Global O-glycoproteome enrichment and analysis enabled by a combinatorial enzymatic workflow.

A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.

Targeted and Self-Adjuvated Nanoglycovaccine Candidate for Cancer Immunotherapy.

Advanced-stage solid primary tumors and metastases often express mucin 16 (MUC16), carrying immature glycans such as the Tn antigen, resulting in specific glycoproteoforms not found in healthy human tissues. This presents a valuable approach for designing targeted therapeutics, including cancer glycovaccines, which could potentially promote antigen recognition and foster the immune response to control disease spread and prevent relapse. In this study, we describe an adjuvant-free poly(lactic-co-glycolic acid) (PLGA)-based nanoglycoantigen delivery approach that outperforms conventional methods by eliminating the need for protein carriers while exhibiting targeted and adjuvant properties. To achieve this, we synthesized a library of MUC16-Tn glycoepitopes through single-pot enzymatic glycosylation, which were then stably engrafted onto the surface of PLGA nanoparticles, generating multivalent constructs that better represent cancer molecular heterogeneity. These glycoconstructs demonstrated affinity for Macrophage Galactose-type Lectin (MGL) receptor, known to be highly expressed by immature antigen-presenting cells, enabling precise targeting of immune cells. Moreover, the glycopeptide-grafted nanovaccine candidate displayed minimal cytotoxicity and induced the activation of dendritic cells in vitro, even in the absence of an adjuvant. In vivo, the formulated nanovaccine candidate was also nontoxic and elicited the production of IgG specifically targeting MUC16 and MUC16-Tn glycoproteoforms in cancer cells and tumors, offering potential for precise cancer targeting, including targeted immunotherapies.

Comparison of sialylated and fucosylated N-glycans attached to Asn 6 and Asn 41 with different roles in hyaluronan and proteoglycan link protein 1 (HAPLN1).

Hyaluronan and proteoglycan link protein 1 (HAPLN1) is an extracellular matrix protein stabilizing interactions between hyaluronan and proteoglycan. Although HAPLN1 is being investigated for various biological roles, its N-glycosylation is poorly understood. In this study, the structure of N-glycopeptides of trypsin-treated recombinant human HAPLN1 (rhHAPLN1) expressed from CHO cells were identified by nano-liquid chromatography-tandem mass spectrometry. A total of 66 N-glycopeptides were obtained, including 16 and 12 N-glycans at sites Asn 6 (located in the N-terminal region) and Asn 41 (located in the Ig-like domain, which interacts with proteoglycan), respectively. The quantities (%) of each N-glycan relative to the totals (100 %) at each site were calculated. Tri- and tetra-sialylation (to resist proteolysis and extend half-life) were more abundant at Asn 6, and di- (core- and terminal-) fucosylation (to increase binding affinity and stability) and sialyl-Lewis X/a epitope (a major ligand for E-selectin) were more abundant at Asn 41. These results indicate that N-glycans attached to Asn 6 (protecting HAPLN1) and Asn 41 (supporting molecular interactions) play different roles in HAPLN1. This is the first study of site-specific N-glycosylation in rhHAPLN1, which will be useful for understanding its molecular interactions in the extracellular matrix.

GlyComb: A novel glycoconjugate data repository that bridges glycomics and proteomics.

The glycosylation of proteins and lipids is known to be closely related to the mechanisms of various diseases such as influenza, cancer, and muscular dystrophy. Therefore, it has become clear that the analysis of post-translational modifications of proteins, including glycosylation, is important to accurately understand the functions of each protein molecule and the interactions among them. In order to conduct large-scale analyses more efficiently, it is essential to promote the accumulation, sharing, and reuse of experimental and analytical data in accordance with the FAIR (Findability, Accessibility, Interoperability, and Re-usability) data principles. However, a FAIR data repository for storing and sharing glycoconjugate information, including glycopeptides and glycoproteins, in a standardized format did not exist. Therefore, we have developed GlyComb (https://glycomb.glycosmos.org) as a new standardized data repository for glycoconjugate data. Currently, GlyComb can assign a unique identifier to a set of glycosylation information associated with a specific peptide sequence or UniProt ID. By standardizing glycoconjugate data via GlyComb identifiers and coordinating with existing web resources such as GlyTouCan and GlycoPOST, a comprehensive system for data submission and data sharing among researchers can be established. Here we introduce how GlyComb is able to integrate the variety of glycoconjugate data already registered in existing data repositories to obtain a better understanding of the available glycopeptides and glycoproteins, and their glycosylation patterns. We also explain how this system can serve as a foundation for a better understanding of glycan function.

