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Pancreatic cancer is one of the deadliest cancers worldwide, mainly due to late diagnosis. Therefore, there is an urgent need for novel diagnostic approaches to identify the disease as early as possible. We have developed a diagnostic assay for pancreatic cancer based on the detection of naturally occurring tumor associated autoantibodies against Mucin‐1 (MUC1) using engineered glycopeptides on nanoparticle probes. We used a structure‐guided approach to develop unnatural glycopeptides as model antigens for tumor‐associated MUC1. We designed a collection of 13 glycopeptides to bind either SM3 or 5E5, two monoclonal antibodies with distinct epitopes known to recognize tumor associated MUC1. Glycopeptide binding to SM3 or 5E5 was confirmed by surface plasmon resonance and rationalized by molecular dynamics simulations. These model antigens were conjugated to gold nanoparticles and used in a dot‐blot assay to detect autoantibodies in serum samples from pancreatic cancer patients and healthy volunteers. Nanoparticle probes with glycopeptides displaying the SM3 epitope did not have diagnostic potential. Instead, nanoparticle probes displaying glycopeptides with high affinity for 5E5 could discriminate between cancer patients and healthy controls. Remarkably, the best‐discriminating probes show significantly better true and false positive rates than the current clinical biomarkers CA19‐9 and carcinoembryonic antigen (CEA). [ABSTRACT FROM AUTHOR]

Introduction: High-risk febrile neutropenia (FN) is one of the main causes of morbidity and mortality in onco-hematology. The initiation of empirical antibiotic therapy is an emergency that can change the prognosis of some patients. Given the emergence of increasingly resistant Gram-positive bacteremia, glycopeptides, as an empirical treatment, have an important place in the management of high-risk FN. The aim of this study is to evaluate the appropriateness of glycopeptide prescription in high-risk FN patients. Methods: This study was conducted in the Hematology Department of Fattouma Bourguiba University Hospital of Monastir, Tunisia. Patients with high-risk FN were enrolled during the period between January 1 and December 31, 2020. Results: Of the 29 patients included in this study, 88 FN episodes were noted of which 39 episodes treated with glycopeptides were evaluated. Twenty-four febrile episodes were empirically treated with glycopeptides (27.3%) of which 17 prescriptions (70.8%) were appropriate according to the European Conference on Infection in Leukemia and the Infectious Diseases Society of America recommendations. A therapeutic escalation using glycopeptides was noted in 17% of cases and appropriately opted in 6 FN episodes (40%). Conclusion: Prescriptions of glycopeptides were appropriate according to the international recommendations in 71% of the empirical prescriptions and in 40% of the therapeutic escalation using glycopeptides. In high-risk FN episodes, glycopeptides prescriptions should be rationalized and limited to the indications detailed in the international guidelines to control the emergence of multidrug-resistant bacteria. [ABSTRACT FROM AUTHOR]

Proteoglycans (PGs), consisting of glycosaminoglycans (GAGs) linked with the core protein through a tetrasaccharide linkage region, play roles in many important biological events. The chemical synthesis of PG glycopeptides is extremely challenging. In this work, the enzymes required for synthesis of chondroitin sulfate (CS) PG (CSPG) have been expressed and the suitable sequence of enzymatic reactions has been established. To expedite CSPG synthesis, the peptide acceptor was immobilized on solid phase and the glycan units were directly installed enzymatically onto the peptide. Subsequent enzymatic chain elongation and sulfation led to the successful synthesis of CSPG glycopeptides. The CS dodecasaccharide glycopeptide was the longest homogeneous CS glycopeptide synthesized to date. The enzymatic synthesis was much more efficient than the chemical synthesis of the corresponding CS glycopeptides, which could reduce the total number of synthetic steps by 80 %. The structures of the CS glycopeptides were confirmed by mass spectrometry analysis and NMR studies. In addition, the interactions between the CS glycopeptides and cathepsin G were studied. The sulfation of glycan chain was found to be important for binding with cathepsin G. This efficient chemoenzymatic strategy opens new avenues to investigate the structures and functions of PGs. [ABSTRACT FROM AUTHOR]

Reported herein is a new HPLC‐based automated synthesizer (HPLC−A) capable of a temperature‐controlled synthesis and purification of carbohydrates. The developed platform allows to perform various protecting group manipulations as well as the synthesis of O‐ and N‐glycosides. A fully automated synthesis and purification was showcased in application to different carbohydrate derivatives including glycosides, oligosaccharides, glycopeptides, glycolipids, and nucleosides. [ABSTRACT FROM AUTHOR]

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The study of glycoproteomics presents a set of unique challenges, primarily due to the low abundance of glycopeptides and their intricate heterogeneity, which is specific to each site. Glycoproteins play a crucial role in numerous biological functions, including cell signaling, adhesion, and intercellular communication, and are increasingly recognized as vital markers in the diagnosis and study of various diseases. Consequently, a quantitative approach to glycopeptide research is essential. One effective strategy to address this need is the use of multiplex glycopeptide labeling. By harnessing the synergies of 15N metabolic labeling via the isotopic detection of amino sugars with glutamine (IDAWG) technique for glycan parts and tandem mass tag (TMT)pro labeling for peptide backbones, we have developed a method that allows for the accurate quantification and comparison of multiple samples simultaneously. The adoption of the liquid chromatography-synchronous precursor selection (LC-SPS-MS3) technique minimizes fragmentation interference, enhancing data reliability, as shown by a 97% TMT labeling efficiency. This method allows for detailed, high-throughput analysis of 32 diverse samples from 231BR cell lines, using both 14N and 15N glycopeptides at a 1:1 ratio. A key component of our methodology was the precise correction for isotope and TMTpro distortions, significantly improving quantification accuracy to less than 5% distortion. This breakthrough enhances the efficiency and accuracy of glycoproteomic studies, increasing our understanding of glycoproteins in health and disease. Its applicability to various cancer cell types sets a new standard in quantitative glycoproteomics, enabling deeper investigation into glycopeptide profiles. [ABSTRACT FROM AUTHOR]

