Presence of rare potential pathogenic variants in subjects under 60 years old with very severe or fatal COVID-19

Background/Objectives: Genetic variants affecting host defense against pathogens may help explain COVID-19 fatal outcomes. Our aim was to identify rare genetic variants related to COVID-19 severity in a selected group of patients under 60 years who required intubation or resulting in death. Method(s): Forty-four very severe COVID-19 patients were selected from the Spanish STOP-Coronavirus cohort, which comprises more than 3,500 COVID-19 patients. Genotype was performed by whole exome sequencing and variants were selected by using a gene panel of 867 candidate genes (immune response, primary immunodeficiencies or coagulation, among other). Variants were filtered, priorized and their potential pathogenicity was assessed following ACGM criteria. Result(s): We detected 44 different variants of interest, in 29 different patients (66%). Some of these variants were previously described as pathogenic (26%). Mostly, the candidate variants were located in genes related to immune response (38%), congenital disorders of glycosylation (14%) or damaged DNA binding genes (9%). A network analysis, showed three main components, consisting of 25 highly interconnected genes related to immune response and two additional networks enriched in carbohydrate metabolism and in DNA metabolism and repair processes. Conclusion(s): The variants identified affect different, but interrelated, functional pathways such as immune response and glycosylation. Further studies are needed for confirming the ultimate role of the new candidate genes described in the present study on COVID-19 severity.

Liver transplantation recovers hepatic N-glycosylation with persistent IgG glycosylation abnormalities: Three-year follow-up in a patient with phosphomannomutase-2-congenital disorder of glycosylation.

Phosphomannomutase-2-congenital disorder of glycosylation (PMM2-CDG) is the most common CDG and presents with highly variable features ranging from isolated neurologic involvement to severe multi-organ dysfunction. Liver abnormalities occur in in almost all patients and frequently include hepatomegaly and elevated aminotransferases, although only a minority of patients develop progressive hepatic fibrosis and liver failure. No curative therapies are currently available for PMM2-CDG, although investigation into several novel therapies is ongoing. We report the first successful liver transplantation in a 4-year-old patient with PMM2-CDG. Over a 3-year follow-up period, she demonstrated improved growth and neurocognitive development and complete normalization of liver enzymes, coagulation parameters, and carbohydrate-deficient transferrin profile, but persistently abnormal IgG glycosylation and recurrent upper airway infections that did not require hospitalization. Liver transplant should be considered as a treatment option for PMM2-CDG patients with end-stage liver disease, however these patients may be at increased risk for recurrent bacterial infections post-transplant.

A Dual-Approach Strategy to Optimize the Safety and Efficacy of Anti-Zika Virus Monoclonal Antibody Therapeutics.

Antibody-dependent enhancement of infection (ADE) is clinically relevant to Dengue virus (DENV) infection and poses a major risk to the application of monoclonal antibody (mAb)-based therapeutics against related flaviviruses such as the Zika virus (ZIKV). Here, we tested a two-tier approach for selecting non-cross-reactive mAbs combined with modulating Fc glycosylation as a strategy to doubly secure the elimination of ADE while preserving Fc effector functions. To this end, we selected a ZIKV-specific mAb (ZV54) and generated three ZV54 variants using Chinese hamster ovary cells and wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants as production hosts (ZV54CHO, ZV54WT, and ZV54ΔXF). The three ZV54 variants shared an identical polypeptide backbone, but each exhibited a distinct Fc N-glycosylation profile. All three ZV54 variants showed similar neutralization potency against ZIKV but no ADE activity for DENV infection, validating the importance of selecting the virus/serotype-specific mAbs for avoiding ADE by related flaviviruses. For ZIKV infection, however, ZV54CHO and ZV54ΔXF showed significant ADE activity while ZV54WT completely forwent ADE, suggesting that Fc glycan modulation may yield mAb glycoforms that abrogate ADE even for homologous viruses. In contrast to the current strategies for Fc mutations that abrogate all effector functions along with ADE, our approach allowed the preservation of effector functions as all ZV54 glycovariants retained antibody-dependent cellular cytotoxicity (ADCC) against the ZIKV-infected cells. Furthermore, the ADE-free ZV54WT demonstrated in vivo efficacy in a ZIKV-infection mouse model. Collectively, our study provides further support for the hypothesis that antibody-viral surface antigen and Fc-mediated host cell interactions are both prerequisites for ADE, and that a dual-approach strategy, as shown herein, contributes to the development of highly safe and efficacious anti-ZIKV mAb therapeutics. Our findings may be impactful to other ADE-prone viruses, including SARS-CoV-2.