Streptomyces-based whole-cell biosensors for detecting diverse cell envelope-targeting antibiotics.

Cell envelope-targeting antibiotics are potent therapeutic agents against various bacterial infections. The emergence of multiple antibiotic-resistant strains underscores the significance of identifying potent antimicrobials specifically targeting the cell envelope. However, current drug screening approaches are tedious and lack sufficient specificity and sensitivity, warranting the development of more efficient methods. Genetic circuit-based whole-cell biosensors hold great promise for targeted drug discovery from natural products. Here, we performed comparative transcriptomic analysis of Streptomyces coelicolor M1146 exposed to diverse cell envelope-targeting antibiotics, aiming to identify regulatory elements involved in perceiving and responding to these compounds. Differential gene expression analysis revealed significant activation of VanS/R two-component system in response to the glycopeptide class of cell envelope-acting antibiotics. Therefore, we engineered a pair of VanS/R-based biosensors that exhibit functional complementarity and possess exceptional sensitivity and specificity for glycopeptides detection. Additionally, through promoter screening and characterization, we expanded the biosensor's detection range to include various cell envelope-acting antibiotics beyond glycopeptides. Our genetically engineered biosensor exhibits superior performance, including a dynamic range of up to 887-fold for detecting subtle antibiotic concentration changes in a rapid 2-h response time, enabling high-throughput screening of natural product libraries for antimicrobial agents targeting the bacterial cell envelope.

Facile Preparation of Carbohydrate-Containing Adjuvants Based on Self-Assembling Glycopeptide Conjugates.

Carbohydrates are intriguing biomolecules possessing diverse biological activities, including immune stimulating capability. However, their biomedical applications have been limited by their complex and heterogeneous structures. In this study, we have utilized a self-assembling glycopeptide conjugate (GPC) system to produce uniform nanoribbons appending homogeneous oligosaccharides with multivalency. This system successfully translates the nontrivial structural differences of oligomannoses into varied binding affinities to C-type lectin receptors (CLRs). We have shown that GPCs could promote the CLR-mediated endocytosis of ovalbumin (OVA) antigen, and two mannotriose-modified peptides F3m2 and F3m5 exhibit potent activity in inducing antigen-presenting cell maturation, as indicated by increased CD86 and MHCII expression. In vivo studies demonstrated that GPCs, combined with OVA antigen, significantly enhanced OVA-specific antibody production. Specifically, F3m2 and F3m5 exhibited the highest immunostimulatory effects, eliciting both Th1- and Th2-biased immune responses and promoting differentiation of CD4+ and CD8+  T cells. These findings highlight the potential of GPCs as vaccine adjuvants, and showcase their versatility in exploiting the biological functions of carbohydrates.

Salivary Proteome and Intact N-Glycopeptides Analysis Reveal Specific Signatures in Periodontitis.

