Search

Aims: Better prediction of outcome after total hip arthroplasty (THA) is warranted. Systemic inflammation and central neuroinflammation are possibly involved in progression of osteoarthritis and pain. We explored whether inflammatory biomarkers in blood and cerebrospinal fluid (CSF) were associated with clinical outcome, and baseline pain or disability, 12 months after THA. Methods: A total of 50 patients from the Danish Pain Research Biobank (DANPAIN-Biobank) between January and June 2018 were included. Postoperative outcome was assessed as change in Oxford Hip Score (OHS) from baseline to 12 months after THA, pain was assessed on a numerical rating scale, and disability using the Pain Disability Index. Multiple regression models for each clinical outcome were included for biomarkers in blood and CSF, respectively, including age, sex, BMI, and Kellgren-Lawrence score. Results: Change in OHS was associated with blood concentrations of tumour necrosis factor (TNF), interleukin-8 (IL-8), interleukin-6 receptor (IL-6R), glycoprotein 130 (gp130), and IL-1β (R2 = 0.28, p = 0.006), but not with CSF biomarkers. Baseline pain was associated with blood concentrations of lymphotoxin alpha (LTα), TNFR1, TNFR2, and IL-6R (R2 = 0.37, p < 0.001) and CSF concentrations of TNFR1, TNFR2, IL-6, IL-6R, and IL-1Ra (R2 = 0.40, p = 0.001). Baseline disability was associated with blood concentrations of TNF, LTα, IL-8, IL-6, and IL-1α (R2 = 0.53, p < 0.001) and CSF concentrations of gp130, TNF, and IL-1β (R2 = 0.26, p = 0.002). Thus, preoperative systemic low-grade inflammation predicted 12-month postoperative outcome after THA, and was associated with preoperative pain and disability. Conclusion: This study highlights the importance of systemic inflammation in osteoarthritis, and presents a possible path for better patient selection for THA in the future. Preoperative central neuroinflammation was associated with preoperative pain and disability, but not change in OHS after THA. Cite this article: Bone Joint Res 2024;13(12):741–749.

Sweet potato is an important crop, and rich in protein and peptide which have great development potential. Sweet potato protein mainly includes dominant storage protein and small amount glycoprotein, different molecular weight sweet potato peptide can be obtained from protein by enzymatic or chemical hydrolysis. Sweet potato protein and peptide contain 18 types of amino acids that are essential for human body, and its diverse composition of amino acids provides unique nutritional value and its biological activity possess prominent role in the prevention and treatment of cancer, hypertension, obesity and other diseases. Sweet potato is usually used to produce starch, vermicellis and other foods, whereas, huge wastewater was produced during the production process, causing environmental pollution and wastage of protein resource. Therefore, the extraction and application of sweet potato protein from waste water is crucial. Currently, effective extraction and purification methods of sweet potato protein and peptide are commonly used, such as alkaline extraction and acid precipitation, enzymatic digestion, ultrafiltration and so on, but still need optimization and innovation to improve the extraction rate and purity to meet the needs of various fields. Future research can combine modern biotechnology and information technology to study the structure-function relationship, and explore more biological activities and application potentials of sweet potato protein and peptide. The current research status of sweet potato protein and peptide are reviewed in terms of structure composition, physicochemical properties, extraction, purification and biological activity in this paper. This review provides a reliable basis for the industrial production of sweet potato protein and peptide, and promotes the sustainable development of sweet potato protein and peptide.

Abstract Human Gb3/CD77 synthase (α1,4-galactosyltransferase, P1/Pk synthase, UDP-galactose: β-d-galactosyl-β1-R 4-α-d-galactosyltransferase, EC 2.4.1.228) forms Galα1 → 4Gal structures on glycosphingolipids and glycoproteins. These glycans are recognized by bacterial adhesins and toxins. Globotriaosylceramide (Gb3), the major product of Gb3/CD77 synthase, is a glycosphingolipid located predominantly in plasma membrane lipid rafts, where it serves as a main receptor for Shiga toxins released by enterohemorrhagic Escherichia coli and Shigella dysenteriae of serotype 1. On the other hand, accumulation of glycans formed by Gb3/CD77 synthase contributes to the symptoms of Anderson–Fabry disease caused by α-galactosidase A deficiency. Moreover, variation in Gb3/CD77 synthase expression and activity underlies the P1PK histo-blood group system. Glycosphingolipids synthesized by the enzyme are overproduced in colorectal, gastric, pancreatic, and ovarian cancer, and elevated Gb3 biosynthesis is associated with cancer cell chemo- and radioresistance. Furthermore, Gb3/CD77 synthase acts as a key glycosyltransferase modulating ovarian cancer cell plasticity. Here, we describe the role of human Gb3/CD77 synthase and its products in the P1PK histo-blood group system, Anderson–Fabry disease, and bacterial infections. Additionally, we provide an overview of emerging evidence that Gb3/CD77 synthase and its glycosphingolipid products are involved in cancer metastasis and chemoresistance. Graphical Abstract

The Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus that causes high mortality in humans. This enveloped virus harbors two surface glycoproteins (GP), Gn and Gc, that are released by processing of a glycoprotein precursor complex whose maturation takes place in the ER and is completed through the secretion pathway. Here, we characterized the trafficking network exploited by CCHFV GPs during viral assembly, envelopment, and/or egress. We identified membrane trafficking motifs in the cytoplasmic domains (CD) of CCHFV GPs and addressed how they impact these late stages of the viral life cycle using infection and biochemical assays, and confocal microscopy in virus-producing cells. We found that several of the identified CD motifs modulate GP transport through the retrograde trafficking network, impacting envelopment and secretion of infectious particles. Finally, we identified PACS-2 as a crucial host factor contributing to CCHFV GPs trafficking required for assembly and release of viral particles.

Rabies is a lethal zoonotic disease that threatens human health. As the only viral surface protein, the rabies virus (RABV) glycoprotein (G) induces main neutralizing antibody (Nab) responses; however, Nab titre is closely correlated with the conformation of G. Virus-like particles (VLP) formed by the co-expression of RABV G and matrix protein (M) improve retention and antigen presentation, inducing broad, durable immune responses. RABV nucleoprotein (N) can elicit humoral and cellular immune responses. Hence, we developed a series of nucleoside-modified RABV mRNA vaccines encoding wild-type G, soluble trimeric RABV G formed by an artificial trimer motif (tG-MTQ), membrane-anchored prefusion-stabilized G (preG). Furthermore, we also developed RABV VLP mRNA vaccine co-expressing preG and M to generate VLPs, and VLP/N mRNA vaccine co-expressing preG, M, and N. The RABV mRNA vaccines induced higher humoral and cellular responses than inactivated rabies vaccine, and completely protected mice against intracerebral challenge. Additionally, the IgG and Nab titres in RABV preG, VLP and VLP/N mRNA groups were significantly higher than those in G and tG-MTQ groups. A single administration of VLP or VLP/N mRNA vaccines elicited protective Nab responses, the Nab titres were significantly higher than that in inactivated rabies vaccine group at day 7. Moreover, RABV VLP and VLP/N mRNA vaccines showed superior capacities to elicit potent germinal centre, long-lived plasma cell and memory B cell responses, which linked to high titre and durable Nab responses. In summary, our data demonstrated that RABV VLP and VLP/N mRNA vaccines could be promising candidates against rabies.