An N-glycopeptide MS/MS data analysis workflow leveraging two complementary glycoproteomic software tools for more confident identification and assignments.

Complete coverage of all N-glycosylation sites on the SARS-CoV2 spike protein would require the use of multiple proteases in addition to trypsin. Subsequent identification of the resulting glycopeptides by searching against database often introduces assignment errors due to similar mass differences between different permutations of amino acids and glycosyl residues. By manually interpreting the individual MS2 spectra, we report here the common sources of errors in assignment, especially those introduced by the use of chymotrypsin. We show that by applying a stringent threshold of acceptance, erroneous assignment by the commonly used Byonic software can be controlled within 15%, which can be reduced further if only those also confidently identified by a different search engine, pGlyco3, were considered. A representative site-specific N-glycosylation pattern could be constructed based on quantifying only the overlapping subset of N-glycopeptides identified at higher confidence. Applying the two complimentary glycoproteomic software in a concerted data analysis workflow, we found and confirmed that glycosylation at several sites of an unstable Omicron spike protein differed significantly from those of the stable trimeric product of the parental D614G variant.

Multifaceted roles of Fcγ receptors in COVID-19 and vaccine responses.

Recent data have revealed various effector functions of FcγRs in immune responses against challenges with SARS-CoV-2 virus. FcγRs act as a bridge between antibody specificity and effector cells. In many cases, IgG/FcγR interactions generate cell-mediated immune protection from infection via ADCP or ADCC. These responses are beneficial, as they may participate in virus elimination and persist longer than neutralizing anti-Spike antibodies. In contrast, these interactions may sometimes prove beneficial to the virus by enhancing viral uptake into phagocytic cells via ADE and causing excessive inflammation. Here, we summarize key features of FcγRs, discuss effector functions, clinical relevance, and factors influencing FcγR-mediated immune responses in COVID-19 and vaccine responses, and consider IVIg and kinase inhibitors for targeting FcγRs signaling in COVID-19.

GlycoEVLR: Glycosylated extracellular vesicle-like receptors for targeting and sensing viral antigen

Biosensors are rapid and portable detection devices with great potential for the instant screening of infectious diseases. Receptors are the critical element of biosensors. They determine the specificity, sensitivity and stability. However, current receptors are mainly limited to antibodies and aptamers. Herein, we developed a glycosylated extracellular vesicle-like receptor (GlycoEVLR) for the rapid detection of virus antigens, specifically using SARS-CoV-2 as a model. The human angiotensin-converting enzyme 2 (ACE2)-overexpressed and heparin-functionalized HEK-293T cell membrane-cloaked Fe3O4 nanoparticles (NPs) were prepared as functionalizing GlycoEVLR. They were characterized as spherical core–shell structures with a diameter of around 100 nm, which were perfectly comparable to natural extracellular vesicles. Binding affinities between GlycoEVLR and spike1 (S1) antigen were demonstrated using surface plasmon resonance (SPR). The GlycoEVLR was fixed on magnetic electrodes to construct electrochemical biosensors. Using electrochemical impedance spectroscopy (EIS) as a measurement technique, the S1 antigen was detected down to 1 pg/mL within 20 min and showed a good linearity range from 1 pg/mL to 1 ng/mL. Also, the GlycoEVLR-based electrochemical biosensors showed excellent antifouling performance and stability. Overall, our work provides a useful methodology for developing extracellular vesicle-like receptors for biosensors. Combining the inherit natural receptor proteins and antifouling lipids from the host cells with engineered glycan motifs to target and sense viral antigens will open a newavenue for biosensors.

How helpful were molecular dynamics simulations in shaping our understanding of SARS-CoV-2 spike protein dynamics?

The SARS-CoV-2 spike protein (S) represents an important viral component that is required for successful viral infection in humans owing to its essential role in recognition of and entry to host cells. The spike is also an appealing target for drug designers who develop vaccines and antivirals. This article is important as it summarizes how molecular simulations successfully shaped our understanding of spike conformational behavior and its role in viral infection. MD simulations found that the higher affinity of SARS-CoV-2-S to ACE2 is linked to its unique residues that add extra electrostatic and van der Waal interactions in comparison to the SARS-CoV S. This illustrates the spread potential of the pandemic SARS-CoV-2 relative to the epidemic SARS-CoV. Different mutations at the S-ACE2 interface, which is believed to increase the transmission of the new variants, affected the behavior and binding interactions in different simulations. The contributions of glycans to the opening of S were revealed via simulations. The immune evasion of S was linked to the spatial distribution of glycans. This help the virus to escape the immune system recognition. This article is important as it summarizes how molecular simulations successfully shaped our understanding of spike conformational behavior and its role in viral infection. This will pave the way to us preparing for the next pandemic as the computational tools are tailored to help fight new challenges.