Periodontitis is a prevalent oral inflammatory disease that can result in tooth loss and is closely linked to type 2 diabetes (T2D). In this study, we analyzed the salivary proteome and intact N-glycopeptides (IGPs) of individuals with mild-moderate, severe, aggressive periodontitis, and periodontitis with T2D, including those treated with antidiabetic drugs, to identify specific signatures associated with the disease. Our results revealed that salivary proteins and glycoproteins were altered in all periodontitis groups (PRIDE ID: 1-20230612-72345), with fucose- and sialic acid-containing N-glycans showing the greatest increase. Additionally, differentially expressed proteins were classified into 9 clusters, including those that were increased in all periodontitis groups and those that were only altered in certain types of periodontitis. Interestingly, treatment with antidiabetic drugs reversed many of the changes observed in the salivary proteome and IGPs in T2D-related periodontitis, suggesting a potential therapeutic approach for managing periodontitis in patients with T2D. Consistent with MS/MS results, the expression of salivary IGHA2 and Fucα1-3/6GlcNAc (AAL) was significantly increased in MP. These findings provide new insights into the pathogenesis of periodontitis and highlight the potential of salivary biomarkers for diagnosis, prognosis, and monitoring of disease progression and treatment response.

Expression of influenza A virus glycan receptor candidates in mallard, chicken, and tufted duck.

Influenza A virus (IAV) pandemics result from interspecies transmission events within the avian reservoir and further into mammals including humans. Receptor incompatibility due to differently expressed glycan structures between species has been suggested to limit zoonotic IAV transmission from the wild bird reservoir as well as between different bird species. Using glycoproteomics, we have studied the repertoires of expressed glycan structures with focus on putative sialic acid-containing glycan receptors for IAV in mallard, chicken and tufted duck; three bird species with different roles in the zoonotic ecology of IAV. The methodology used pinpoints specific glycan structures to specific glycosylation sites of identified glycoproteins and was also used to successfully discriminate α2-3- from α2-6-linked terminal sialic acids by careful analysis of oxonium ions released from glycopeptides in tandem MS/MS (MS2), and MS/MS/MS (MS3). Our analysis clearly demonstrated that all three bird species can produce complex N-glycans including α2-3-linked sialyl Lewis structures, as well as both N- and O- glycans terminated with both α2-3- and α2-6-linked Neu5Ac. We also found the recently identified putative IAV receptor structures, Man-6P N-glycopeptides, in all tissues of the three bird species. Furthermore, we found many similarities in the repertoires of expressed receptors both between the bird species investigated and to previously published data from pigs and humans. Our findings of sialylated glycan structures, previously anticipated to be mammalian specific, in all three bird species may have major implications for our understanding of the role of receptor incompatibility in interspecies transmission of IAV.

Multiplexed Antibody Glycosylation Profiling Using Dual Enzyme Digestion and Liquid Chromatography-Triple Quadrupole Mass Spectrometry Method.

Antibody glycosylation plays a crucial role in the humoral immune response by regulating effector functions and influencing the binding affinity to immune cell receptors. Previous studies have focused mainly on the immunoglobulin G (IgG) isotype owing to the analytical challenges associated with other isotypes. Thus, the development of a sensitive and accurate analytical platform is necessary to characterize antibody glycosylation across multiple isotypes. In this study, we have developed an analytical workflow using antibody-light-chain affinity beads to purify IgG, IgA, and IgM from 16 µL of human plasma. Dual enzymes, trypsin and Glu-C, were used during on-bead digestion to obtain enzymatic glycopeptides and protein-specific surrogate peptides. Ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry was used in order to determine the sensitivity and specificity. Our platform targets 95 glycopeptides across the IgG, IgA, and IgM isotypes, as well as eight surrogate peptides representing total IgG, four IgG classes, two IgA classes, and IgM. Four stable isotope-labeled internal standards were added after antibody purification to calibrate the preparation and instrumental bias during analysis. Calibration curves constructed using serially diluted plasma samples showed good curve fitting (R2 > 0.959). The intrabatch and interbatch precision for all the targets had relative standard deviation of less than 29.6%. This method was applied to 19 human plasma samples, and the glycosylation percentages were calculated, which were comparable to those reported in the literature. The developed method is sensitive and accurate for Ig glycosylation profiling. It can be used in clinical investigations, particularly for detailed humoral immune profiling.

Site-Specific Profiling of N-Glycans in Drosophila melanogaster.