Background: Respiratory syncytial virus (RSV) infection imposes substantial health burden and disproportionally affects young infants, elderly, and immunocompromised hosts. RSV harbors key surface glycoproteins F and G, both crucial for viral infection and evolution. Methods: In this study, we examined the genetic characteaistics of 179 RSV isolates collected between 2017 and 2021 in Taiwan. G ectodomain and whole F gene were sequenced and aligned with available references from GenBank. Results: RSV ON1 and BA9 were two predominant genotypes throughout the study period. Genetic variations of G protein accumulated over time. New ON1 strains containing E257K and K204R-V225A-T238I-Y280H in combination emerged in 2019 and contributed to a local endemic in 2020. RSV-B strain with A131T and T137I substitution in G protein emerged in 2018. On the other hand, F protein of both RSV genotypes was generally conserved but some feature changes should be noted: RSV-B in Taiwan harbored 100% of I206M and Q209R in site Ø, and L172Q and S173L in site V. These amino acid changes do not affect the susceptibility of Nirsevimab but imply no effectiveness of Suptavumab. Conclusion: RSV continuously evolves in Taiwan and accumulated signature genetic changes over time. Vigilant RSV genomic surveillance is important to monitor the viral evolution in the upcoming future of new RSV vaccines and prophylaxis.

Objective: The study aimed to investigate the effects of Periplaneta americana glycoprotein (PAG) on the growth and metabolites of Lactobacillus plantarum and Bifidobacterium adolescentis. Methods: With inulin as a positive control, the probiotic bacteria were cultured in medium containing different concentrations of PAG (MRS medium with PAG concentrations of 0.5, 1.0, 2.0, and 4.0 mg/mL, respectively. TPY medium with PAG concentrations of 1.0, 2.0, 4.0, and 8.0 mg/mL, respectively) to determine the OD600 and pH, and the contents of lactic acid, short-chain fatty acid (SCFA, snamely acetic acid, propionic acid and butyric acid) in the bacterial solution. Results: The growth of Lactobacillus plantarum was greatest when the concentration of PAG reached 1.0 mg/mL, while Bifidobacterium adolescentis grew best with a PAG concentration of 2.0 mg/mL. The OD600 value, production of lactic acid and SCFAs of Lactobacillus plantarum solution at 1.0 mg/mL PAG were higher than those of inulin, while the pH value was significantly lower. In the bacterial solution of Bifidobacterium adolescentis with 2.0 mg/mL PAG, the values of OD600 and the production of propionic acid and butyric acid increased compared to the same concentration of inulin, the lactic acid production was better than that of inulin at 12 h, the acetic acid production was better than that of inulin at 24 h, and the pH value decreased, indicating that PAG and inulin had different effects on promoting Bifidobacterium adolescentis to produce lactic acid and SCFAs. Conclusion: The PAG could promote the proliferation and organic acid production of Lactobacillus plantarum and Bifidobacterium adolescentis, and the effect was better than that of inulin.

Background: Glycoprotein (GP)VI is a platelet-specific collagen receptor required for platelet activation during hemostasis. Platelet reactivity toward collagen is routinely assessed during diagnostic workup of platelet disorders. GPVI can be activated by inducing receptor clustering with suspensions of fibrillar collagen or synthetic cross-linked collagen-related peptide (CRP-XL). However, these suspensions are poorly standardized or difficult to produce. Nanobodies are small recombinant camelid-derived heavy-chain antibody variable regions. They are highly stable, specific, and ideal candidates for developing a stable GPVI agonist for diagnostic assays. Objectives: Develop a stable nanobody-based GPVI agonist. Methods: Nanobody D2 (NbD2) was produced as dimers and purified. Tetramers were generated via C-terminal fusion of dimers with click chemistry. Nanobody constructs were functionally characterized with light transmission aggregometry (LTA) in platelet-rich plasma and whole blood flow cytometry. Diagnostic performance was assessed in patients with inherited platelet function disorders with LTA and flow cytometry. Results: NbD2 was specific for human platelet GPVI. Dimers did not result in platelet activation in LTA or flow cytometry settings and fully inhibited CRP-XL-induced P-selectin expression and fibrinogen binding in whole blood and attenuated collagen-induced platelet aggregation in platelet-rich plasma. However, NbD2 tetramers caused full platelet aggregation, as well as P-selectin expression and fibrinogen binding. NbD2 tetramers were able to discriminate between inherited platelet function disorder patients and healthy controls based on fibrinogen binding, similar to CRP-XL. Conclusion: Nanobody tetramers to GPVI induce platelet activation and can be used to assess the GPVI pathway in diagnostic assays.

Ebola virus disease is a deadly pathogenic disease with a fatality rate of 25–90 % as recorded in previous outbreaks. The Ebola Virus glycoprotein (EBOV-GP) plays a crucial role in the entry of viruses into human cells, making it an interesting target for therapeutic discovery. Therefore, inhibiting this protein can directly limit the virus replication and disease progression at an early stage of infection. The present study focuses on the design of novel potent EBOV-GP inhibitors using multiple computational techniques. In this context, two QSAR models were built from a set of 86 amodiaquine derivatives as anti-EBOV-GP using Monte Carlo and genetic algorithm multiple linear regression methods. Both models confirmed their predictive performance with satisfactory statistical parameters of the validation (R2 = 0.9129; Q2 = 0.8848 for the CORAL model and R2 = 0.8848; Q2 = 0.8148 for the GA-MLR model). From the outputs of the CORAL model, the structural fragments responsible for increasing and decreasing the inhibition activity were extracted and interpreted. This molecular information was used to design 26 new potentially safe and active candidate drugs. Molecular docking and dynamics simulations have affirmed the efficacy of the designed compounds. Specifically, compounds D2 (pIC50_coral = 7.12; pIC50_GA-MLR = 7.07), D3 (pIC50_coral = 7.83; pIC50_GA-MLR = 7.10), and D5 (pIC50_coral = 7.26; pIC50_GA-MLR = 7.55) displayed notable predicted inhibitory activity, according to both models. These compounds also exhibited conformational and structural stability, as well as a favorable binding profile. Furthermore, these potential drug candidates were found to be non-toxicity and have acceptable pharmacological properties.

Severe fever with thrombocytopenia syndrome virus (SFTSV) or Dabie bandavirus is an emerging pathogen responsible for SFTS. It is considered a novel threat to human health, given the high associated fatality. SFTSV is a segmented negative-strand RNA virus containing three single-stranded RNAs, with the M segment encoding the glycoproteins Gn and Gc. Gc is vital for viral entry into the host cell surface, along with the Gn protein. As the Gc is the surface-exposable antigen from virions, it is a critical diagnostic marker of infection. Although various SFTSV Gn or N protein-based sero-diagnostic methods have been developed, there are no commercially available sero-diagnostic kits. Therefore, we generated monoclonal antibodies (mAbs) against SFTSV Gc and explored their application in serum diagnostic tests to develop sensitive serodiagnostic tools covering broad-range genotypes (A to F). First, 10 SFTSV Gc antibody-binding fragments (Fabs) were isolated using a phage display system and converted into human IgGs. Enzyme-linked immunosorbent assays (ELISA) of the SFTSV and Rift Valley fever virus (RVFV: same genus as SFTSV) Gc antigens showed that all antibodies attached to the SFTSV Gc protein had high affinity. An immunofluorescence assay (IFA), to verify the cross-reactivity of seven antibodies with high affinities for various SFTSV genotypes (A, B2, B3, D, and F) and detect mAb binding with intact Gc proteins, revealed that five IgG type mAbs were bound to intact Gc proteins of various genotypes. Six high-affinity antibodies were selected using ELISA and IFA. The binding capacity of the six antibodies against the SFTSV Gc antigen was measured using surface plasmon resonance. All antibodies had high binding capacity. Consequently, these antibodies serve as valuable markers in the serological diagnosis of SFTSV.