Self‐assembly in living cells represents one versatile strategy for drug delivery; however, it suffers from the limited precision and efficiency. Inspired by viral traits, we here report a cascade targeting‐hydrolysis‐transformation (THT) assembly of glycosylated peptides in living cells holistically resembling viral infection for efficient cargo delivery and combined tumor therapy. We design a glycosylated peptide via incorporating a β‐galactose‐serine residue into bola‐amphiphilic sequences. Co‐assembling of the glycosylated peptide with two counterparts containing irinotecan (IRI) or ligand TSFAEYWNLLSP (PMI) results in formation of the glycosylated co‐assemblies SgVEIP, which target cancer cells via β‐galactose‐galectin‐1 association and undergo galactosidase‐induced morphological transformation. While GSH‐reduction causes release of IRI from the co‐assemblies, the PMI moieties release p53 and facilitate cell death via binding with protein MDM2. Cellular experiments show membrane targeting, endo‐/lysosome‐mediated internalization and in situ formation of nanofibers in cytoplasm by SgVEIP. This cascade THT process enables efficient delivery of IRI and PMI into cancer cells secreting Gal‐1 and overexpressing β‐galactosidase. In vivo studies illustrate enhanced tumor accumulation and retention of the glycosylated co‐assemblies, thereby suppressing tumor growth. Our findings demonstrate an in situ assembly strategy mimicking viral infection, thus providing a new route for drug delivery and cancer therapy in the future. [ABSTRACT FROM AUTHOR]

In this work, a new type of FE-1 COF material is prepared by a reversible imine condensation reaction with diaminoferrocene and diaminodiformaldehyde as materials. The material is connected by imine bonds to form a COF skeleton, and the presence of plenty of nitrogen-containing groups gives the material good hydrophilicity; the presence of metal Fe ions provides the material application potential in the enrichment of phosphopeptides. According to the different binding abilities of N-glycopeptide and phosphopeptide on FE-1 COF, it can simultaneously enrich N-glycopeptide and phosphopeptide through different elution conditions to realize its controllable and selective enrichment. Using the above characteristics, 18 phosphopeptides were detected from α-casein hydrolysate, 8 phosphopeptides were detected from β-casein hydrolysate and 21 glycopeptides were detected from IgG hydrolysate. Finally, the gradual elution strategy was used; 16 phosphopeptides and 19 glycopeptides were detected from the α-casein hydrolysate and IgG hydrolysate. The corresponding glycopeptides and phosphopeptides were identified from the human serum. It proves that the FE-1 COF material has a good enrichment effect on phosphopeptides and glycopeptides. [ABSTRACT FROM AUTHOR]

Endogenous peptides, particularly those with post-translational modifications, are increasingly being studied as biomarkers for diagnosing various diseases. However, they are weakly ionizable, have a low abundance in biological samples, and may be interfered with by high levels of proteins, peptides, and other macromolecular impurities, resulting in a high limit of detection and insufficient amounts of post-translationally modified peptides in real biological samples to be examined. Therefore, separation and enrichment are necessary before analyzing these biomarkers using mass spectrometry. Mesoporous materials have regular adjustable pores that can eliminate large proteins and impurities, and their large specific surface area can bind more target peptides, but this may result in the partial loss or destruction of target peptides during centrifugal separation. On the other hand, magnetic mesoporous materials can be used to separate the target using an external magnetic field, which improves the separation efficiency and yield. Core–shell magnetic mesoporous materials are widely utilized for peptide separation and enrichment due to their biocompatibility, efficient enrichment capability, and excellent recoverability. This paper provides a review of the latest progress in core–shell magnetic mesoporous materials for enriching glycopeptides and phosphopeptides and compares their enrichment performance with different types of functionalization methods. [ABSTRACT FROM AUTHOR]

Infections due to multidrug-resistant organisms (MDRO) are a serious concern for public health with high morbidity and mortality. Though many antibiotics have been introduced to manage these infections, there are remaining concerns regarding the optimal management of Gram-positive MDROs. A literature search on the PubMed/Medline database was conducted. We applied no language and time limits for the search strategy. In this narrative review, we discuss the current options for managing Gram-positive MDROs as well as non-traditional antibacterial agents in development. Despite their introduction more than 70 years ago, glycopeptides are still the cornerstone in treating Gram-positive infections: all registrative studies of new antibiotics have glycopeptides as control; these studies are designed as not inferior studies, therefore it is almost impossible to give recommendations other than the use of glycopeptides in the treatment of Gram-positive infections. The best evidence on treatments different from glycopeptides comes from post-hoc analysis and meta-analysis. Non-traditional antibacterial agents are being studied to aid in short and effective antibiotic therapies. The use of non-traditional antibacterial agents is not restricted to replacing traditional antibacterial agents with alternative therapies; instead, they should be used in combination with antibiotic therapies. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Expressed protein ligation (EPL) provides a powerful tool to access large‐size proteins with precise structures. Existing methods for constructing the critical protein thioester for EPL have predominantly relied on the recombinant intein fusion expressed in Escherichia coli (E. coli). Despite its powerful applications, the expression of thioester derived from eukaryotic protein in E. coli inherently suffers from its limited solubility, the inactivity of intein, premature hydrolysis and low yields. To overcome these obstacles, we present herein the facile one‐flask synthesis of inaccessible protein α‐thioester via a SUMO‐protein‐intein (SPI) sandwich model. The utility of SUMO enhances the protein fusion yield and solubility, prevents premature hydrolysis and simplifies the purification process. The inaccessible protein thioester with internal Cys residues can be readily produced and is compatible with the EPL‐desulfurization protocol used to prepare complex proteins, which is otherwise difficult to obtain using traditional methods. Its utility has been highlighted through the synthesis of human granulocyte colony‐stimulating factor (G‐CSF). [ABSTRACT FROM AUTHOR]

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The emergence of antibiotic resistance poses a significant threat to public health worldwide, affecting various medical fields, including ophthalmology. Eye infections, ranging from conjunctivitis to more severe conditions like keratitis, are commonly treated with antibiotics. However, the misuse and overuse of these drugs have led to the development of resistant strains of bacteria, allowing traditional treatments ineffective. This paper aims to examine the current situation of antibiotic resistance in eye infections globally, with a specific focus on a large group of hospitals located in Milan (Italy) with considerable experience in cataract and cornea surgery as well as in retinopathy. The results of the study show the prevalence of Gram-positives in the tested samples and a low resistance of fluoroquinolones and glycopeptides. The results also highlight the need to implement sample collection methods for ocular infections, as the quantity of positive samples is rather low compared to the total number of samples. In conclusion, the study, although with limited data, shows that resistance to aminoglycosides and cephalosporins is a situation to be monitored. These data also show the critical need to improve and guide the biological sample collection modalities in order to make the diagnosis more reliable. [ABSTRACT FROM AUTHOR]

SUMMARY: The primary proteins in egg whites are glycoproteins, which are hydrolyzed by digestive enzymes to produce substantial egg white glycopeptides (EWGs). However, the potential role of EWG has not been reported. In this study, EWG was obtained through simulated digestion in vitro and affinity chromatography enrichment, and we further examined the effect of EWG on protein expression in mouse macrophages (RAW264.7) through quantitative proteomic analysis. Compared to the control group, 149 differentially expressed proteins (DEPs) were upregulated, and 35 DEPs were downregulated in RAW264.7 cells by treatment with EWG (125 µg/mL). These DEPs were related to 76 KEGG pathways (such as “NF-kappa B signaling pathway,” “TNF signaling pathway,” “C-type lectin receptor signaling pathway”) and 12 protein domains (such as “Lectin C-type domain,” “Immunoglobulin domain,” “Immunoglobulin V-set domain”), which suggested that EWG could exert immunomodulatory effects through immune signaling pathways and protein domains. Our findings provide promising signaling pathways for in-depth exploration of the immunomodulation of EWG and contribute to the development of EWG as a potential immunoactivity ingredient.