BACKGROUND: Drosophila melanogaster is a well-studied and highly tractable genetic model system for deciphering the molecular mechanisms underlying various biological processes. Although being one of the most critical post-translational modifications of proteins, the understanding of glycosylation in Drosophila is still lagging behind compared with that of other model organisms. METHODS: In this study, we systematically investigated the site-specific N-glycan profile of Drosophila melanogaster using intact glycopeptide analysis technique. This approach identified the glycans, proteins, and their glycosites in Drosophila, as well as information on site-specific glycosylation, which allowed us to know which glycans are attached to which glycosylation sites. RESULTS: The results showed that the majority of N-glycans in Drosophila were high-mannose type (69.3%), consistent with reports in other insects. Meanwhile, fucosylated N-glycans were also highly abundant (22.7%), and the majority of them were mono-fucosylated. In addition, 24 different sialylated glycans attached with 16 glycoproteins were identified, and these proteins were mainly associated with developmental processes. Gene ontology analysis showed that N-glycosylated proteins in Drosophila were involved in multiple biological processes, such as axon guidance, N-linked glycosylation, cell migration, cell spreading, and tissue development. Interestingly, we found that seven glycosyltransferases and four glycosidases were N-glycosylated, which suggested that N-glycans may play a regulatory role in the synthesis and degradation of N-glycans and glycoproteins. CONCLUSIONS: To our knowledge, this work represents the first comprehensive analysis of site-specific N-glycosylation in Drosophila, thereby providing new perspectives for the understanding of biological functions of glycosylation in insects.

IGF2 Peptide-Based LYTACs for Targeted Degradation of Extracellular and Transmembrane Proteins.

Lysosome-targeting chimeras (LYTACs) have recently been developed to facilitate the lysosomal degradation of specific extracellular and transmembrane molecular targets. However, the LYTAC particles described to date are based on glycopeptide conjugates, which are difficult to prepare and produce on a large scale. Here, we report on the development of pure protein LYTACs based on the non-glycosylated IGF2 peptides, which can be readily produced in virtually any facility capable of monoclonal antibody production. These chimeras utilize the IGF2R/CI-M6PR pathway for lysosomal shuttling and, in our illustrative example, target programmed death ligand 1 (PD-L1), eliciting physiological effects analogous to immune checkpoint blockade. Results from in vitro assays significantly exceed the effects of anti-PD-L1 antibodies alone.

Molecular mechanism of decision-making in glycosaminoglycan biosynthesis.

Two major glycosaminoglycan types, heparan sulfate (HS) and chondroitin sulfate (CS), control many aspects of development and physiology in a type-specific manner. HS and CS are attached to core proteins via a common linker tetrasaccharide, but differ in their polymer backbones. How core proteins are specifically modified with HS or CS has been an enduring mystery. By reconstituting glycosaminoglycan biosynthesis in vitro, we establish that the CS-initiating N-acetylgalactosaminyltransferase CSGALNACT2 modifies all glycopeptide substrates equally, whereas the HS-initiating N-acetylglucosaminyltransferase EXTL3 is selective. Structure-function analysis reveals that acidic residues in the glycopeptide substrate and a basic exosite in EXTL3 are critical for specifying HS biosynthesis. Linker phosphorylation by the xylose kinase FAM20B accelerates linker synthesis and initiation of both HS and CS, but has no effect on the subsequent polymerisation of the backbone. Our results demonstrate that modification with CS occurs by default and must be overridden by EXTL3 to produce HS.

Glycan/Protein-Stable Isotope Labeling in Cell Culture for Enabling Concurrent Quantitative Glycomics/Proteomics/Glycoproteomics.