Understanding how transmembrane domains regulate interactions between human BST-2 and the SARS-CoV-2 accessory protein ORF7a.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID, replicates at intracellular membranes. Bone marrow stromal antigen 2 (BST-2; tetherin) is an antiviral response protein that inhibits transport of viral particles after budding within infected cells. RNA viruses such as SARS-CoV-2 use various strategies to disable BST-2, including use of transmembrane 'accessory' proteins that interfere with BST-2 oligomerization. ORF7a is a small, transmembrane protein present in SARS-CoV-2 shown previously to alter BST-2 glycosylation and function. In this study, we investigated the structural basis for BST-2 ORF7a interactions, with a particular focus on transmembrane and juxtamembrane interactions. Our results indicate that transmembrane domains play an important role in BST-2 ORF7a interactions and mutations to the transmembrane domain of BST-2 can alter these interactions, particularly single-nucleotide polymorphisms in BST-2 that result in mutations such as I28S. Using molecular dynamics simulations, we identified specific interfaces and interactions between BST-2 and ORF7a to develop a structural basis for the transmembrane interactions. Differences in glycosylation are observed for BST-2 transmembrane mutants interacting with ORF7a, consistent with the idea that transmembrane domains play a key role in their heterooligomerization. Overall, our results indicate that ORF7a transmembrane domain interactions play a key role along with extracellular and juxtamembrane domains in modulating BST-2 function.

In planta deglycosylation improves the SARS-CoV-2 neutralization activity of recombinant ACE2-Fc.

SARS-CoV-2 infects human cells via binding of the viral spike glycoprotein to its main cellular receptor, angiotensin-converting enzyme 2 (ACE2). The spike protein-ACE2 receptor interaction is therefore a major target for the development of therapeutic or prophylactic drugs to combat coronavirus infections. Various engineered soluble ACE2 variants (decoys) have been designed and shown to exhibit virus neutralization capacity in cell-based assays and in vivo models. Human ACE2 is heavily glycosylated and some of its glycans impair binding to the SARS-CoV-2 spike protein. Therefore, glycan-engineered recombinant soluble ACE2 variants might display enhanced virus-neutralization potencies. Here, we transiently co-expressed the extracellular domain of ACE2 fused to human Fc (ACE2-Fc) with a bacterial endoglycosidase in Nicotiana benthamiana to produce ACE2-Fc decorated with N-glycans consisting of single GlcNAc residues. The endoglycosidase was targeted to the Golgi apparatus with the intention to avoid any interference of glycan removal with concomitant ACE2-Fc protein folding and quality control in the endoplasmic reticulum. The in vivo deglycosylated ACE2-Fc carrying single GlcNAc residues displayed increased affinity to the receptor-binding domain (RBD) of SARS-CoV-2 as well as improved virus neutralization activity and thus is a promising drug candidate to block coronavirus infection.

The Impact of N-butyldeoxynojirimycin and 1-deoxymannojirimycin on SARS-CoV-2 S1 Subunit and Angiotensin-Converting Enzyme 2 Interaction in Intestinal Epithelial Cells

The severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2) targets mainly the respiratory tract. In addition to respiratory symptoms, many extrapulmonary manifestations were observed in the gastrointestinal tract and reported by SARS-CoV-2 patients, including abdominal pain, nausea, and diarrhea. SARS-CoV-2 binds initially to angiotensin-converting enzyme 2 (ACE2) on the host cell surface via its spike (S) protein before it undergoes endocytosis and fusion with the lysosomal membrane. The spike protein of SARS-CoV-2 is a heavily N- and O-glycosylated trimer. Glycosylation is an essential posttranslational modification in the life cycle of membrane and secretory proteins that affects their structural and functional characteristics as well as their trafficking and sorting patterns. This study aimed at elucidating the impact of glycosylation modulation on the trafficking of both S1 subunit and ACE2 as well as their interaction at the cell surface of intestinal epithelial cells. For this purpose, the S1 protein was expressed in COS-1 cells and its glycosylation modified using N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of ER-located alpha-glucosidases I and II, and or 1-deoxymannojirimycin (dMM), an inhibitor of the Golgi-located alpha-mannosidase I. The intracellular and secreted S1 proteins were analyzed by endoglycosidase H treatment. Similarly, ACE2 trafficking to the brush border membrane of intestinal Caco-2 cells was also assessed in the presence or absence of the inhibitors. Finally, the interaction between the S1 protein and ACE2 was investigated at the surface of Caco-2 cells by co-immunoprecipitation. Our data show that NB-DNJ significantly reduced the secretion of S1 proteins in COS-1 cells, while dMM affected S1 secretion to a lesser extent. Moreover, NB-DNJ and dMM differentially affected ACE2 trafficking and sorting to the brush border membrane of intestinal Caco-2 cells. Strikingly, the interaction between S1 and ACE2 was significantly reduced when both proteins were processed by the glycosylation inhibitors, rendering glycosylation and its inhibitors potential candidates for SARS-CoV-2 treatment. This work has been supported by a grant from the German Research Foundation (DFG) grant NA331/15-1 to HYN. M.K. was supported by a scholarship from the Hannover Graduate School for Veterinary Pathobiology, Neuroinfectiology, and Translational Medicine (HGNI) and by the DFG grant NA331/15-1.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

SARS-CoV-2 infection alters the breast milk glycomic profile

Breast milk is generally accepted as the perfect source of nutrition for the health and development of infants. It also assists in infant innate and adaptive immunity through many proteins that are decorated with glycans. Examples of these glycoproteins include IgA, IgG, and innate immune lectins. Maternal diet and environmental exposure such as pathogens and pollutants affect human milk composition including its glycoprofile. Despite altered glycosylation can have a consequence on the nursing infant's health and immunity, the current knowledge is still emerging in this area of study. COVID-19 has gained attention in recent years by causing severe morbidity and mortality. Similar to other infectious diseases such as influenza, our lab recently revealed alterations in glycome of plasma and different tissue samples of COVID-19 infected patients. Inspired by these findings, we are interested in disclosing the effect of SARS-CoV-2 on glycosylation of breast milk proteins. Toward this, we performed a large-scale systematic study using our high-throughput lectin microarray analysis technology. We analyzed 132 control samples (breast milk collected pre- COVID) and breast milk from 78 COVID-19 infected mothers. Our data showed there is a 4-fold increase in -2,3 sialic acid on glycoproteins that is associated with SARS-CoV-2 infection in lactating mothers. Lectin pulldown experiments further testified to these findings. Given the significance of -2,3 sialic acid glycan signature in infectious diseases, our finding could provide valuable insight into therapeutic development.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Etiological identification and genetic source tracking of a cluster of cases with fever during the period of regular prevention and control of COVID-19 epidemic

Objective To identify the pathogen and track the genetic source of a cluster of cases with fever in a kindergarten in Fengtai district during the normalization of COVID-19 prevention and control in Beijing.Methods A descriptive analysis method was used to investigate this cluster of cases with fever in April 2021.Pharyngeal swabs were collected and viral nucleic acid was extracted, real-time PCR was performed to identify SARS-CoV-2 and other common respiratory virus. G gene of human metapneumovirus（hMPV） was amplified by RT-PCR and was then sequenced. BioEdit was used for G gene sequence analysis and the Neighbor-Joining model in MEGA 5. 0 software was used to construct the phylogenic tree of G gene. Results A total of 16 cases were reported in one class with the incidence of 53. 3%（16/30） during 8 days of a cluster outbreak. All pharyngeal swabs collected from 12 cases were tested SARS-CoV-2 negative, six were found to be hMPV positive by multiplex-PCR, and one was positive for both human adenovirus and hMPV. Full-length sequences of G genes were obtained from 2 strains of hMPV. Sequence analysis showed that both strains were hMPV B2 and the nucleic acid homology of G gene was 96. 73%-98. 01% with strains from Japan（LC337940, LC337935, LC1922349） in 2016 and over 98. 40%with strains from Shandong（OL625642, OL625644） in 2019, Henan MN944096 in 2019.Compared with the amino acid sequence of hMPV-B2 reference strain（AY297748）, six amino acid insertions containing EKEKEK were identified between 161-166 amino acid location and N-glycosylation of G protein analysis showed that the two strains had four N-glycosylation sites. Conclusions The leading pathogen for this cluster outbreak is found to be hMPV-B2, which are highly homologous with strains from Japan, Shandong and Henan. Therefore, a non-stop surveillance of hMPV is necessary during the normalization control and prevention period for COVID-19.