Abstract Introduction Long-acting lipoglycopeptides such as dalbavancin may have utility in patients with Gram-positive bloodstream infections (BSI), particularly in those with barriers to discharge or who require prolonged parenteral antibiotic courses. A retrospective cohort study was performed to provide further multicenter real-world evidence on dalbavancin use as a sequential therapy for Gram-positive BSI. Methods One hundred fifteen patients received dalbavancin with Gram-positive BSI, defined as any positive blood culture or diagnosed with infective endocarditis, from 13 centers geographically spread across the United States between July 2015 and July 2021. Results Patients had a mean (SD) age of 48.5 (17.5) years, the majority were male (54%), with many who injected drugs (40%). The most common infection sources (non-exclusive) were primary BSI (89%), skin and soft tissue infection (SSTI) (25%), infective endocarditis (19%), and bone and joint infection (17%). Staphylococcus aureus accounted for 72% of index cultures, coagulase-negative Staphylococcus accounted for 18%, and Streptococcus species in 16%. Dalbavancin started a median (Q1–Q3) of 10 (6–19) days after index culture collection. The most common regimen administered was dalbavancin 1500 mg as one dose for 50% of cases. The primary outcome of composite clinical failure occurred at 12.2%, with 90-day mortality at 7.0% and 90-day BSI recurrence at 3.5%. Conclusions Dalbavancin may serve as a useful tool in facilitating hospital discharge in patients with Gram-positive BSI. Randomized controlled trials are anticipated to validate dalbavancin as a surrogate to current treatment standards.

Carbohydrates play pivotal roles in an array of essential biological processes and are consequently involved in many diseases. To meet the needs of glycobiology research, chemical enzymatic and non‐enzymatic methods have been developed to generate glycoconjugates with well‐defined structures. Herein, harnessing the unique properties of C6‐oxidized glycans, we report a straightforward and robust strategy for site‐ and stereoselective glycomodification of biomolecules with N‐terminal tryptophan residues by a carbohydrate‐promoted Pictet–Spengler reaction, which is not adapted to typical aldehyde substrates under biocompatible conditions. This method reliably delivers highly homogeneous glycoconjugates with stable linkages and thus has great potential for functional modulation of peptides and proteins in glycobiology research. Moreover, this reaction can be performed at the glycosites of glycopeptides, glycoproteins and living‐cell surfaces in a site‐specific manner. Control experiments indicated that the protected α‐O atom of aldehyde donors and free N−H bond of the tryptamine motif are crucial for this reaction. Mechanistic investigations demonstrated that the reaction exhibited a first‐order dependence on both tryptophan and glycan, and deprotonation/rearomatization of the pentahydro‐β‐carbolinium ion intermediate might be the rate‐determining step. [ABSTRACT FROM AUTHOR]

In the context of peptide drug development, glycosylation plays a pivotal role. Accordingly, L-type peptides were synthesized predicated upon the PD-1/PD-L1 blocker DPPA-1. Subsequent glycosylation resulted in the production of two distinct glycopeptides, D-glu-LPPA-1 and D-gal-LPPA-1, by using D-glucose (D-glu) and D-galactose (D-gal), respectively, during glycosylation. Both glycopeptides significantly inhibited the interaction between PD-1 and PD-L1, and the measured half maximal inhibitory concentrations (IC50s) were 75.5 μM and 101.9 μM for D-glu-LPPA-1 and D-gal-LPPA-1, respectively. Furthermore, D-gal-LPPA-1 displayed a pronounced ability to restore T-cell functionality. In an MC38 tumor-bearing mouse model, D-gal-LPPA-1 demonstrated a significant inhibitory effect. Notably, D-gal-LPPA-1 substantially augmented the abundance and functionality of CD8+ T cells in the tumor microenvironment. Additionally, in the lymph nodes and spleens, D-gal-LPPA-1 significantly increased the proportion of CD8+ T cells secreting interferon-gamma (IFN-γ). These strong findings position D-gal-LPPA-1 as a potent enhancer of the antitumor immune response in MC38 tumor-bearing mice, underscoring its potential as a formidable PD-1/PD-L1 blocking agent. [ABSTRACT FROM AUTHOR]

Objectives This study aims to elucidate the genomic dynamics driving the emergence of antimicrobial resistance (AMR), with a specific focus on the interplay between AMR and antimicrobial usage. Methods We conducted a comprehensive analysis using a ST239 methicillin-resistant Staphylococcus aureus (MRSA) dataset over a continuous 12-year period from a single hospital. Genomic analyses were performed tracking the changes in MRSA populations, particularly the emergence of reduced vancomycin susceptibility, and assessing the impact of glycopeptide use on these emergence events. Results Our findings reveal a significant correlation between hospital glycopeptide usage and the selection of MRSA strains with reduced vancomycin susceptibility. Genomic analyses provided insights into the molecular mechanisms driving resistance emergence, including the slowing of the molecular clock rate in response to heightened antimicrobial consumption. Conclusions In conclusion, this study the highlights the complex dynamics between AMR and antimicrobial use at the hospital level. The observed correlation between antimicrobial consumption and the development of less susceptible MRSA strains underscores the importance of antimicrobial stewardship programmes and the establishment of optimal consumption thresholds for mitigating AMR effectively. [ABSTRACT FROM AUTHOR]

Modern glycoproteomics experiments require the use of search engines due to the generation of countless spectra. While these tools are valuable, manual validation of search engine results is often required for detailed analysis of glycopeptides as false-discovery rates are often not reliable for glycopeptide data. Near-isobaric mismatches are a common source of misidentifications for the popular glycopeptide-focused search engine pGlyco3.0, and in this technical note we share a strategy and script that improves the accuracy of the search utilizing two manually validated datasets of the glycoproteins CD16a and HIV-1 Env as proof-of-principle. [ABSTRACT FROM AUTHOR]