The complexity and heterogeneity of protein glycosylation present an analytical challenge to the studies of characterization and quantitation. Various LC-MS-based quantitation strategies have emerged in recent decades. Metabolic stable isotope labeling has been developed to enhance the accurate LC/MS-based quantitation between different cell lines. Stable isotope labeling by amino acids in a cell culture (SILAC) is the most widely used metabolic labeling method in proteomic analysis. However, it can only label the peptide backbone and is thus limited in glycomic studies. Here, we present a metabolic isotope labeling strategy, named GlyProSILC (Glycan Protein Stable Isotope Labeling in Cell Culture), that can label both the glycan motif and peptide backbone from the same batch of cells. It was performed by feeding cells with a heavy medium containing amide-15N-glutamine, 13C6-arginine (Arg6), and 13C6-15N2-lysine (Lys8). No significant change of cell line metabolism after GlyProSILC labeling was observed based on transcriptomic, glycomic, and proteomic data. The labeling conditions, labeling efficiency, and quantitation accuracy were investigated. After quantitation correction, we simultaneously quantified 62 N-glycans, 574 proteins, and 344 glycopeptides using the same batch of mixed 231BR/231 cell lines. So far, GlyProSILC provides an accurate and effective quantitation approach for glycomics, proteomics, and glycoproteomics in a cell culture system.

N-linked glycoproteome analysis reveals central glycosylated proteins involved in response to wheat yellow mosaic virus in wheat.

Glycosylation is an important proteins post-translational modification and is involved in protein folding, stability and enzymatic activity, which plays a crucial role in regulating protein function in plants. Here, we report for the first time on the changes of N-glycoproteome in wheat response to wheat yellow mosaic virus (WYMV) infection. Quantitative analyses of N-linked glycoproteome were performed in wheat without and with WYMV infection by ZIC-HILIC enrichment method combined with LC-MS/MS. Altogether 1160 N-glycopeptides and 971 N-glycosylated sites corresponding to 734 N-glycoproteins were identified, of which 64 N-glycopeptides and 64 N-glycosylated sites in 60 N-glycoproteins were significantly differentially expressed. Two conserved typical N-glycosylation motifs N-X-T and N-X-S and a nontypical motifs N-X-C were enriched in wheat. Gene Ontology analysis showed that most differentially expressed proteins were mainly enriched in metabolic process, catalytic activity and response to stress. Kyoto Encyclopedia of Genes and Genomes analysis indicated that two significantly changed glycoproteins were specifically related to plant-pathogen interaction. Furthermore, we found that over-expression of TaCERK reduced WYMV accumulation. Glycosylation site mutation further suggested that N-glycosylation of TaCERK could regulate wheat resistance to WYMV. This study provides a new insight for the regulation of protein N-glycosylation in defense response of plant.

Identification of the glycopeptide epitope recognized by a protective Cryptosporidium monoclonal antibody.

Cryptosporidium species are a leading cause of pediatric diarrheal disease and death in low- and middle-income countries and pose a particular threat to immunocompromised individuals. As a zoonotic pathogen, Cryptosporidium can have devastating effects on the health of neonatal calves. Despite its impact on human and animal health, consistently effective drug treatments for cryptosporidiosis are lacking and no vaccine is available. We previously showed that C. parvum mucin-like glycoproteins, gp40, and gp900 express an epitope identified by a monoclonal antibody 4E9. 4E9 neutralized C. parvum infection in vitro as did glycan-binding proteins specific for the Tn antigen (GalNAc-α1-S/T). Here, we show that 4E9 ameliorates disease in vivo in a calf challenge model. The 4E9 epitope is present on C. hominis in addition to C. parvum gp40 and gp900 and localizes to the plasma membrane and dense granules of invasive and intracellular stages. To characterize the epitope recognized by 4E9, we probed a glycan array containing over 500 defined glycans together with a custom-made glycopeptide microarray containing glycopeptides from native mucins or C. parvum gp40 and gp15. 4E9 exhibited no binding to the glycan array but bound strongly to glycopeptides from native mucins or gp40 on the glycopeptide array, suggesting that the antibody epitope contains both peptide and glycan moieties. 4E9 only recognized glycopeptides with adjacent S or T residues in the motif S*/T*-X-S*/T* where X = 0 or 1. These data define the 4E9 epitope and have implications for the inclusion of the epitope in the development of vaccines or other immune-based therapies.

Short-term association between outdoor temperature and the hydration-marker copeptin: a pooled analysis in five cohorts.