IntroductionEpstein-Barr virus (EBV) is an oncogenic human herpesvirus associated with ~350,000 cases of lymphoid and epithelial malignancies every year, and is etiologically linked to infectious mononucleosis and multiple sclerosis. Despite four decades of research, no EBV vaccine candidate has yet reached licensure. Most previous vaccine attempts focused on a single viral entry glycoprotein, gp350, but recent data from clinical and pre-clinical studies, and the elucidation of viral entry mechanisms, support the inclusion of multiple entry glycoproteins in EBV vaccine design.MethodsHere we generated a modified vaccinia Ankara (MVA)-vectored EBV vaccine, MVA-EBV5-2, that targets five EBV entry glycoproteins, gp350, gB, and the gp42gHgL complex. We characterized the genetic and translational stability of the vaccine, followed by immunogenicity assessment in BALB/c mice and rhesus lymphocryptovirus-negative rhesus macaques as compared to a gp350-based MVA vaccine. Finally, we assessed the efficacy of MVA-EBV5-2-immune rhesus serum at preventing EBV infection in human CD34+ hematopoietic stem cell-reconstituted NSG mice, under two EBV challenge doses.ResultsThe MVA-EBV5-2 vaccine was genetically and translationally stable over 10 viral passages as shown by genetic and protein expression analysis, and when administered to female and male BALB/c mice, elicited serum EBV-specific IgG of both IgG1 and IgG2a subtypes with neutralizing activity in vitro. In Raji B cells, this neutralizing activity outperformed that of serum from mice immunized with a monovalent MVA-vectored gp350 vaccine. Similarly, MVA-EBV5-2 elicited EBV-specific IgG in rhesus macaques that were detected in both serum and saliva of immunized animals, with serum antibodies demonstrating neutralizing activity in vitro that outperformed serum from MVA-gp350-immunized macaques. Finally, pre-treatment with serum from MVA-EBV5-2-immunized macaques resulted in fewer EBV-infected mice in the two challenge experiments than pretreatment with serum from pre-immune macaques or macaques immunized with the monovalent gp350-based vaccine.DiscussionThese results support the inclusion of multiple entry glycoproteins in EBV vaccine design and position our vaccine as a strong candidate for clinical translation.

Objective: This study was conducted to investigate the purification process aimed at obtaining a shiitake glycoprotein with lead eliminating effect, and to explore its preventive and therapeutic efficacy against lead acetate-induced lead toxicity in a rat model. Methods: The crude polysaccharide was extracted from shiitake mushrooms using a hot water extraction method, followed by further purification involving protein removal and chromatography techniques to obtain the bioactive glycoprotein. The molecular characteristics of the glycoprotein were determined through various analytical methods, including infrared spectroscopy analysis, monosaccharide composition analysis, N-terminal amino acid sequencing, and internal amino acid sequence analysis. To evaluate the bioactive potential of shiitake polysaccharide-peptide in addressing lead poisoning in rats, 63 male Sprague-Dawley rats were randomly distributed into nine groups, including a control group, a model group, a positive control group, and shiitake glycoprotein treatment groups at doses of 4, 8 and 160 mg/kg, as well as shiitake fruiting body treatment groups at doses of 4, 12 and 36 g/kg. Lead poisoning was induced in the rats via intraperitoneal injection of lead acetate. The rats were administered polysaccharide-peptide and shiitake fruiting body treatments for a duration of 30 days, with observations made regarding their physical condition. Parameters assessed included body weight, lead content in blood and various organs, as well as biochemical markers in serum. Results: The monosaccharide composition analysis showed that the purified shiitake glycoprotein (LEPP) was composed of glucose, mannose, galactose, glucuronic acid, xylose, fucose, ribose, galacturonic acid, arabinose and rhamnose. The amino acid sequence analysis showed that the N-terminal sequence was MPEQVVVADA, indicating that the shiitake glycoprotein had polysaccharide and protein components. The findings indicated that shiitake glycoprotein effectively promoted weight gain in rats afflicted with lead poisoning. It significantly reduced lead concentrations in both blood and liver, mitigated lead deposition within the liver, enhanced the activities of superoxide dismutase (SOD) and catalase (CAT) in the serum, lowered the levels of malondialdehyde (MDA) （P

Introduction: Platelet-rich Fibrin (PRF) is an autologous platelet concentrate preparation containing several proteins that aid in healing. Vitronectin is one of these proteins that has not been quantified in all types of PRF. Various protocols have been suggested to alter the yield of different components of PRF to enhance wound healing. Hence, it is beneficial to know the vitronectin levels in PRF. Aim: To detect, estimate, and compare the levels of vitronectin in two PRF protocols and serum. Materials and Methods: The present cross-sectional study conducted in the Department of Periodontics at the SDM College of Dental Sciences and Hospital. Dharwad, Karnataka, India from January 2019 to June 2020 involved 12 systemically and periodontally healthy volunteers. Blood was obtained from each volunteer to collect and prepare serum, Leukocyte-PRF (L-PRF), and injectable-PRF (i-PRF), respectively. Three distinct samples-supernatant, exudate, and clot-were collected and categorised into seven groups (L-PRF supernatant, L-PRF exudate, L-PRF clot, i-PRF supernatant, i-PRF exudate, i-PRF clot, blood serum) that were assayed for levels of vitronectin. The data were statistically analysed using the independent t-test, one-way Analysis of Variance (ANOVA), and Newman-Keuls Post-hoc procedures. Results: The mean age was 24.92±2.57 years. Vitronectin was detected and estimated in all the samples. Vitronectin levels ranged from 64.09±0.04 ng/mL to 64.20±0.21 ng/mL. One-way ANOVA applied to test between and within groups was significant (p=0.049). A statistically significant difference was observed only between L-PRF exudate and serum (p=0.05). Conclusion: The comparable levels of vitronectin in L-PRF and i-PRF observed in present study suggest that vitronectin in these two PRF protocols may aid wound healing.

Proteins found within the fungal cell wall usually contain both N- and O-oligosaccharides. N-glycosylation is the process where these oligosaccharides (hereinafter: glycans) are attached to asparagine residues, while in O-glycosylation the glycans are covalently bound to serine or threonine residues. The PMT family is grouped into PMT1, PMT2, and PMT4 subfamilies. Using bioinformatics analysis within the Botrytis cinerea genome database, an ortholog to Saccharomyces cerevisiae Pmt4 and other fungal species was identified. The aim of this study was to assess the relevance of the bcpmt4 gene in B. cinerea glycosylation. For this purpose, the bcpmt4 gene was disrupted by homologous recombination in the B05.10 strain using a hygromycin B resistance cassette. Expression of bcpmt4 in S. cerevisiae ΔScpmt4 or ΔScpmt3 null mutants restored glycan levels like those observed in the parental strain. The phenotypic analysis showed that Δbcpmt4 null mutants exhibited significant changes in hyphal cell wall composition, including reduced mannan levels and increased amounts of chitin and glucan. Furthermore, the loss of bcpmt4 led to decreased glycosylation of glycoproteins in the B. cinerea cell wall. The null mutant lacking PMT4 was hypersensitive to a range of cell wall perturbing agents, antifungal drugs, and high hydrostatic pressure. Thus, in addition to their role in glycosylation, the PMT4 is required to virulence, biofilm formation, and membrane integrity. This study adds to our knowledge of the role of the B. cinerea bcpmt4 gene, which is involved in glycosylation and cell biology, cell wall formation, and antifungal response.