The sample preparation step is pivotal in glycoproteomic analysis. An effective approach in glycoprotein sample preparation involves enriching glycopeptides by solid-phase extraction (SPE) using polar stationary phases in hydrophilic interaction liquid chromatography (HILIC) mode. The aim of this work is to show how different experimental conditions influence the enrichment efficiency of glycopeptides from human immunoglobulin G (IgG) on an aminopropyl-modified SPE column. Different compositions of the elution solvent (acetonitrile, methanol, and isopropanol), along with varying concentrations of elution solvent acidifiers (formic and acetic acid), and different concentrations of acetonitrile for the conditioning and washing solvents (65%, 75%, and 85% acetonitrile) were tested to observe their effects on the glycopeptide enrichment process. Isopropanol proved less effective in enriching glycopeptides, while acetonitrile was the most efficient, with methanol in between. Higher formic acid concentrations in the elution solvent weakened the ionic interactions, particularly with sialylated glycopeptides. Substituting formic acid with acetic acid led to earlier elution of more glycopeptides. The acetonitrile concentration in conditioning and washing solutions played a key role; at 65% acetonitrile, glycopeptides were not retained on the SPE column and were detected in the flow-through fraction. Ultimately, it was proven that the enrichment method was applicable to human plasma samples, resulting in a significant decrease in the abundances of non-glycosylated peptides. To the best of our knowledge, this study represents the first systematic investigation into the impact of the mobile phase on glycopeptide enrichment using an aminopropyl-modified SPE column in HILIC mode. This study demonstrates the substantial impact of even minor variations in experimental conditions, which have not yet been considered in the literature, on SPE-HILIC glycopeptide enrichment. Consequently, meticulous optimization of these conditions is imperative to enhance the specificity and selectivity of glycoproteomic analysis, ensuring accurate and reliable quantification. [ABSTRACT FROM AUTHOR]

Deep learning has achieved a notable success in mass spectrometry-based proteomics and is now emerging in glycoproteomics. While various deep learning models can predict fragment mass spectra of peptides with good accuracy, they cannot cope with the non-linear glycan structure in an intact glycopeptide. Herein, we present DeepGlyco, a deep learning-based approach for the prediction of fragment spectra of intact glycopeptides. Our model adopts tree-structured long-short term memory networks to process the glycan moiety and a graph neural network architecture to incorporate potential fragmentation pathways of a specific glycan structure. This feature is beneficial to model explainability and differentiation ability of glycan structural isomers. We further demonstrate that predicted spectral libraries can be used for data-independent acquisition glycoproteomics as a supplement for library completeness. We expect that this work will provide a valuable deep learning resource for glycoproteomics. Deep learning has achieved a notable success in proteomics and is now emerging in glycoproteomics. Here, the authors develop a neural network-based method to predict mass spectra of intact glycopeptides and demonstrate its potential in data-dependent and data-independent acquisition glycoproteomics. [ABSTRACT FROM AUTHOR]

Rationale: Hydrogen/deuterium exchange mass spectrometry (HDX‐MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat‐denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX‐MS, but one challenge for complete sequence coverage is N‐glycosylation sites. The deuteration of peptides post‐translationally modified by asparagine‐bound glycans (glycopeptides) has not always been identified in previous reports of HDX‐MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein. Methods: We detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data‐dependent acquisition. An MS scan was used to identify precursor ions; if high‐energy collision‐induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low‐energy collision‐induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX‐MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2O. Results: We report HDX‐MS analysis of the SARS‐CoV‐2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N‐glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements. Conclusion: Inclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to identify glycopeptide resistance genes and virulence factors in vancomycin-resistant Enterococcus species and to investigate the influence of the microbiota and hospital environment on glycopeptide resistance. Materials and Methods: A total of 107 enterococcal isolates were collected from patients' rectal swab cultures and environmental samples taken for surveillance purposes. Multiplex Polymerase Chain Reaction (PCR) analysis was conducted to investigate specific virulence genes (esp, hyl, asa1, cyl, and galE) and glycopeptide resistance genes (vanA, vanB, van C1-C2, van D, vanE, and vanG). Additionally, perirectal swab cultures were obtained from patients without vancomycin-resistant enterococcal colonization to investigate the presence of glycopeptide resistance genes in their microbiota. Results: Seven isolates (6.5%) were identified as infectious agents. The most common vancomycin resistance genes were vanA (23.3%), followed by vanA + vanB (14%) and vanB + vanD (14%), respectively. The Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR) method showed that patient surveillance and environmental isolates were clonally related. Moreover, microbiota analysis of patients without vancomycin-resistant enterococcal colonization revealed Clostridium spp. in two patients and Lactobacillus spp. in one patient, with the vanG gene found in the microbiota of only one (2.5%) patient. Conclusion: The detection of genes responsible for dissemination indicates that colonized isolates also have the potential for infection, and the hospital environment plays a primary role in the acquisition of vancomycin-resistant enterococci. [ABSTRACT FROM AUTHOR]

Introduction: Long-acting lipoglycopeptides such as dalbavancin may have utility in patients with Gram-positive bloodstream infections (BSI), particularly in those with barriers to discharge or who require prolonged parenteral antibiotic courses. A retrospective cohort study was performed to provide further multicenter real-world evidence on dalbavancin use as a sequential therapy for Gram-positive BSI. Methods: One hundred fifteen patients received dalbavancin with Gram-positive BSI, defined as any positive blood culture or diagnosed with infective endocarditis, from 13 centers geographically spread across the United States between July 2015 and July 2021. Results: Patients had a mean (SD) age of 48.5 (17.5) years, the majority were male (54%), with many who injected drugs (40%). The most common infection sources (non-exclusive) were primary BSI (89%), skin and soft tissue infection (SSTI) (25%), infective endocarditis (19%), and bone and joint infection (17%). Staphylococcus aureus accounted for 72% of index cultures, coagulase-negative Staphylococcus accounted for 18%, and Streptococcus species in 16%. Dalbavancin started a median (Q1–Q3) of 10 (6–19) days after index culture collection. The most common regimen administered was dalbavancin 1500 mg as one dose for 50% of cases. The primary outcome of composite clinical failure occurred at 12.2%, with 90-day mortality at 7.0% and 90-day BSI recurrence at 3.5%. Conclusions: Dalbavancin may serve as a useful tool in facilitating hospital discharge in patients with Gram-positive BSI. Randomized controlled trials are anticipated to validate dalbavancin as a surrogate to current treatment standards. [ABSTRACT FROM AUTHOR]