Significance of 1,25-Dihydroxyvitamin D3 on Overall Mortality in Peritoneal Dialysis Patients with COVID-19.

In previous publications, we pointed out the importance of mannosylation of fibrinogen for the development of cardiovascular complications and fucosylation as a predictor of peritoneal membrane dysfunction in patients on peritoneal dialysis (PD). After a follow-up period of 30 months from the onset of the COVID-19 pandemic, we evaluated the significance of 1,25-dihydroxyvitamin D3 (calcitriol) therapy, primary disease, biochemical and hematologic analyzes, and previously performed glycan analysis by lectin-based microarray as predictors of mortality in this patient group. After univariate Cox regression analysis, diabetes mellitus (DM) and calcitriol therapy were found to be potential predictors of mortality. Additional multivariate Cox regression analysis confirmed that only DM was a predictor of mortality. Nevertheless, the use of calcitriol in therapy significantly reduced mortality in this patient group, as shown by the Kaplan-Meier survival curve. The presence of DM as a concomitant disease proved to be a strong predictor of fatal outcome in PD patients infected with SARS-CoV-2. This is the first study to indicate the importance and beneficial effect of calcitriol therapy on survival in PD patients with COVID-19 infection. In addition, this study points to the possibility that adverse thrombogenic events observed in PD patients during the pandemic may be caused by aberrant fibrinogen glycosylation.

QTQTN motif upstream of the furin-cleavage site plays a key role in SARS-CoV-2 infection and pathogenesis.

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site-the FCS, loop length, and glycosylation-are required for efficient SARS-CoV-2 replication and pathogenesis.

Plasma markers of Covid-19 severity: A pilot study

SARS-CoV-2 infected patients show heterogeneous clinical presentations ranging from mild symptoms to severe respiratory failure and death. Consequently, various markers reflect certain disease presentations. Our cohort included moderate (n = 10) and severe (n = 10) COVID-19 patients, and 10 healthy controls. We determined plasma levels of nine acute phase proteins by nephelometry, full-length (M65), caspase-cleaved (M30) cytokeratin 18, and ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type-1 motif 13) by ELISA. In addition, we examined whole plasma N-glycosylation by capillary gel electrophoresis coupled to laser-induced fluorescence detection. When compared to healthy controls, COVID-19 patients had significantly lower concentrations of ADAMTS13 and albumin (ALB) but higher M30, M65, alpha-1-acid glycoprotein, alpha1-antitrypsin (AAT), ceruloplasmin, haptoglobin, and highsensitivity C-reactive protein. The concentrations of alpha1-antichymotrypsin, alpha2-macroglobulin and serum amyloid A proteins did not differ. We found significantly higher levels of AAT and M65 but lower ALB in severe compared to moderate COVID-19 patients. N-glycan analysis of the serum proteome revealed increased levels of oligomannose and sialylated di-antennary glycans, while the non-sialylated di-antennary glycan A2G2 significantly decreased in COVID-19 patients compared to controls. COVID-19-associated changes in levels and N-glycosylation of specific plasma proteins highlight involvement of different pathophysiological mechanisms and grant further investigations.

Post-translational modifications: Host defence mechanism, pathogenic weapon, and emerged target of anti-infective drugs

Post-translational modifications are changes introduced to proteins after their translation. They are the means to generate molecular diversity, expand protein function, control catalytic activity and trigger quick responses to a wide range of stimuli. Moreover, they regulate numerous biological processes, including pathogen invasion and host defence mechanisms. It is well established that bacteria and viruses utilize post-translational modifications on their own or their host's proteins to advance their pathogenicity. Doing so, they evade immune responses, target signaling pathways and manipulate host cytoskeleton to achieve survival, replication and propagation. Many bacterial species secrete virulence factors into the host and mediate hostpathogen interactions by inducing post-translational modifications that subvert fundamental cellular processes. Viral pathogens also utilize post translational modifications in order to overcome the host defence mechanisms and hijack its cellular machinery for their replication and propagation. For example, many coronavirus proteins are modified to achieve host invasion, evasion of immune responses and utilization of the host translational machinery. PTMs are also considered potential targets for the development of novel therapeutics from natural products with antibiotic properties, like lasso peptides and lantibiotics. The last decade, significant progress was made in understanding the mechanisms that govern PTMs and mediate regulation of protein structure and function. This urges the identification of relevant molecular targets, the design of specific drugs and the discovery of PTM-based medicine. Therefore, PTMs emerge as a highly promising field for the investigation and discovery of new therapeutics for many infectious diseases.Copyright © 2021 Bentham Science Publishers.