BACKGROUND: Whereas outdoor temperature is linked to both mortality and hydration status, the hormone vasopressin, measured through the surrogate copeptin, is a marker of cardiometabolic risk and hydration. We recently showed that copeptin has a seasonal pattern with higher plasma concentration in winter. Here, we aimed to investigate the association between outdoor temperature and copeptin. METHODS: Copeptin was analysed in fasting plasma from five cohorts in Malmö, Sweden (n = 26,753, 49.7% men, age 18-86 years). We utilized a multivariable adjusted non-linear spline model with four knots to investigate the association between short-term temperature (24 h mean apparent) and log copeptin z-score. FINDINGS: We found a distinct non-linear association between temperature and log copeptin z-score, with both moderately low and high temperatures linked to higher copeptin concentration (p < 0.0001). Between 0 °C and nadir at the 75th temperature percentile (corresponding to 14.3 °C), log copeptin decreased 0.13 z-scores (95% CI 0.096; 0.16), which also inversely corresponded to the increase in z-score log copeptin between the nadir and 21.3 °C. INTERPRETATION: The J-shaped association between short-term temperature and copeptin resembles the J-shaped association between temperature and mortality. Whereas the untangling of temperature from other seasonal effects on hydration warrants further study, moderately increased water intake constitutes a feasible intervention to lower vasopressin and might mitigate adverse health effects of both moderately cold and hot outdoor temperatures. FUNDING: Swedish Research Council, Å Wiberg, M Stephen, A Påhlsson, Crafoord and Swedish Heart-Lung Foundations, Swedish Society for Medical Research and Swedish Society of Medicine.

Comprehensive Glycoprofiling of Oral Tumors Associates N-Glycosylation With Lymph Node Metastasis and Patient Survival.

While altered protein glycosylation is regarded a trait of oral squamous cell carcinoma (OSCC), the heterogeneous and dynamic glycoproteome of tumor tissues from OSCC patients remain unmapped. To this end, we here employ an integrated multi-omics approach comprising unbiased and quantitative glycomics and glycoproteomics applied to a cohort of resected primary tumor tissues from OSCC patients with (n = 19) and without (n = 12) lymph node metastasis. While all tumor tissues displayed relatively uniform N-glycome profiles suggesting overall stable global N-glycosylation during disease progression, altered expression of six sialylated N-glycans was found to correlate with lymph node metastasis. Notably, glycoproteomics and advanced statistical analyses uncovered altered site-specific N-glycosylation revealing previously unknown associations with several clinicopathological features. Importantly, the glycomics and glycoproteomics data unveiled that comparatively high abundance of two core-fucosylated and sialylated N-glycans (Glycan 40a and Glycan 46a) and one N-glycopeptide from fibronectin were associated with low patient survival, while a relatively low abundance of N-glycopeptides from both afamin and CD59 were also associated with poor survival. This study provides insight into the complex OSCC tissue N-glycoproteome, thereby forming an important resource to further explore the underpinning disease mechanisms and uncover new prognostic glycomarkers for OSCC.

Supramolecular Assemblies of Glycopeptides Enhance Gap Junction Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes via Inducing Spheroids Formation to Optimize Cardiac Repair.

Stem cell-based therapies have demonstrated significant potential for use in heart regeneration. An effective paradigm for heart repair in rodent and large animal models is the transplantation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Despite this, the functional and phenotypical immaturity of 2D-cultured hiPSC-CMs, particularly their low electrical integration, poses a caveat for clinical translation. In this study, a supramolecular assembly of a glycopeptide containing a cell adhesion motif-RGD, and saccharide-glucose (Bio-Gluc-RGD) is designed to enable the 3D spheroid formation of hiPSC-CMs, promoting cell-cell and cell-matrix interactions that occur during spontaneous morphogenesis. HiPSC-CMs in spheroids are prone to be phenotypically mature and developed robust gap junctions via activation of the integrin/ILK/p-AKT/Gata4 pathway. Monodispersed hiPSC-CMs encapsulated in the Bio-Gluc-RGD hydrogel are more likely to form aggregates and, therefore, survive in the infarcted myocardium of mice, accompanied by more robust gap junction formation in the transplanted cells, and hiPSC-CMs delivered with the hydrogels also displayed angiogenic effect and anti-apoptosis capacity in the peri-infarct area, enhancing their overall therapeutic efficacy in myocardial infarction. Collectively, the findings illustrate a novel concept for modulating hiPSC-CM maturation by spheroid induction, which has the potential for post-MI heart regeneration.