To realize the high-value utilization of buckwheat protein resources, α-glucosidase-inhibiting glycopeptide (α-GIG) was prepared by fermentation of Lactobacillus plantarum and Streptococcus thermophilus which were selected from 6 strains. One-factor-at-a-time method aimed to determine the level of experimental factors and a further response surface experiment was used to optimize fermentation. The fermentation broth was separated and purified by Sephadex G-25 and reversed phase-high performance liquid chromatography (RP-HPLC) to prepare a-GIG. The method of β-elimination reaction was used to determine the type of glycopeptide bond and infrared spectroscopy was used to analyze the secondary structure. The inhibitory effects of α-GIG on α-glucosidase was investigated by enzyme kinetics. The results showed that when the inoculum was 2%, pH was 7.5, solid-liquid ratio was 1:14.8 and fermentation time was 2.8 d, the α-glucosidase inhibition rate was 70.83%. I1, I2, I3 were separated from Sephadex G-25, and I2 with the best enzyme inhibitory effect whose IC50 is 1.72 mg/mL. I2 was further separated and purified by RP-HPLC to obtain α-GIG with a purity of 94.17%. Fourier transform infrared spectroscopy suggest that the secondary structure composition was estimated as 70.51% β-folding, 18.95% α-helices and 10.54% β-corner α-elimination reaction demonstrated the existence of 0-glycosidic linkage in "-GIG. The results of the enzyme inhibition kinetics study showed that α-GIG exhibited a mixed-type non-competitive inhibition of α-glucosidase. Therefore, α-GIG had the potential to be developed as a natural α-glucosidase inhibitor. [ABSTRACT FROM AUTHOR]

Glycoproteins in urine have the potential to provide a rich class of informative molecules for studying human health and disease. Despite this promise, the urine glycoproteome has been largely uncharacterized. Here, we present the analysis of glycoproteins in human urine using LC–MS/MS-based intact glycopeptide analysis, providing both the identification of protein glycosites and characterization of the glycan composition at specific glycosites. Gene enrichment analysis reveals differences in biological processes, cellular components, and molecular functions in the urine glycoproteome versus the urine proteome, as well as differences based on the major glycan class observed on proteins. Meta-heterogeneity of glycosylation is examined on proteins to determine the variation in glycosylation across multiple sites of a given protein with specific examples of individual sites differing from the glycosylation trends in the overall protein. Taken together, this dataset represents a potentially valuable resource as a baseline characterization of glycoproteins in human urine for future urine glycoproteomics studies. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) based on micro/nanostructured materials with different natures has received increasing attention for the analysis of a wide variety of analytes. However, up to now, only a few studies have shown the application of simple platforms in MALDI-MS for the identification of intact proteins. The present work reports on the application of copper oxide particles (Cu2O PS), obtained by a greener route, in combination with low amounts of 2,5-dihydroxybenzoic acid (DHB) as a novel hybrid platform. The combined Cu2O PS@DHB matrix, containing only 2.5 mg mL−1 of particles and 10 mg mL−1 of DHB, was easily applicable in MALDI-MS without surface modification of target plates. Under optimal conditions, the analysis of intact proteins up to 150,000 Da was possible, including immunoglobulin G, bovine serum albumin, and cytochrome C with adequate spot-to-spot signal reproducibility (RSD < 10%). In addition, the analysis of glycopeptides from IgG digests was carried out to prove the multipurpose application of the Cu2O PS@DHB platform in the low m/z range (2500–3000 Da). From the obtained results, it can be concluded that the optical and surface properties of as-synthesized Cu2O PS are likely to be responsible for the superior performance of Cu2O PS@DHB in comparison with conventional matrices. In this sense, the proposed user-friendly methodology opens up the prospect for possible implementation in bioanalysis and diagnostic research. [ABSTRACT FROM AUTHOR]

Bacterial toxin-antitoxin (TA) systems are widespread in chromosomes and plasmids of free-living microorganisms, but only a few have been identified in obligate intracellular species. We found seven putative type II TA modules in Waddlia chondrophila, a Chlamydia-related species that is able to infect a very broad series of eukaryotic hosts, ranging from protists to mammalian cells. The RNA levels of Waddlia TA systems are significantly upregulated by iron starvation and novobiocin, but they are not affected by antibiotics such as ß-lactams and glycopeptides, which suggests different mechanisms underlying stress responses. Five of the identified TA modules, including HigBA1 and MazEF1, encoded on the Waddlia cryptic plasmid, proved to be functional when expressed in a heterologous host. TA systems have been associated with the maintenance of mobile genetic elements, bacterial defense against bacteriophages, and persistence upon exposure to adverse conditions. As their RNA levels are upregulated upon exposure to adverse conditions, Waddlia TA modules may be involved in survival to stress. Moreover, as Waddlia can infect a wide range of hosts including free-living amoebae, TA modules could also represent an innate immunity system to fight against bacteriophages and other microorganisms with which Waddlia has to share its replicative niche. [ABSTRACT FROM AUTHOR]

Optimizing antibiotic therapy is imperative with rising bacterial resistance and high infection mortality. Extended infusion defined as a continuous infusion (COI) or prolonged infusion (PI) of beta-lactams and glycopeptides might improve efficacy and safety compared to their intermittent administration (IA). This study aimed to evaluate the efficacy and safety of extended infusion in pediatric patients. Adhering to Cochrane standards, we conducted a systematic review with meta-analysis investigating the efficacy and safety of COI (24 h/d) and PI (>1 h/dose) compared to IA (≤1 h/dose) of beta-lactams and glycopeptides in pediatrics. Primary outcomes included mortality, clinical success, and microbiological eradication. Five studies could be included for the outcome mortality, investigating meropenem, piperacillin/tazobactam, cefepime, or combinations of these. The pooled relative risk estimate was 0.48 (95% CI 0.26–0.89, p = 0.02). No significant differences between the administration modes were found for the outcomes of clinical success, microbiological eradication (beta-lactams; glycopeptides), and mortality (glycopeptides). No study reported additional safety issues, e.g., adverse drug reactions when using COI/PI vs. IA. Our findings suggest that the administration of beta-lactams by extended infusion leads to a reduction in mortality for pediatric patients. [ABSTRACT FROM AUTHOR]

Objective: To analyze and describe the pharmacokinetic aspects of vancomycin usage in a cohort of critically ill children and to construct a pharmacokinetic model for this population. Method: We conducted an observational study in a pediatric intensive care unit from September 2017 to March 2019. Children receiving vancomycin with at least one serum measurement were included. Variables with a p-value lower than 0.2 in univariate analysis, and biologically plausible for inducing nephrotoxicity and not correlated with other predictors, were incorporated into logistic regression. Additionally, pharmacokinetic modeling was performed using the PMETRICS® package for patients with creatinine clearance (CLCR) > 30 mL/min. Result: The study included 70 children, with an average vancomycin dose of 60 mg/kg/day. Only eleven children achieved vancomycin levels within the target range (15-20 mg/L). No significant differences in doses/mg/kg/day were observed among children above, within, or below the vancomycin target range. In the multivariate model, children above the recommended serum range had an odds ratio of 4.6 [95% CI 1.4 – 17.2] for nephrotoxicity. A pharmacokinetic model was proposed using data from 15 children, estimating PK parameters for CLCR and V as 0.94 L/h and 5.71 L, respectively. Conclusion: Nephrotoxicity was associated with vancomycin plasma concentrations equal to or exceeding 15 mg/L. The developed model enhanced understanding of the drug’s behavior within this population, potentially aiding clinical practice in dose calculations and estimation of the area under the curve – a recommended parameter for vancomycin monitoring.