ABSTRACT Symbiotic interactions between humans and our communities of resident gut microbes (microbiota) play many roles in health and disease. Some gut bacteria utilize mucus as a nutrient source and can under certain conditions damage the protective barrier it forms, increasing disease susceptibility. We investigated how Ruminococcus torques—a known mucin degrader that has been implicated in inflammatory bowel diseases (IBDs)—degrades mucin glycoproteins or their component O-linked glycans to understand its effects on the availability of mucin-derived nutrients for other bacteria. We found that R. torques utilizes both mucin glycoproteins and released oligosaccharides from gastric and colonic mucins, degrading these substrates with a panoply of mostly constitutively expressed, secreted enzymes. Investigation of mucin oligosaccharide degradation by R. torques revealed strong α-L-fucosidase, sialidase and β1,4-galactosidase activities. There was a lack of detectable sulfatase and weak β1,3-galactosidase degradation, resulting in accumulation of glycans containing these structures on mucin polypeptides. While the Gram-negative symbiont, Bacteroides thetaiotaomicron grows poorly on mucin glycoproteins, we demonstrate a clear ability of R. torques to liberate products from mucins, making them accessible to B. thetaiotaomicron. This work underscores the diversity of mucin-degrading mechanisms in different bacterial species and the probability that some species are contingent on others for the ability to more fully access mucin-derived nutrients. The ability of R. torques to directly degrade a variety of mucin and mucin glycan structures and unlock released glycans for other species suggests that it is a keystone mucin degrader, which might contribute to its association with IBD.IMPORTANCEAn important facet of maintaining healthy symbiosis between host and intestinal microbes is the mucus layer, the first defense protecting the epithelium from lumenal bacteria. Some gut bacteria degrade the various components of intestinal mucins, but detailed mechanisms used by different species are still emerging. It is imperative to understand these mechanisms as they likely dictate interspecies interactions and may illuminate species associated with bacterial mucus damage and subsequent disease susceptibility. Ruminococcus torques is positively associated with IBD in multiple studies. We identified mucin glycan-degrading enzymes in R. torques and found that it shares mucin degradation products with another species of gut bacteria, Bacteroides thetaiotaomicron. Our findings underscore the importance of understanding mucin degradation mechanisms in different gut bacteria and their consequences on interspecies interactions, which may identify keystone bacteria that disproportionately affect mucus damage and could therefore be key players in effects that result from reductions in mucus integrity.

ABSTRACT: The transmission electron microscopy revealed a dendritic cell in the medulla of the chicken bursal follicle. This dendritic cell has a classical secretory machinery; therefore, it has been named a bursal secretory dendritic cell (BSDC). The corticomedullary epithelial arch (CMEA) encloses lymphoid-like cells, which can proliferate and after entering the medulla, begin to differentiate to immature, then mature BSDC, which discharges glycoprotein (gp). With the exhaustion of gp production, the BSDC rapidly transforms into a macrophage-like cell (Mal), which is an activated endocytic cell of innate immunity. The Mal drifts through the follicle-associated epithelium (FAE)-supporting cells into the FAE, and via FAE, the Mal is eliminated in the bursal lumen. The infectious bursal disease virus (IBDV) infection accelerates the maturation process of BSDC precursors, which results in acute emptying of CMEA and subsequently, numerous immature BSDC(s) emerge. The IBDV infection stops the gp discharge, and the gp appears in the virus-containing Mal. The Movat pentachrome staining recognizes the gp in the extracellular spaces of the medulla and after infection in the Mal. The BSDC is the primary target of the IBDV. During IBDV infection, a large number of suddenly formed Mal actively migrate into the cortex, initiating cytokine storm and recruiting heterophil granulocytes. During embryogenesis, the vimentin-positive, possibly embryonic dendritic cells provide a microenvironment for carbohydrate switch. Around hatching, these embryonic, temporary dendritic cells get the Fc receptor, which bind maternal IgY. The posthatched forms of BSDC(s) gradually replace the embryonic ones and bind their own IgY.

Abstract Background Cryptosporidium spp. are common protozoa causing diarrhea in humans and animals. There are currently only one FDA-approved drug and no vaccines for cryptosporidiosis, largely due to the limited knowledge of the molecular mechanisms involved in the invasion of the pathogens. Previous studies have shown that GP60, which is cleaved into GP40 and GP15 after expression, is an immunodominant mucin protein involved in the invasion of Cryptosporidium. The protein is highly O-glycosylated, and recombinant proteins expressed in prokaryotic systems are non-functional. Therefore, few studies have investigated the function of GP40 and GP15. Methods To obtain recombinant GP40 with correct post-translational modifications, we used CRISPR/Cas9 technology to insert GP40 and GP15 into the UPRT locus of Toxoplasma gondii, allowing heterologous expression of Cryptosporidium proteins. In addition, the Twin-Strep tag was inserted after GP40 for efficient purification of GP40. Results Western blotting and immunofluorescent microscopic analyses both indicated that GP40 and GP15 were stably expressed in T. gondii mutants. GP40 localized not only in the cytoplasm of tachyzoites but also in the parasitophorous vacuoles, while GP15 without the GPI anchor was expressed only in the cytoplasm. In addition, a large amount of recTgGP40 was purified using Strep-TactinXT supported by a visible band of ~ 50 kDa in SDS-PAGE. Conclusions The establishment of a robust and efficient heterologous expression system of GP40 in T. gondii represents a novel approach and concept for investigating Cryptosporidium mucins, overcoming the limitations of previous studies that relied on unstable transient transfection, which involved complex steps and high costs. Graphical Abstract

Abstract Background The Junín virus (JUNV) is well known for causing argentine haemorrhagic fever (AHF), a severe endemic disease in farming premises. The glycoprotein of JUNV is an important therapeutic target in vaccine design. Despite using drugs and neutralizing weakened antibodies being used in the medication, neither the severity reduced nor eradicated the infection. However, this constraint can be resolved by immunoinformatic approaches. Results The glycoprotein fasta sequence was retrieved from NCBI to anticipate the B cell and T cell epitopes through the Immune Epitope Database. Furthermore, each epitope underwent validation in Vaxijen 2.0, Aller Top, and Toxin Pred to find antigenic, nonallergic, and non-toxic peptides. Moreover, the vaccine is designed with appropriate adjuvants and linkers. Subsequently, physicochemical properties were determined in ProtParam including solubility and disulphide bonds in the SCRATCH server. The vaccine 3D structure was built using I-TASSER and refined in ModRefine. Docking between JUNV glycoprotein (PDB ID:5NUZ) with a built vaccine revealed a balanced docked complex visualized in the Drug Discovery studio, identified 280 hydrogen bonds between them. The docking score of − 15.5 kcal/mol was determined in the MM/GBSA analysis in HawkDock. MD simulations employed using the GROMACS at 20 ns resulted in minimal deviation and fewer fluctuations, particularly with high hydrogen bond-forming residues. Conclusion However, these findings present a potential vaccine for developing against JUNV glycoprotein after validating the epitopes and 3D vaccine construct through in silico methods. Therefore, further investigation in the wet laboratory is necessary to confirm the potentiality of the predicted vaccine.

Glycoproteins are special proteins and important nutrients for hypoglycemic activity. However, the structure of Auricularia Auricula glycoprotein (AAG) and the stability of its hypoglycemic activity during simulated digestion (including saliva, gastral and intestine digestion) in vitro are still unknown. In this study, AAG-3 was isolated from Auricularia Auricula. SDS-PAGE, UV spectrum, FTIR, amino acid composition, dichroic spectrum and SEM were used to characterize its structure. The hypoglycemic activity of AAG-3 during in vitro digestion was investigated via inhibition of α-amylase and α-glucosidase activities, as well as glucose consumption, glycogen content and related enzyme activity in insulin-resistant HepG2 cells. Structural characterization showed that AAG-3 with a Mw of 18.21 kDa had an O-type glycopeptide bond and typical functional groups of glycoproteins. AAG-3 contained 18 kinds of amino acid and many α-helixes and β-turns, and its microstructure was sheet-like. With the simulated digestion of AAG-3 in vitro, the inhibition of α-amylase and α-glucosidase activity as well as the glucose consumption, glycogen content and HK and PK enzyme activities in insulin-resistant HepG2 cells were significantly increased. Therefore, AAG-3 has a potential role in reducing blood glucose levels and improving insulin resistance and can be used as a potential micronutritional supplement for diabetic patients.