Dynamic expression of mucins and the genes controlling mucin-type O-glycosylation within the mouse respiratory system.

The COVID-19 global pandemic has underscored the need to understand how viruses and other pathogens are able to infect and replicate within the respiratory system. Recent studies have highlighted the role of highly O-glycosylated mucins in the protection of the respiratory system as well as how mucin-type O-glycosylation may be able to modify viral infectivity. Therefore, we set out to identify the specific genes controlling mucin-type O-glycosylation throughout the mouse respiratory system as well as determine how their expression and the expression of respiratory mucins is influenced by infection or injury. Here, we show that certain mucins and members of the Galnt family are abundantly expressed in specific respiratory tissues/cells and demonstrate unique patterns of O-glycosylation across diverse respiratory tissues. Moreover, we find that the expression of certain Galnts and mucins is altered during lung infection and injury in experimental mice challenged with infectious agents, toxins, and allergens. Finally, we examine gene expression changes of Galnts and mucins in a mouse model of SARS-CoV-2 infection. Our work provides foundational knowledge regarding the specific expression of Galnt enzyme family members and mucins throughout the respiratory system, and how their expression is altered upon lung infection and injury.

The Alterations of Serum N-glycome in Response to SARS-CoV-2 Vaccination.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has caused a global concern since its outbreak in 2019, with one of the main solutions being vaccination. Altered glycosylation has been described in patients after SARS-CoV-2 infection, while the effect of vaccination on serum glycoproteins remained unexplored. In this study, total serum glycosylation was analyzed in patients after SARS-CoV-2 infection and/or mRNA vaccination in order to identify potential glycosylation-based alterations. Enzyme-linked immunosorbent assay was applied to identify post-COVID-19 and post-Vaccinated patients and rule out potential outliers. Serum samples were deglycosylated by PNGase F digestion, and the released glycans were fluorescently derivatized using procainamide labeling. Solid-phase extraction was used to purify the labeled glycans followed by the analysis of hydrophilic-interaction liquid chromatography with fluorescence and mass-spectrometric detection. Alterations of serum N-glycome in response to SARS-CoV-2 infection and mRNA vaccination were revealed by linear discriminant analysis.

Comparison of Activity and Safety of DSPAα1 and Its N-Glycosylation Mutants.

DSPAα1 is a potent rude thrombolytic protein with high medicative value. DSPAα1 has two natural N-glycan sites (N153Q-S154-S155, N398Q-K399-T400) that may lead to immune responses when administered in vivo. We aimed to study the effect of its N-glycosylation sites on DSPAα1 in vitro and in vivo by mutating these N-glycosylation sites. In this experiment, four single mutants and one double mutant were predicted and expressed in Pichia pastoris. When the N398Q-K399-T400 site was mutated, the fibrinolytic activity of the mutant was reduced by 75%. When the N153Q-S154-S155 sites were inactivated as described above, the plasminogen activating activity of its mutant was reduced by 40%, and fibrin selectivity was significantly reduced by 21-fold. The introduction of N-glycosylation on N184-G185-A186T and K368N-S369-S370 also considerably reduced the activity and fibrin selectivity of DSPAα1. The pH tolerance and thermotolerance of all mutants did not change significantly. In vivo experiments also confirmed that N-glycosylation mutations can reduce the safety of DSPAα1, lead to prolonged bleeding time, non-physiological reduction of coagulation factor (α2-AP, PAI) concentration, and increase the risk of irregular bleeding. This study ultimately demonstrated the effect of N-glycosylation mutations on the activity and safety of DSPAα1.

COVID-19 Pandemic: An Overview of its Origin, Current Status, and Ongoing Clinical Trials

The COVID-19 pandemic is raging across the globe, with the total active cases increas-ing each day. Globally over 63 million COVID-19cases and more than 1.4 million deaths have been reported to WHO. Throughout the world, academicians, clinicians and scientists are working tirelessly on developing a treatment to combat this pandemic. The origin of novel SARS-CoV-2 virus still remains foggy but is believed to have originated from a bat coronavirus RaTG13 with which it shares approximately 96% sequence similarity. In the present review, the authors have pro-vided an overview of the COVID-19 pandemic, epidemiology, transmission, developments related to diagnosis, drugs and vaccines, along with the genetic diversity and lifecycle of the SARS-CoV-2 based on the current studies and information available.Copyright © 2022 Bentham Science Publishers.