Therapeutic drug monitoring (TDM) is a critical clinical tool used to optimize the safety and effectiveness of drugs by measuring their concentration in biological fluids. These fluids are primarily plasma or blood. TDM, together with real-time dosage adjustment, contributes highly to the successful management of glycopeptide antimicrobial therapies. Understanding pharmacokinetic/pharmacodynamic (PK/PD) properties is vital for optimizing antimicrobial therapies, as the efficacy of these therapies depends on both the exposure of the patient to the drug (PK) and pharmacodynamic (PD) parameters such as the in vitro estimated minimum drug concentration that inhibits bacterial growth (MIC). Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is widely recognized as the gold standard for measuring small molecules, such as antibiotics. This review provides a comprehensive overview of LC-MS/MS methods available for TDM of glycopeptide antibiotics, including vancomycin, teicoplanin, dalbavancin, oritavancin, and telavancin.

Copyright of Health Sciences Journal / Revista Ciências em Saúde is the property of Hospital de Clinicas de Itajuba - Associacao de Integracao Social de Itajuba and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Annali di Igiene, Medicina Preventiva e di Comunità is the property of Societa Editrice Universo s.r.l. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Arquivos de Ciências da Saúde da UNIPAR is the property of Associacao Paranaense de Ensino e Cultura and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

3-Amino-4-hydroxybenzoic acid (AHB) was the first matrix identified by glycoprotein glycan analysis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). However, compared to commonly used matrices, such as 2,5-dihydroxybenzoic acid (DHB), AHB is less efficient at glycan ionization and lacks the ability to ionize other molecular species, such as peptides, and thus is no longer used. In this study, we focused on the glycan-selective ionization ability of AHB and its low-noise properties in the low-molecular-weight region, as we expected that these properties could be enhanced by adding sodium to AHB. Sodium-doped AHB (AHB/Na) selectively imparts sodium adduct ions onto O-glycan fragments generated by the in-source decay (ISD) of glycopeptides and glycoproteins containing O-glycans that occurs during intense laser irradiation, enabling direct O-glycan analysis. Furthermore, we demonstrated that it is possible to investigate the internal structure of each O-glycan fragment with pseudo-MS/MS/MS using the sodium adduct ion of the O-glycan-derived ISD fragments from an intact mucin mixture. [ABSTRACT FROM AUTHOR]

Vancomycin is not appreciably passaged via the colonic membrane to the gastrointestinal (GI) tract in persons with an intact gut epithelium due to its large chemical structure. However; hospitalized patients with diarrhea often have a disrupted GI tract. The aim of this study was to determine the frequency of detectable vancomycin concentrations in the stool of patients with antibiotic-associated diarrhea receiving IV vancomycin. This was a multicenter cohort study of hospitalized patients with stool samples collected for Clostridioides difficile testing. Leftover stool samples were collected from patients who had received at least 3 days of IV vancomycin. Fecal vancomycin was quantified by high-performance liquid chromatography. The study cohort included 33 unique patients, majority female (54.5%) aged 60 years (range 23–84). Eighteen of thirty-three patients (54.5%) tested positive for C. difficile toxins. The average duration of systemic vancomycin administration prior to stool collection was 3.5 (range 2–15) days. Three of 33 (9%) stool samples had a detectable vancomycin concentration (range 1.2–13.2 mcg/mL). These concentrations may promote the development of vancomycin-resistant Enterococcus or van mutations in C. difficile, leading to vancomycin resistance. Further studies on implications are warranted. [ABSTRACT FROM AUTHOR]

The interaction between quinoa glycopeptides (QGs) and three polyphenols [(−)-epigallocatechin-3-gallate (EGCG), gallic acid (GA) and tannic acid (TA)] was investigated by particle size, potential, fluorescence spectrum, and Fourier infrared spectrum, and the changes of antioxidant activity after compound were investigated by in vitro antioxidant experiments. The results showed that the average particle size of quinoa glycopeptide decreased and its distribution became more uniform after it was combined with three polyphenols. The intrinsic fluorescence was quenched and decreased with the increase of polyphenol content, and the maximum emission wavelength of the intrinsic fluorescence spectrum was redshifted. When combined with EGCG, GA and TA, the surface hydrophobicity of quinoa glycopeptides decreased significantly. The quenching type of EGCG, GA, and TA on quinoa glycopeptides was static quenching. The addition of polyphenols changed the hydrophobic amino acid groups in quinoa glycopeptides, and the hydrophobic interaction was the main force. Additionally, the secondary structure of QG was slightly changed after the binding of polyphenols to QG. Moreover, DPPH and ABTS radical scavenging abilities and ferric reducing antioxidant power analysis showed that EGCG, GA, and TA enhanced significantly the antioxidant capacity of QG. Therefore, this study can guide the preparation of quinoa functional food. [ABSTRACT FROM AUTHOR]

Antibiotics are central to modern medicine, and yet they are mainly the products of intra and inter-kingdom evolutionary warfare. To understand how nature evolves antibiotics around a common mechanism of action, we investigated the origins of an extremely valuable class of compounds, lipid II targeting glycopeptide antibiotics (GPAs, exemplified by teicoplanin and vancomycin), which are used as last resort for the treatment of antibiotic resistant bacterial infections. Using a molecule-centred approach and computational techniques, we first predicted the nonribosomal peptide synthetase assembly line of paleomycin, the ancestral parent of lipid II targeting GPAs. Subsequently, we employed synthetic biology techniques to produce the predicted peptide and validated its antibiotic activity. We revealed the structure of paleomycin, which enabled us to address how nature morphs a peptide antibiotic scaffold through evolution. In doing so, we obtained temporal snapshots of key selection domains in nonribosomal peptide synthesis during the biosynthetic journey from ancestral, teicoplanin-like GPAs to modern GPAs such as vancomycin. Our study demonstrates the synergy of computational techniques and synthetic biology approaches enabling us to journey back in time, trace the temporal evolution of antibiotics, and revive these ancestral molecules. It also reveals the optimisation strategies nature has applied to evolve modern GPAs, laying the foundation for future efforts to engineer this important class of antimicrobial agents. Glycopeptide antibiotics (GPAs) are microbial natural products synthesized by multiple enzymes, including a nonribosomal peptide synthetase for assembly of the peptide core. Here, the authors use computational techniques to infer a gene set for biosynthesis of an ancestral GPA, produce the peptide in a microbial host, and provide insights into the evolution of key enzymatic domains. [ABSTRACT FROM AUTHOR]