N-glycans have recently emerged as highly varied elements of Chlorella strains and products. Four years and eighty samples later, the increasing N-glycan diversity calls for a re-examination in the light of concepts of species designations and product authenticity. N-glycans of commercial products were analyzed by matrix-assisted time-of-flight mass spectrometry (MALDI-TOF MS) supported by chromatography on porous graphitic carbon with mass spectrometric detection. Although 36% of 172 products were labeled C. vulgaris, only 9% presented what could be taken as a C. vulgaris type N-glycan pattern. Respectively, 5 and 20% of the products matched with C. sorokiniana strains SAG 211-8k and SAG 211-34, which, however, carry entirely different structures. Furthermore, 41% presented with one of four frequently occurring glyco-types while 26% of the samples showed unique or rare N-glycan patterns. These glycan signatures thus profoundly challenge the stated species designations. By no means do we want to question the presumed health benefits of the products or the sincerity of manufacturers. We rather aim to raise awareness of the fascinating but also concerning diversity of microalgal N-glycans and suggest it as a means for defining product identity and taxonomic classifications.

Objective To screen, identify and verify the immunoreactive epitopes on Hantaan virus glycoprotein C-terminal (HTNV Gc). Methods On the basis of predicting the major histocompatibility complex (MHC) gene in more than 98% of the global population, the affinity of 9-peptide and 15-peptide epitopes on HTNV Gc was systematically analyzed by bioinformatics cutting-edge algorithms such as Immune Epitope Database (IEDB) and Technical University of Denmark (DTU). Their immunogenicity was analyzed by using the Vaxijen tool, and the interspecies intraspecific conservation was analyzed with the Blastp tool. Bidirectional hierarchical clustering was employed to compare the cross-immunoreactivity of MHC-class Ⅰ and class Ⅱ restricted epitopes. Possible results of dominant epitopes docking with MHC were simulated by using molecular docking. Enzyme-linked immunospot (ELISpot) assay was performed to validate the results of computer analyses. Results After integrating the results of various bioinformatics algorithms, we obtained 8 9-peptide epitopes and 80 15-peptide epitopes with high affinity, intraspecific conservation and strong immunogenicity, including 8 human-mouse common dominant epitopes. Cluster analysis showed that H-2 genotype had certain similarity as HLA genotype. The results of molecular docking revealed that dominant epitopes had good docking with various MHC genotypes, which were consistent with the results of affinity analysis. The amount of IFN-γ secreted from the spleen cells was higher in the mice with dominant epitopes than those with non-dominant epitopes. Conclusion With the aids of various bioinformatics algorithms, we successfully screen out the dominant epitopes on HTNV Gc, and validate the cellular immunoreactivity of these epitopes through ELISpot assay.

Transductional targeting of herpes simplex virus (HSV)-based gene therapy vectors offers the potential for improved tissue-specific delivery and can be achieved by modification of the viral entry machinery to incorporate ligands that bind the desired cell surface proteins. The interaction of nerve growth factor (NGF) with tropomyosin receptor kinase A (TrkA) is essential for survival of sensory neurons during development and is involved in chronic pain signaling. We targeted HSV infection to TrkA-bearing cells by replacing the signal peptide and HVEM binding domain of glycoprotein D (gD) with pre-pro-NGF. This TrkA-targeted virus (KNGF) infected cells via both nectin-1 and TrkA. However, infection through TrkA was inefficient, prompting a genetic search for KNGF mutants showing enhanced infection following repeat passage on TrkA-expressing cells. These studies revealed unique point mutations in envelope glycoprotein gH and in UL24, a factor absent from mature particles. Together these mutations rescued efficient infection of TrkA-expressing cells, including neurons, and facilitated the production of a completely retargeted KNGF derivative. These studies provide insight into HSV vector improvements that will allow production of replication-defective TrkA-targeted HSV for delivery to the peripheral nervous system and may be applied to other retargeted vector studies in the central nervous system.

N-glycans in many proteins are of great concern because of their strong association with food allergies. Triticum aestivum (bread wheat), a major food crop, is known as one of the “Big Eight” allergenic groups. However, little research has been done about N-glycans in wheat glycoproteins. In this study, a soluble wheat glycoprotein was purified from wheat and further identified as globulin-1 S allele (GSA). The wheat GSA displayed significant IgE-binding activity. Moreover, one N-glycosylation site and 6 kinds of N-glycans were identified by mass spectrometry, including 3 high mannose types and 3 complex types. Furthermore, the IgE-binding activity of wheat GSA is proved to be reduced by the removal of N-glycan, thermal treatment (temperatures > 80 °C), and strong acidic treatment (pH 3.0). These findings would provide a better understanding of the effects of N-glycosylation, thermal treatment, and acidic treatment on the molecular characteristics of GSA, and further provide new insights into the development of hypoallergenic wheat products.

Rabies virus (RABV) causes a fatal neurological disease, consisting of unsegmented negative-strand RNA, which encodes five structural proteins (3′-N-P-M-G-L-5′). Apolipoprotein D (ApoD), a lipocalin, is upregulated in the nervous system after injury or pathological changes. Few studies have focused on the role of ApoD during virus infection so far. This study demonstrated that ApoD is upregulated in the mouse brain (in vivo) and C8-D1A cells (in vitro) after RABV infection. By upregulating ApoD expression in C8-D1A cells, we found that ApoD facilitated RABV replication. Additionally, Co-immunoprecipitation demonstrated that ApoD interacted with RABV glycoprotein (G protein). The interaction could promote RABV replication by upregulating the cholesterol level. These findings revealed a novel role of ApoD in promoting RABV replication and provided a potential therapeutic target for rabies.

Oncolytic herpes simplex viruses (oHSVs) have emerged as leading cancer therapeutic agents. Effective oHSV virotherapy may ultimately require both intratumoral and systemic vector administration to target the primary tumor and distant metastases. An attractive approach to enhancing oHSV tumor specificity is engineering the virus envelope glycoproteins for selective recognition of and infection via tumor-specific cell surface proteins. We previously demonstrated that oHSVs could be retargeted to EGFR-expressing cells by the incorporation of a single-chain antibody (scFv) at the N terminus of glycoprotein D (gD). Here, we compared retargeted oHSVs generated by the insertion of scFv, affibody molecule, or VHH antibody ligands at different positions within the N terminus of gD. When compared to the scFv-directed oHSVs, VHH and affibody molecules mediated enhanced EGFR-specific tumor cell entry, spread and cell killing in vitro, and enabled long-term tumor-specific virus replication following intravenous delivery in vivo. Moreover, oHSVs retargeted via a VHH ligand reduced tumor growth upon intravenous injection and achieved complete tumor destruction after intratumoral injection. Systemic oHSV delivery is important for the treatment of metastatic disease, and our enhancements in targeted oHSV design are a critical step in creating an effective tumor-specific oHSVs for safe administration via the bloodstream.

