STEM students due to scientific literatures unique formatting and use of specialized jargon. Generative AI tools such as ChatGPT offer students the ability to ask questions about what they are reading interactively. Previously, we reported the development of a ChatGPT-assisted reading guide that combined structured, active reading strategies with using ChatGPT to clarify unfamiliar words and concepts in real time. In the initial study, undergraduates found the use of the ChatGPT-assisted reading guide helpful in their understanding of an abstract and introduction of a journal article. Here, the ChatGPT-assisted reading guide was used in a journal club assignment for an undergraduate chemistry course. ChatGPT transcripts were analyzed for common types of interactions, and students were surveyed about their experience. Overall, students reported that using the ChatGPT-assisted reading guide was helpful in understanding the article and helped them have more productive class discussions. However, some students also expressed skepticism about using AI tools, citing concerns about accuracy of AI-generated information and the effect of using AI on their own learning.

Listeria monocytogenes relies on a tightly controlled set of surface-associated and secreted proteins to mediate host interaction and infection. The correct localization and exposure of these proteins at the bacterial surface are critical for virulence, yet the role of cell wall components in organizing this process remains incompletely understood. In particular, wall teichoic acid (WTA) glycosylation has been implicated in anchoring and function of selected surface proteins, but its global impact on protein distribution across the bacterial cell envelope is unclear. Here, we performed a comprehensive proteomic analysis to investigate how WTA glycosylation influences protein distribution in L. monocytogenes. Using isogenic mutants lacking rhamnose ({Delta}rmlT) or GlcNAc ({Delta}lmo1079) WTA glycosylation, we compared the exoproteome, the surface-accessible proteome and the surface-exposed proteome. Loss of WTA glycosylation did not result in a global disruption of the surface proteome but instead induced a redistribution of proteins across extracellular and surface-associated fractions. This effect was dependent on protein anchoring mechanisms, with limited changes observed for LPXTG-anchored proteins, moderate effects on non-covalently associated proteins, and a marked enrichment of lipoproteins in the surface-exposed proteome, particularly in the {Delta}lmo1079 mutant. In parallel, virulence-associated proteins displayed altered accessibility and exposure, with a progressive shift towards increased surface localization and a combination of shared and mutant-specific responses. This global effect was supported by functional annotation, which revealed that the affected proteins were associated with similar biological processes across fractions, highlighting a broad rather than pathway-specific impact of WTA glycosylation loss Together, these findings indicate that WTA glycosylation plays a key role in organizing the bacterial surface by modulating protein retention, exposure and release. Rather than affecting specific proteins, WTA glycosylation broadly shapes the spatial distribution of proteins across the cell envelope, with potential consequences for host- pathogen interactions.

In yeast and humans, the conserved DENN-domain (Differentially Expressed in Normal and Neoplastic tissue) protein Avl9 is thought to play roles in membrane traffic and secretion, but its precise function remains poorly defined. Since DENN-containing proteins are associated with Rab GTPase function, we sought to understand Avl9 function in the context of Rab regulation. Here, we show that Avl9 localizes to peripheral punctae that are consistent with secretory vesicles. Moreover, we demonstrate genetic interactions and co-localization between Avl9 and numerous Rabs in the secretory and endosomal pathways, suggesting a potential function at the interface of secretion and recycling. Consistent with this role, avl9{Delta} results in defective recycling of the endosomal cargo Snc1 but does not alter plasma membrane delivery of an endocytosis-defective Snc1EN- mutant, suggesting that Avl9 is not directly involved in secretory traffic from the TGN to the plasma membrane. The avl9{Delta} recycling defect is exacerbated by the additional loss of RCY1 or SNX4, but not VPS35. Each of these three genes contributes to a distinct endosomal recycling pathway, indicating that Avl9 acts in conjunction with multiple recycling pathways. Summary StatementIn this study, Rioux et al. describe a role for the DENN domain protein Avl9, previously thought to regulate secretion, as a novel factor involved in recycling of cargos from endosomal compartments.

The secretion of glucagon from the pancreatic alpha () cell within the islets of Langerhans is physiologically regulated by nutrients (glucose, amino acids, fatty acids), neurotransmitters, and paracrine hormones. Insulin and somatostatin form an intra-islet paracrine network to control glucagon secretion through direct inhibitory effects on cell secretory granule exocytosis. In a potential new cellular pathway for the regulation of glucagon secretion, we have previously identified the neuronal trafficking protein Stathmin-2 (Stmn2) as a negative regulator of glucagon trafficking and secretion by directing glucagon to degradative lysosomes. In this study, we examined if insulin and somatostatin direct glucagon to lysosomes in a Stmn2-dependent manner as part of their paracrine mechanisms. Using the TC1-6 glucagon-secreting cell line and confocal microscopy of both fixed and live cells, we show that insulin and somatostatin direct glucagon, glucagon+LAMP1+ vesicles, and LAMP1-RFP to the intracellular region, away from sites of exocytosis. As visualized in live cells, insulin treatment resulted in the rapid retrograde transport of lysosomes from the cell periphery, and this effect was lost under siRNA-mediated silencing of Stmn2. Somatostatin appeared to enhance the intracellular retention of lysosomes, also in a Stmn2-dependent manner. We determined a possible mechanism for Stmn2 in the regulation of lysosome transport in TC1-6 cells through the Arf-like small GTPase Arl8, indicating that Stmn2 may function in lysosomal positioning along microtubules. We propose that Stmn2-mediated lysosomal transport may be a potential new pathway, in addition to inhibition of secretory granule exocytosis, through which insulin and somatostatin regulate glucagon secretion.