Metabolic Control of Glycosylation Forms for Establishing Glycan-Dependent Protein Interaction Networks.

Protein-protein interactions (PPIs) provide essential insights into the complex molecular mechanisms and signaling pathways within cells that regulate development and disease-related phenotypes. However, for membrane proteins, the impact of various forms of glycosylation has often been overlooked in PPI studies. In this study, we introduce a novel approach, glycan-dependent affinity purification followed by mass spectrometry (GAP-MS), to assess variations in PPIs for any glycoprotein of interest under different glycosylation conditions.

Absorption, anti-inflammatory, antioxidant, and cardioprotective impacts of a novel fasting mimetic containing spermidine, nicotinamide, palmitoylethanolamide, and oleoylethanolamide: A pilot dose-escalation study in healthy young adult men.

This pilot dose-escalation study evaluated the absorption and metabolism of a novel fasting mimetic formulation containing spermidine, nicotinamide, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) taken as oral supplements in young adults. Five healthy men consumed a standardized breakfast, followed by control (wheat flour) or low, medium, or high doses of supplements containing spermidine, nicotinamide, PEA, and OEA 2 hours later. Blood was drawn at 0, 1, 2, and 4 hours after the supplement (2, 3, 4, and 6 hours postprandial).

A multi-glycomic platform for the analysis of food carbohydrates.

Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures.

-Glycan profile of the cell membrane as a probe for lipopolysaccharide-induced microglial neuroinflammation uncovers the effects of common fatty acid supplementation.

Altered -glycosylation of proteins on the cell membrane is associated with several neurodegenerative diseases. Microglia are an ideal model for studying glycosylation and neuroinflammation, but whether aberrant -glycosylation in microglia can be restored by diet remains unknown. Herein, we profiled the -glycome, proteome, and glycoproteome of the human microglia following lipopolysaccharide (LPS) induction to probe the impact of dietary and gut microbe-derived fatty acids-oleic acid, lauric acid, palmitic acid, valeric acid, butyric acid, isobutyric acid, and propionic acid-on neuroinflammation using liquid chromatography-tandem mass spectrometry.

Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates which support the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the approximately 200 structurally diverse HMOs at scale has proved difficult. Here we produce a diversity of HMOs by leveraging the robust carbohydrate anabolism of plants.

Mucus production, host-microbiome interactions, hormone sensitivity, and innate immune responses modeled in human cervix chips.

Modulation of the cervix by steroid hormones and commensal microbiome play a central role in the health of the female reproductive tract. Here we describe organ-on-a-chip (Organ Chip) models that recreate the human cervical epithelial-stromal interface with a functional epithelial barrier and production of mucus with biochemical and hormone-responsive properties similar to living cervix. When Cervix Chips are populated with optimal healthy versus dysbiotic microbial communities (dominated by Lactobacillus crispatus and Gardnerella vaginalis, respectively), significant differences in tissue innate immune responses, barrier function, cell viability, proteome, and mucus composition are observed that are similar to those seen in vivo.

Lutein and Zeaxanthin Enhance, Whereas Oxidation, Fructosylation, and Low pH Damage High-Density Lipoprotein Biological Functionality.

High-density lipoproteins (HDLs) are key regulators of cellular cholesterol homeostasis but are functionally altered in many chronic diseases. The factors that cause HDL functional loss in chronic disease are not fully understood. It is also unknown what roles antioxidant carotenoids play in protecting HDL against functional loss.

Structural proteomics of a bacterial mega membrane protein complex: FtsH-HflK-HflC.

Recent advances in mass spectrometry (MS) yielding sensitive and accurate measurements along with developments in software tools have enabled the characterization of complex systems routinely. Thus, structural proteomics and cross-linking mass spectrometry (XL-MS) have become a useful method for structural modeling of protein complexes. Here, we utilized commonly used XL-MS software tools to elucidate the protein interactions within a membrane protein complex containing FtsH, HflK, and HflC, over-expressed in E.

The effects of immortalization on the N-glycome and proteome of CDK4-transformed lung cancer cells.

Biological experiments are often conducted in vitro using immortalized cells due to their accessibility and ease of propagation compared to primary cells and live animals. However, immortalized cells may present different proteomic and glycoproteomic characteristics from the primary cell source due to the introduction of genes that enhance proliferation (e.g.

Protein oxidation of fucose environments (POFE) reveals fucose-protein interactions.

Cell membrane glycoproteins are generally highly fucosylated and sialylated, and post-translational modifications play important roles in the proteins' functions of signaling, binding and cellular processing. For these reasons, methods for measuring sialic acid-mediated protein-protein interactions have been developed. However, determining the role of fucose in these interactions has been limited by technological barriers that have thus far hindered the ability to characterize and observe fucose-mediated protein-protein interactions.

Profiling Intact Glycosphingolipids with Automated Structural Annotation and Quantitation from Human Samples with Nanoflow Liquid Chromatography Mass Spectrometry.