Proteoglycans are a small but diverse family of proteins that play a wide variety of roles at the cell surface and in the extracellular matrix. In addition to their glycosaminoglycan (GAG) chains, they are N- and O-glycosylated. All of these types of glycosylation are crucial to their function but present a considerable analytical challenge. We describe the combination of serial proteolysis followed by the application of higher-energy collisional dissociation (HCD) and electron transfer/higher-energy collisional dissociation (EThcD) to optimize protein sequence coverage and glycopeptide identification from proteoglycans. In many cases, the use of HCD alone allows the identification of more glycopeptides. However, the localization of glycoforms on multiply glycosylated peptides has remained elusive. We demonstrate the use of EThcD for the confident assignment of glycan compositions on multiply glycosylated peptides. Dense glycosylation on proteoglycans is key to their biological function; thus, developing tools to identify and quantify doubly glycosylated peptides is of interest. Additionally, glycoproteomics searches identify glycopeptides in otherwise poorly covered regions of proteoglycans. The development of these and other analytical tools may permit glycoproteomic similarity comparisons in biological samples. [ABSTRACT FROM AUTHOR]

Low abundance and heterogeneity of N-glycosylation at the peptide level poses a great challenge to the structural and functional analysis of glycosylation in the field of glycobiology. Solving this conundrum requires a sufficient and specific method for intact N-glycopeptide enrichment. Using the C18 or HLB desalting column followed by the mixed-mode strong anion exchange (MAX) or hydrophilic interaction chromatography (HILIC) glycopeptide enrichment column are commonly applied approaches for sample preparation of intact N-glycopeptides from complex samples. Herein, we compared the effects of different combinations of two desalting columns and two enrichment columns using equal amounts of mouse brain tissues from the same source. The results revealed the C18 column was a bit superior to the HLB column, and the MAX and HILIC columns were complementary on intact N-glycopeptides enrichment. Additionally, the results also demonstrated that enriching glycopeptides using a HILIC column followed by a MAX column from the flow-through solution got a better enrichment performance than the reversed order. Based on these results, the sequential enrichment of glycopeptides using HILIC and then MAX columns could maximize the enrichment performance of intact N-glycopeptides, and therefore is an option for in-depth analysis of site-specific glycoproteome. [ABSTRACT FROM AUTHOR]

Background: There is a need for new serum biomarkers for early detection of hepatocellular carcinoma (HCC). Haptoglobin (Hp) N-glycosylation is altered in HCC, but the diagnostic value of site-specific Hp glycobiomarkers is rarely reported. We aimed to determine the site-specific glycosylation profile of Hp for early-stage HCC diagnosis. Method: Hp glycosylation was analyzed in the plasma of patients with liver diseases (n=57; controls), early-stage HCC (n=50) and late-stage HCC (n=32). Hp phenotype was determined by immunoblotting. Hp was immunoisolated and digested into peptides. N-glycopeptides were identified and quantified using liquid chromatography-mass spectrometry. Cohort samples were compared using Wilcoxon rank-sum (Mann-Whitney U) tests. Diagnostic performance was assessed using receiver operating characteristic (ROC) curves and area under curve (AUC). Results: Significantly higher fucosylation, branching and sialylation of Hp glycans, and expression of high-mannose glycans, was observed as disease progressed from cirrhosis to early- and late-stage HCC. Several glycopeptides demonstrated high values for early diagnosis of HCC, with an AUC of 93% (n=1), >80% (n=3), >75% (n=13) and >70% (n=11), compared with alpha-fetoprotein (AFP; AUC of 79%). The diagnostic performance of the identified biomarkers was only slightly affected by Hp phenotype. Conclusion: We identified a panel of Hp glycopeptides that are significantly differentially regulated in early- and late-stage HCC. Some glycobiomarkers exceeded the diagnostic value of AFP (the most commonly used biomarker for HCC diagnosis). Our findings provide evidence that glycobiomarkers can be effective in the diagnosis of early HCC - individually, as a panel of glycopeptides or combined with conventional serological biomarkers. [ABSTRACT FROM AUTHOR]

Vancomycin is not appreciably passaged via the colonic membrane to the gastrointestinal (GI) tract in persons with an intact gut epithelium due to its large chemical structure. However; hospitalized patients with diarrhea often have a disrupted GI tract. The aim of this study was to determine the frequency of detectable vancomycin concentrations in the stool of patients with antibiotic-associated diarrhea receiving IV vancomycin. This was a multicenter cohort study of hospitalized patients with stool samples collected for Clostridioides difficile testing. Leftover stool samples were collected from patients who had received at least 3 days of IV vancomycin. Fecal vancomycin was quantified by high-performance liquid chromatography. The study cohort included 33 unique patients, majority female (54.5%) aged 60 years (range 23–84). Eighteen of thirty-three patients (54.5%) tested positive for C. difficile toxins. The average duration of systemic vancomycin administration prior to stool collection was 3.5 (range 2–15) days. Three of 33 (9%) stool samples had a detectable vancomycin concentration (range 1.2–13.2 mcg/mL). These concentrations may promote the development of vancomycin-resistant Enterococcus or van mutations in C. difficile, leading to vancomycin resistance. Further studies on implications are warranted.

Abstract Background Herein, we analyzed the efficacy of main antibiotic therapy regimens in the treatment of healthcare-associated meningitis (HCAM). Materials/methods This retrospective cohort study was conducted in 18 tertiary-care academic hospitals Turkey, India, Egypt and Romania. We extracted data and outcomes of all patients with post-neurosurgical meningitis cases fulfilling the study inclusion criteria and treated with empirical therapy between December 2006-September 2018. Results Twenty patients in the cefepime + vancomycin-(CV) group, 31 patients in the ceftazidime + vancomycin-(CFV) group, and 119 patients in the meropenem + vancomycin-(MV) group met the inclusion criteria. The MV subgroup had a significantly higher mean Glasgow Coma Score, a higher rate of admission to the intensive care unit within the previous month, and a higher rate of antibiot herapy within the previous month before the meningitis episode (p 0.05) among the three cohorts. No regimen was effective against carbapenem-resistant bacteria, and vancomycin resulted in an EOT clinical success rate of 60.6% in the methicillin-resistant staphylococci or ampicillin-resistant enterococci subgroup (n = 34). Conclusions Our study showed no significant difference in terms of clinical success and mortality among the three treatment options. All regimens were ineffective against carbapenem-resistant bacteria. Vancomycin was unsuccessful in approximately 40% of cases involving methicillin-resistant staphylococci or ampicillin-resistant enterococci.