Comparative Evaluation of Serum Copeptin in Obesity with Non Alcoholic Fatty Liver Disease.

INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) is rapidly becoming a major cause of liver disease in India. NAFLD is regarded as the metabolic syndrome's hepatic component. AVP has a significant impact on glucose and lipid metabolism by stimulating hepatic glycogenolysis, gluconeogenesis, and fat production by modulating insulin and glucagon release from the pancreatic Langerhans' islets. AVP has a relatively short half-life of about 20 minutes, and more than 90% of AVP in circulation is bound to platelets. As a result, AVP is ineffective as a biomarker in clinical practice. Copeptin is a cleavage product of the AVP precursor, pre pro-vasopressin, which is secreted by the posterior pituitary gland in response to hypotension and hyperosmolality. We aimed to compare the serum copeptin level in obese with NAFLD patients with obese without NAFLD patients. MATERIALS: We collected data from 80 obese patients which were divided into two groups based on the presence and absence of NAFLD. Serum copeptin levels were measured by a Human Copeptin ELISA kit (Shanghai Coon Koon). The level of serum Copeptin was compared between two groups and also in three different grades of NAFLD. RESULT: Out of 80 subjects in our study, the mean value of serum copeptin in the group of obese with NAFLD was 24.96 pmol/L and in the group of obese without NAFLD was 15.38 pmol/L. The median (IQR) of serum copeptin in obese with NAFLD group was 24.5 pmol/L and in obese without NAFLD group was 12.25 pmol/L. The highest median S. Copeptin in pmol/L was seen in the obese with NAFLD group with a significant difference between 2 groups (W = 1388.500, p = ≤ 0.001). Strength of Association (Point-Biserial Correlation) = 0.44 (Large Effect Size). The mean value of serum copeptin was 20.00, 24.14, and 31.77 pmol/L in NAFLD grade 1, grade 2, and grade 3 respectively. The highest median S. Copeptin in pmol/L is seen in NAFLD Grade 3 group with a significant difference between 3 groups (χ2 = 23.446, p = ≤ 0.001). CONCLUSION: Serum copeptin can be used as an additional tool to predict the severity of the nonalcoholic fatty liver disease. Measurement of serum copeptin levels can be used as an additional, simple, non-invasive, easily accessible, and cost effective parameter to predict the severity of the nonalcoholic fatty liver disease.

Lycium barbarum glycopeptide targets PER2 to inhibit lipogenesis in glioblastoma by downregulating SREBP1c.

Lycium barbarum polysaccharide (LBP) is a substance with various biological activities extracted from Lycium barbarum. LbGPs are peptidoglycans with a short peptide backbone and a complex, branched glycan moiety, which is further extracted and isolated from LBPs. Previous studies have shown that LbGP can inhibit cancer cell growth, but its specific mechanism is not completely clear. In this study, we found that LbGP could inhibit the proliferation of glioma cells and promote the expression of period 2 (PER2) through the PKA-CREB pathway. In addition, LbGP could inhibit the de novo synthesis of lipids by downregulating SREBP1c and its target genes, which depended on the expression of PER2. Moreover, PER2 negatively regulated the expression of SREBP1c via suppressing PI3K/AKT/mTOR pathway. In summary, LbGP may upregulate the expression of PER2 to reduce the expression of SREBP1c, inhibit lipid synthesis in glioblastoma, and inhibit glioblastoma cell proliferation. This study provides an alternative drug for the treatment of glioma and elucidates its potential mechanism.