Introduction: Prolyl-4-hydroxylases (P4H) catalyse the irreversible conversion of proline to hydroxyproline, constituting a common posttranslational modification of proteins found in humans, plants, and microbes. Hydroxyproline residues can be further modified in plants to yield glycoproteins containing characteristic O-glycans. It is currently unknown how these plant endogenous modifications impact protein functionality and they cause considerable concerns for the recombinant production of therapeutic proteins in plants. In this study, we carried out host engineering to generate a therapeutic glycoprotein largely devoid of plant-endogenous O-glycans for functional characterization.Methods: Genome editing was used to inactivate two genes coding for enzymes of the P4H10 subfamily in the widely used expression host Nicotiana benthamiana. Using glycoengineering in plants and expression in human HEK293 cells we generated four variants of a potent, SARS-CoV-2 neutralizing antibody, COVA2-15 IgA1. The variants that differed in the number of modified proline residues and O-glycan compositions of their hinge region were assessed regarding their physicochemical properties and functionality.Results: We found that plant endogenous O-glycan formation was strongly reduced on IgA1 when transiently expressed in the P4H10 double mutant N. benthamiana plant line. The IgA1 glycoforms displayed differences in proteolytic stability and minor differences in receptor binding thus highlighting the importance of O-glycosylation in the hinge region of human IgA1.Discussion: This work reports the successful protein O-glycan engineering of an important plant host for recombinant protein expression. While the complete removal of endogenous hydroxyproline residues from the hinge region of plant-produced IgA1 is yet to be achieved, our engineered line is suitable for structure-function studies of O-glycosylated recombinant glycoproteins produced in plants.

The production of N-linked glycoproteins in genetically engineered Escherichia coli holds significant potential for reducing costs, streamlining bioprocesses, and enhancing customization. However, the construction of a stable and low-cost microbial cell factory for the efficient production of humanized N-glycosylated recombinant proteins remains a formidable challenge. In this study, we developed a glyco-engineered E. coli chassis to produce N-glycosylated proteins with the human-like glycan Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc-, containing the human glycoform Gal-β-1,4-GlcNAc-β-1,3-. Our initial efforts were to replace various loci in the genome of the E. coli XL1-Blue strain with oligosaccharyltransferase PglB and the glycosyltransferases LsgCDEF to construct the E. coli chassis. In addition, we systematically optimized the promoter regions in the genome to regulate transcription levels. Subsequently, utilizing a plasmid carrying the target protein, we have successfully obtained N-glycosylated proteins with 100% tetrasaccharide modification at a yield of approximately 320 mg/L. Furthermore, we constructed the metabolic pathway for sialylation using a plasmid containing a dual-expression cassette of the target protein and CMP-sialic acid synthesis in the tetrasaccharide chassis cell, resulting in a 40% efficiency of terminal α-2,3- sialylation and a production of 65 mg/L of homogeneously sialylated glycoproteins in flasks. Our findings pave the way for further exploration of producing different linkages (α-2,3/α-2,6/α-2,8) of sialylated human-like N-glycoproteins in the periplasm of the plug-and-play E. coli chassis, laying a strong foundation for industrial-scale production.

The modularity of carbohydrate-active enzymes facilitates that enzymes with different functions have similar fragments. However, because of the complex structure of the enzyme active sites and the epistatic effects of various mutations on enzyme activity, it is difficult to design enzymes with multiple mutation sites using conventional methods. In this study, we designed multi-point mutants by fragment replacement in the donor-acceptor binding pocket of Actinobacillus pleuropneumoniae N-glycosyltransferase (ApNGT) to obtain novel properties. Candidate fragments were selected from a customized glycosyltransferase database. The stability and substrate-binding energy of the three fragment replacement mutants were calculated in comparison with wild-type ApNGT, and mutants with top-ranking stability and middle-ranking substrate-binding energy were chosen for priority experimental verification. We found that a mutant called F13, which increased the glycosylation efficiency of the natural substrate by 1.44 times, the relative conversion of UDP-galactose by 14.2 times, and the relative conversion of UDP-xylose from almost 0 to 78.6%. Most importantly, F13 mutant acquired an entirely new property, the ability to utilize UDP-glucuronic acid. On one hand, this work shows that replacing similar fragments in the donor-acceptor binding pocket of the enzyme might provide new ideas for designing mutants with new properties; on the other hand, F13 mutant is expected to play an important role in targeted drug delivery.

Marburg virus (MARV) is one of the filovirus species that cause deadly hemorrhagic fever in humans, with mortality rates up to 90%. Neutralizing antibodies represent ideal candidates to prevent or treat virus disease. However, no antibody has been approved for MARV treatment to date. In this study, we identified a novel human antibody named AF-03 that targeted MARV glycoprotein (GP). AF-03 possessed a high binding affinity to MARV GP and showed neutralizing and protective activities against the pseudotyped MARV in vitro and in vivo. Epitope identification, including molecular docking and experiment-based analysis of mutated species, revealed that AF-03 recognized the Niemann-Pick C1 (NPC1) binding domain within GP1. Interestingly, we found the neutralizing activity of AF-03 to pseudotyped Ebola viruses (EBOV, SUDV, and BDBV) harboring cleaved GP instead of full-length GP. Furthermore, NPC2-fused AF-03 exhibited neutralizing activity to several filovirus species and EBOV mutants via binding to CI-MPR. In conclusion, this work demonstrates that AF-03 represents a promising therapeutic cargo for filovirus-caused disease.

This study aimed to assess the anti-inflammatory properties of a bioactive glutamic-alanine rich glycoprotein (GP) derived from Undaria pinnatifida on both LPS-stimulated RAW264.7 cells, peritoneal macrophages, and mouse models of carrageenan- and xylene-induced inflammation, investigating the underlying molecular mechanisms. In both in-vitro and in-vivo settings, GP was found to reduce the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) while also inhibiting the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in response to lipopolysaccharide (LPS) stimulation. GP treatment significantly impeded the nuclear translocation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway by blocking the phosphorylation of IKKα and IκBα, leading to a reduction in proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). Additionally, GP effectively inhibited the activation of mitogen-activated protein kinases (MAPKs), with specific inhibitors of p38 and extra-cellular signal regulated kinase (ERK) enhancing GP’s anti-inflammatory efficacy. Notably, GP administration at 10 mg/kg/day (p.o.) markedly reduced carrageenan-induced paw inflammation and xylene-induced ear edema by preventing the infiltration of inflammatory cells into targeted tissues. GP treatment also downregulated key inflammatory markers, including iNOS, COX-2, IκBα, and NF-κB, by suppressing the phosphorylation of p38 and ERK, thereby improving the inflammatory index in both carrageenan- and xylene-induced mouse models. These findings suggest that marine resources, particularly seaweeds like U. pinnatifida, could serve as valuable sources of natural anti-inflammatory proteins for the effective treatment of inflammation and related conditions.

Abrin and ricin, both type II ribosome-inactivating proteins, are toxins of significant concern and are under international restriction by the Chemical Weapons Convention and the Biological and Toxin Weapons Convention. The development of a rapid and sensitive detection method for these toxins is of the utmost importance for the first emergency response. Emerging rapid detection techniques, such as surface-enhanced Raman spectroscopy (SERS) and lateral flow assay (LFA), have garnered attention due to their high sensitivity, good selectivity, ease of operation, low cost, and disposability. In this work, we generated stable and high-affinity nanotags, via an efficient freezing method, to serve as the capture module for SERS-LFA. We then constructed a sandwich-style lateral flow test strip using a pair of glycoproteins, asialofetuin and concanavalin A, as the core affinity recognition molecules, capable of trace measurement for both abrin and ricin. The limit of detection for abrin and ricin was 0.1 and 0.3 ng/mL, respectively. This method was applied to analyze eight spiked white powder samples, one juice sample, and three actual botanic samples, aligning well with cytotoxicity assay outcomes. It demonstrated good inter-batch and intra-batch reproducibility among the test strips, and the detection could be completed within 15 min, indicating the suitability of this SERS-LFA method for the on-site rapid detection of abrin and ricin toxins.