Glycosylation is the most common form of post-translational modification of proteins, critically affecting their structure and function. Using liquid chromatography and mass spectrometry for high-resolution site-specific quantification of glycopeptides coupled with high-throughput artificial intelligence-powered data processing, we analyzed differential protein glycoisoform distributions of 597 abundant serum glycopeptides and nonglycosylated peptides in 50 individuals who had been seriously ill with COVID-19 and in 22 individuals who had recovered after an asymptomatic course of COVID-19. As additional comparison reference phenotypes, we included 12 individuals with a history of infection with a common cold coronavirus, 16 patients with bacterial sepsis, and 15 healthy subjects without history of coronavirus exposure. We found statistically significant differences, at FDR < 0.05, for normalized abundances of 374 of the 597 peptides and glycopeptides interrogated between symptomatic and asymptomatic COVID-19 patients. Similar statistically significant differences were seen when comparing symptomatic COVID-19 patients to healthy controls (350 differentially abundant peptides and glycopeptides) and common cold coronavirus seropositive subjects (353 differentially abundant peptides and glycopeptides). Among healthy controls and sepsis patients, 326 peptides and glycopeptides were found to be differentially abundant, of which 277 overlapped with biomarkers that showed differential expression between symptomatic COVID-19 cases and healthy controls. Among symptomatic COVID-19 cases and sepsis patients, 101 glycopeptide and peptide biomarkers were found to be statistically significantly abundant. Using both supervised and unsupervised machine learning techniques, we found specific glycoprotein profiles to be strongly predictive of symptomatic COVID-19 infection. LASSO-regularized multivariable logistic regression and K-means clustering yielded accuracies of 100% in an independent test set and of 96% overall, respectively. Our findings are consistent with the interpretation that a majority of glycoprotein modifications observed which are shared among symptomatic COVID-19 and sepsis patients likely represent a generic consequence of a severe systemic immune and inflammatory state. However, there are glycoisoform changes that are specific and particular to severe COVID-19 infection. These may be representative of either COVID-19-specific consequences or susceptibility to or predisposition for a severe course of the disease. Our findings support the potential value of glycoproteomic biomarkers in the biomedical understanding and, potentially, the clinical management of serious acute infectious conditions.

BackgroundGlycoproteins are important tear components that participate in the stability of the ocular surface. However, the glycopeptides that are present in the tears of wild animals have not yet been described. This work aimed to describe the glycoproteomic profile of roadside hawk (Rupornis magnirostris) and caiman (Caiman latirostris) tears.MethodsTears collected from 10 hawks and 70 caimans using Schirmer tear test strips were used in this study. The samples were submitted to trypsin digestion and separated using a reverse-phase column coupled to a mass spectrometer associated to a nanospray ionization source. The glycoproteins were categorized as: cellular components, biological processes and molecular function, according to the UniProt Knowledgebase.ResultsAs shown by the liquid chromatography-mass spectrometry, all glycopeptides found were classified as N-type. Of the 51 glycoproteins that were identified in the hawk tear film, the most abundant were ovotransferrin, globulins and complement system proteins. In the caiman tear film, 29 glycoproteins were identified. The most abundant caiman glycoproteins were uncharacterized proteins, ATPases, globulins and proteasome components. Ontological characterization revealed that the glycoproteins were extracellular, and the most identified molecular function was endopeptidase activity for both species.ConclusionGlycoproteins are abundant in the tear film of the bird and reptile species studied herein, and all these molecules were shown to have N-type modifications. Location at the extracellular space and an endopeptidase inhibitor activity were the main cell component and molecular function for both species, respectively. These profiles showed differences when compared to human tears, are possibly linked to adaptive processes and can be the basis for further studies on the search of disease biomarkers.

Glycoproteomics is a powerful yet analytically challenging research tool. Software packages aiding the interpretation of complex glycopeptide tandem mass spectra have appeared, but their relative performance remains untested. Conducted through the HUPO Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates solutions for system-wide glycopeptide analysis. The same mass spectrometrybased glycoproteomics datasets from human serum were shared with participants and the relative team performance for N- and O-glycopeptide data analysis was comprehensively established by orthogonal performance tests. Although the results were variable, several high-performance glycoproteomics informatics strategies were identified. Deep analysis of the data revealed key performance-associated search parameters and led to recommendations for improved 'high-coverage' and 'high-accuracy' glycoproteomics search solutions. This study concludes that diverse software packages for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies and specifies key variables that will guide future software developments and assist informatics decision-making in glycoproteomics.

In this work, a new type of FE-1 COF material is prepared by a reversible imine condensation reaction with diaminoferrocene and diaminodiformaldehyde as materials. The material is connected by imine bonds to form a COF skeleton, and the presence of plenty of nitrogen-containing groups gives the material good hydrophilicity; the presence of metal Fe ions provides the material application potential in the enrichment of phosphopeptides. According to the different binding abilities of N-glycopeptide and phosphopeptide on FE-1 COF, it can simultaneously enrich N-glycopeptide and phosphopeptide through different elution conditions to realize its controllable and selective enrichment. Using the above characteristics, 18 phosphopeptides were detected from α-casein hydrolysate, 8 phosphopeptides were detected from β-casein hydrolysate and 21 glycopeptides were detected from IgG hydrolysate. Finally, the gradual elution strategy was used; 16 phosphopeptides and 19 glycopeptides were detected from the α-casein hydrolysate and IgG hydrolysate. The corresponding glycopeptides and phosphopeptides were identified from the human serum. It proves that the FE-1 COF material has a good enrichment effect on phosphopeptides and glycopeptides.

We have recently introduced multiple reaction monitoring (MRM) mass spectrometry as a novel tool for glycan biomarker research and discovery. Herein, we employ this technique to characterize the site-specific glycan alterations associated with primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC). Glycopeptides associated with disease severity were also identified. Multinomial regression modelling was employed to construct and validate multi-analyte diagnostic models capable of accurately distinguishing PBC, PSC, and healthy controls from one another (AUC = 0.93 ± 0.03). Finally, to investigate how disease-relevant environmental factors can influence glycosylation, we characterized the ability of bile acids known to be differentially expressed in PBC to alter glycosylation. We hypothesize that this could be a mechanism by which altered self-antigens are generated and become targets for immune attack. This work demonstrates the utility of the MRM method to identify diagnostic site-specific glycan classifiers capable of distinguishing even related autoimmune diseases from one another.