Sphingolipids are an essential subset of bioactive lipids found in most eukaryotic cells that contribute to membrane biophysical properties and are involved in cellular differentiation, recognition, and mediating interactions. The described nanoHPLC-ESI-Q/ToF methodology utilizes known biosynthetic pathways, accurate mass detection, optimized collision-induced disassociation, and a robust nanoflow chromatographic separation for the analysis of intact sphingolipids found in human tissue, cells, and serum. The methodology was developed and validated with an emphasis on addressing the common issues experienced in profiling these amphipathic lipids, which are part of the glycocalyx and lipidome.

Development and application of GlycanDIA workflow for glycomic analysis.

Glycans modify protein, lipid, and even RNA molecules to form the regulatory outer coat on cells called the glycocalyx. The changes in glycosylation have been linked to the initiation and progression of many diseases. Thus, while the significance of glycosylation is well established, a lack of accessible methods to characterize glycans has hindered the ability to understand their biological functions.

Prevotella copri and microbiota members mediate the beneficial effects of a therapeutic food for malnutrition.

Microbiota-directed complementary food (MDCF) formulations have been designed to repair the gut communities of malnourished children. A randomized controlled trial demonstrated that one formulation, MDCF-2, improved weight gain in malnourished Bangladeshi children compared to a more calorically dense standard nutritional intervention. Metagenome-assembled genomes from study participants revealed a correlation between ponderal growth and expression of MDCF-2 glycan utilization pathways by Prevotella copri strains.

Bioactive glycans in a microbiome-directed food for children with malnutrition.

Evidence is accumulating that perturbed postnatal development of the gut microbiome contributes to childhood malnutrition. Here we analyse biospecimens from a randomized, controlled trial of a microbiome-directed complementary food (MDCF-2) that produced superior rates of weight gain compared with a calorically more dense conventional ready-to-use supplementary food in 12-18-month-old Bangladeshi children with moderate acute malnutrition. We reconstructed 1,000 bacterial genomes (metagenome-assembled genomes (MAGs)) from the faecal microbiomes of trial participants, identified 75 MAGs of which the abundances were positively associated with ponderal growth (change in weight-for-length Z score (WLZ)), characterized changes in MAG gene expression as a function of treatment type and WLZ response, and quantified carbohydrate structures in MDCF-2 and faeces.

Eat your beets: Conversion of polysaccharides into oligosaccharides for enhanced bioactivity.

Bioactive oligosaccharides with the potential to improve human health, especially in modulating gut microbiota via prebiotic activity, are available from few natural sources. This work uses polysaccharide oxidative cleavage to generate oligosaccharides from beet pulp, an agroindustry by-product. A scalable membrane filtration approach was applied to purify the oligosaccharides for subsequent in vitro functional testing.

Resident microbes shape the vaginal epithelial glycan landscape.

Epithelial cells are covered in carbohydrates (glycans). This glycan coat or "glycocalyx" interfaces directly with microbes, providing a protective barrier against potential pathogens. Bacterial vaginosis (BV) is a condition associated with adverse health outcomes in which bacteria reside in direct proximity to the vaginal epithelium.

Effects of Blanching, Freezing and Canning on the Carbohydrates in Sweet Corn.

Sweet corn is frequently consumed in the US and contains carbohydrates as major macronutrients. This study examined the effects of blanching, freezing, and canning on carbohydrates in sweet corn. Fresh bi-color sweet corn was picked in the field and processed immediately into frozen and canned samples.

Combined analysis of secreted proteins and glycosylation identifies prognostic features in cholangiocarcinoma.

Secreted proteins are overexpressed in cholangiocarcinoma (CCA) and actively involved in promoting metastatic spread. Many of these proteins possess one or more sites of glycosylation and their various glycoforms have potential utility as prognostic or diagnostic biomarkers. To evaluate the effects of secretome glycosylation on patient outcome, we elucidated the glycosylation patterns of proteins secreted by parental and metastatic CCA cells using liquid chromatography-mass spectrometry.

Plant-based production of diverse human milk oligosaccharides.

Human milk oligosaccharides (HMOs) are a diverse class of carbohydrates that aid in the health and development of infants. The vast health benefits of HMOs have made them a commercial target for microbial production; however, producing the ∼130 structurally diverse HMOs at scale has proven difficult. Here, we produce a vast diversity of HMOs by leveraging the robust carbohydrate anabolism of plants.

Inducible CRISPR-targeted "knockdown" of human gut in gnotobiotic mice discloses glycan utilization strategies.

Understanding how members of the human gut microbiota prioritize nutrient resources is one component of a larger effort to decipher the mechanisms defining microbial community robustness and resiliency in health and disease. This knowledge is foundational for development of microbiota-directed therapeutics. To model how bacteria prioritize glycans in the gut, germfree mice were colonized with 13 human gut bacterial strains, including seven saccharolytic species.