There are currently no prophylactic vaccines licensed to protect against Lassa fever caused by Lassa virus (LASV) infection. The Emergent BioSolutions (EBS) vaccine candidate, EBS-LASV, is being developed for the prevention of Lassa fever. EBS-LASV is a live-attenuated recombinant Vesicular Stomatitis Virus (rVSV)-vectored vaccine encoding the surface glycoprotein complex (GPC) from LASV and has two attenuating vector modifications: a gene shuffle of the VSV N gene and a deletion of the VSV G gene. Preclinical studies were performed to evaluate EBS-LASV’s neurovirulence potential following intracranial (IC) injection and to determine the biodistribution and vector replication following intramuscular (IM) inoculation in mice. In addition, the potential EBS-LASV toxicity was assessed using repeated-dose IM EBS-LASV administration to rabbits. All mice receiving the IC injection of EBS-LASV survived, while mice administered the unattenuated control vector did not. The vaccine was only detected in the muscle at the injection site, draining lymph nodes, and the spleen over the first week following IM EBS-LASV injection in mice, with no detectable plasma viremia. No toxicity was observed in rabbits receiving a three-dose regimen of EBS-LASV. These studies demonstrate that EBS-LASV is safe when administered to animals and supported a first-in-human dose-escalation, safety, and immunogenicity clinical study.

Tuberculosis (TB) is a major global health threat despite its virtual elimination in developed countries. Issues such as drug accessibility, emergence of multidrug-resistant strains, and limitations of the current BCG vaccine highlight the urgent need for more effective TB control measures. This study constructed BCG strains overexpressing Rv1002c and found that the rBCG-Rv1002c strain secreted more glycosylated proteins, significantly enhancing macrophage activation and immune protection against Mycobacterium tuberculosis (M. tb). These results indicate that Rv1002c overexpression promotes elevated levels of O-glycosylation in BCG bacteriophages, enhancing their phagocytic and antigenic presentation functions. Moreover, rBCG-Rv1002c significantly upregulated immune regulatory molecules on the macrophage surface, activated the NF-κB pathway, and facilitated the release of large amounts of NO and H2O2, thereby enhancing bacterial control. In mice, rBCG-Rv1002c immunization induced greater innate and adaptive immune responses, including increased production of multifunctional and long-term memory T cells. Furthermore, rBCG-Rv1002c-immunized mice exhibited reduced lung bacterial load and histological damage upon M. tb infection. This result shows that it has the potential to be an excellent candidate for a preventive vaccine against TB.

Glycoproteins, essential for cellular functions, contain monosaccharides like Levo-fucose, crucial for cell communication. Recent research highlights serum L-fucose as a potential biomarker for early detection of malignancies. Typically, serum L-fucose levels are low but rise with malignancy. This study evaluates serum L-fucose as an early biomarker in oral submucous fibrosis (OSMF) patients. Aim: Assess serum L-fucose's diagnostic potential for dysplasia in OSMF patients. Objectives: Determine the Association between Serum L Fucose Glycoprotein Levels and Dysplasia in OSF Patients.Evaluate the Diagnostic Accuracy of Serum L Fucose Glycoprotein as a Biomarker for OSF-Related Dysplasia. Methodology: Over a span of two years, this study encompassed 80 subjects, aged between 18 and 60 years, who were clinically and histopathologically identified as OSMF patients, with or without dysplastic alterations. From each participant, 5 ml of blood was collected. Following centrifugation to separate the serum, the samples were analyzed to determine the levels of Levo-fucose. Statistical analysis: Using SPSS (version 17.0), serum L-Fucose levels of the case group were compared to the control group using ANOVA. Frequencies were analyzed with the chi-square test, and Tukey’s Test was used for multiple comparisons. Significance was set at p

The effect of milk fat globule (MFG) size and species (sheep versus cow) on the lipid and protein compositions of sheep and cow milks was studied. The MFGs in raw cow and sheep milks were separated into six significantly different-sized (1.5–5.5 μm) groups by a gravity-based separation method, and their fatty acids, their lipidomes and the protein compositions of their MFG membranes were determined. The proportions of polar lipids increased but glycoproteins decreased with decreasing MFG size in both sheep milk and cow milk; the fatty acid composition showed few differences among the MFG groups. The average size of each MFG group was comparable between sheep milk and cow milk. Sheep milk contained higher proportions of short-chain fatty acids, medium-chain fatty acids and sphingomyelin than cow milk in all MFG groups. The proportion of glycoproteins was higher in cow MFG membrane than in sheep MFG membrane. The results suggested that the lipid and protein compositions were markedly species and size dependent.

While performed by all three domains of life, N-glycosylation in Archaea is less well described than are the parallel eukaryal and bacterial processes. Still, what is known of the archaeal version of this universal post-translational modification reveals numerous seemingly domain-specific traits. Specifically, the biosynthesis of archaeal N-linked glycans relies on distinct pathway steps and components, rare sugars and sugar modifications, as well as unique lipid carriers upon which N-linked glycans are assembled. At the same time, Archaea possess the apparently unique ability to simultaneously modify their glycoproteins with very different N-linked glycans. In addition to these biochemical aspects of archaeal N-glycosylation, such post-translational modification has been found to serve a wide range of roles possibly unique to Archaea, including allowing these microorganisms to not only cope with the harsh physical conditions of the niches they can inhabit but also providing the ability to adapt to transient changes in such environments.

Arabinogalactan-proteins (AGPs) are cell wall glycoproteins that make up a relatively small component of the extracellular matrix of plants yet have significant influence on wall mechanics and signalling. Present in walls of algae, bryophytes and angiosperms, AGPs have a wide range of functional roles, from signalling, cell expansion and division, embryogenesis, responses to abiotic and biotic stress, plant growth and development. AGPs interact with and influence wall matrix components and plasma membrane proteins to regulate developmental pathways and growth responses, yet the exact mechanisms remain elusive. Comprising a large gene family that is highly diverse, from minimally to highly glycosylated members, varying in their glycan heterogeneity, can be plasma membrane bound or secreted into the extracellular matrix, have members that are highly tissue specific to those with constitutive expression; all these factors have made it extremely challenging to categorise AGPs many qualities and roles. Here we attempt to define some key features of AGPs and their biological functions.

Salmonid novirhabdovirus (IHNV) causes infectious haematopoietic necrosis (IHN) in salmonid species. Despite an injectable plasmid-based DNA vaccine of the glycoprotein (G) gene is effective, there are no oral vaccines for mass vaccination of rainbow trout (Oncorhynchus mykiss) fry. Recombinant baculoviruses were generated, used in cabbage looper (Trichoplusia ni) insect larvae to produce IHNV G and IHNV G-C5a proteins. Western blotting and chemiluminescence assays confirmed the expression of recombinant proteins, which were added to the fish feeding and top-coated with unflavored gelatin binder. Commercial rainbow trout were fed with experimental diets containing either IHNV G or IHNV G-C5a proteins for 2 weeks, and boosted 4 weeks after. Four weeks post-booster, fish were challenged with IHNV by immersion. Survival upon the infection challenge was evaluated. Spleen were sampled at 7 and 14 days post infection (dpi). Non-vaccinated and IHNV G fed trout reached a mortality of 91.7 and 97.6%, and 70.9 and 88.4%, respectively at 8 and 15 dpi. The IHNV G-C5a fed group exhibited a reduced mortality of 51.2% at 8 dpi, reaching 81.7% at 15 dpi, suggesting some level of antiviral protection. The individual viral load was measured by RT-qPCR detection of IHNV N gene, showing no significant difference across experimental groups. The transcription modulation of selected immune response markers was evaluated across experimental groups, including Type I IFN-a, Mx-1, CD4, and IgM. Further study is needed to assess how new oral vaccines may become effective to mitigate IHNV pathogenesis in juvenile trout by modulating the host immune response to protect towards IHNV exposure.

Abstract Background Increased glucose uptake and utilization via aerobic glycolysis are among the most prominent hallmarks of tumor cell metabolism. Accumulating evidence suggests that similar metabolic changes are also triggered in many virus-infected cells. Viral propagation, like highly proliferative tumor cells, increases the demand for energy and macromolecular synthesis, leading to high bioenergetic and biosynthetic requirements. Although significant progress has been made in understanding the metabolic changes induced by viruses, the interaction between host cell metabolism and arenavirus infection remains unclear. Our study sheds light on these processes during lymphocytic choriomeningitis virus (LCMV) infection, a model representative of the Arenaviridae family. Methods The impact of LCMV on glucose metabolism in MRC-5 cells was studied using reverse transcription-quantitative PCR and biochemical assays. A focus-forming assay and western blot analysis were used to determine the effects of glucose deficiency and glycolysis inhibition on the production of infectious LCMV particles. Results Despite changes in the expression of glucose transporters and glycolytic enzymes, LCMV infection did not result in increased glucose uptake or lactate excretion. Accordingly, depriving LCMV-infected cells of extracellular glucose or inhibiting lactate production had no impact on viral propagation. However, treatment with the commonly used glycolytic inhibitor 2-deoxy-D-glucose (2-DG) profoundly reduced the production of infectious LCMV particles. This effect of 2-DG was further shown to be the result of suppressed N-linked glycosylation of the viral glycoprotein. Conclusions Although our results showed that the LCMV life cycle is not dependent on glucose supply or utilization, they did confirm the importance of N-glycosylation of LCMV GP-C. 2-DG potently reduces LCMV propagation not by disrupting glycolytic flux but by inhibiting N-linked protein glycosylation. These findings highlight the potential for developing new, targeted antiviral therapies that could be relevant to a wider range of arenaviruses.

In order to obtain high-quality Auricularia auricula glycoprotein and clarify its monosaccharide and amino acid composition, the process conditions of ultrasonic assisted alkaline extraction of Auricularia auricula glycoprotein were optimized by single factor experiment and response surface methodology, and the glycoprotein was separated and purified by AKTAgo purifier and DEAE GE Healthcare chromatographic column, and its monosaccharide and amino acid composition were analyzed by high performance liquid chromatography (HPLC). The optimum extraction conditions of Auricularia auricula glycoprotein were: ultrasonic time 90 min, material liquid ratio 1:90 g/L, ultrasonic power 200 W, pH8.4, extraction temperature 45 ℃, and the comprehensive score of glycoprotein extraction was 55.93. After separation and purification, the purity of the glycoprotein was 74.1% and the protein recovery rate was 37.5%. The monosaccharide composition of the glycoprotein was mainly glucose (9.95 mg/g) and mannose (5.24 mg/g), and the amino acids were mainly L-aspartic acid/L-asparagine (1.16 mg/g), L-phenylalanine (1.14 mg/g) and L-glutamic acid/L-glutamine (1.10 mg/g). Therefore, the ultrasonic alkali method can effectively improve the extraction efficiency of Auricularia auricula glycoprotein, and the Auricularia auricula glycoprotein contains acidic heteropolysaccharides and the necessary amino acids are complete, which provides a scientific reference for the in-depth research and application development of Auricularia auricula glycoprotein.

Abstract Equine coital rash (ECE) is a highly contagious benign infection that induces lesions on external genitals, and it is caused by the equine herpesvirus type 3 (EHV‐3). Although the disease is globally distributed, its presence in Chile has not been documented from a genetic point of view. Here, we performed polymerase chain reaction screenings for EHV‐3 in lesions of external genitals in four horses belonging to a riding station at Bulnes, Ñuble Region, Chile. We sequenced a fragment of the glycoprotein G (gG) gene from three horses with clinical signs of ECE. The sequences were identical between them and 99.7% similar to a haplotype of EHV‐3 detected in Brazil, and phylogenetically related with homologue from Japan, Russia and Brazil. Our results show the presence of EHV‐3 for the first time in horses with ECE in Chile.

Abstract Phaeodactylum tricornutum (Pt) is a critical microbial cell factory to produce a wide spectrum of marketable products including recombinant biopharmaceutical N-glycoproteins. N-glycosylation modification of proteins is important for their activity, stability, and half-life, especially some special modifications, such as fucose-modification by fucosyltransferase (FucT). Three PtFucTs were annotated in the genome of P. tricornutum, PtFucT1 was located on the medial/trans-Golgi apparatus and PtFucT2-3 in the plastid stroma. Algal growth, biomass and photosynthesis efficiency were significantly inhibited in a knockout mutant of PtFucT1 (PtFucT1-KO). PtFucT1 played a role in non-core fucose modification of N-glycans. The knockout of PtFucT1 might affect the activity of PtGnTI in the complex and change the complex N-glycan to mannose type N-glycan. The study provided critical information for understanding the mechanism of protein N-glycosylation modification and using microalgae as an alternative ecofriendly cell factory to produce biopharmaceuticals.

The swiftlet’s nest is an edible bird’s nest (EBN) produced from dried gelatinized saliva secreted by swiftlets during the breeding season. It is widely used in industries for its high nutritional values and health benefits. However, the utilization of ESN is restricted due to its physicochemical properties, such as insolubility. The recently invented bioactive ESN hydrolyzate is reported to improve the ESN’s solubility, functional and nutritional properties to broaden its application. This study has produced bioactive ESN hydrolyzates with different hydrolysis periods to investigate the optimum conditions to achieve maximized degree of hydrolysis (DH) and to compare the physicochemical differences. The result revealed that optimum ESN hydrolysis was at 90 minutes, in which the DH and enhanced solubility (three folds) indicated a complete breakdown of glycoprotein into bioactive glycopeptide. This result is in line with the physicochemical analyses result. The hydrolysis has significantly decreased (p ≤ .05) the ESN protein content, while the levels for peptide, glycopeptide, polysaccharides, and sialic acid showed the opposite. Interestingly, the amino acid from raw ESN and hydrolyzates in all incubation periods showed no significant difference (p ≤ .05). This result suggests that the ESN biological properties remained unchanged with alcalase hydrolysis. Therefore, the findings in this study confirm the feasibility of bioactive ESN hydrolyzate as a versatile product suitable for various industries in utilizing ESN in whole instead of as extracts.

Three or four intramuscular doses of the inactivated human rabies virus vaccines are needed for pre- or post-exposure prophylaxis in humans. This procedure has made a great contribution to prevent human rabies deaths, which bring huge economic burdens in developing countries. Herein, a recombinant adeno-associated virus serotype 9, AAV9-RABVG, harbouring a RABV G gene, was generated to serve as a single dose rabies vaccine candidate. The RABV G protein was stably expressed in the 293T cells infected with AAV9-RABVG. A single dose of 2 × 1011 v.p. of AAV9-RABVG induced robust and long-term positive seroconversions in BALB/c mice with a 100% survival from a lethal RABV challenge. In Cynomolgus Macaques vaccinated with a single dose of 1 × 1013 v.p. of AAV9-RABVG, the titres of rabies VNAs increased remarkably from 2 weeks after immunity, and maintained over 31.525 IU/ml at 52 weeks. More DCs were activated significantly for efficient antigen presentations of RABV G protein, and more B cells were activated to be responsible for antibody responses. Significantly more RABV G specific IFN-γ-secreting CD4+ and CD8+ T cells, and IL-4-secreting CD4+ T cells were activated, and significantly higher levels of IL-2, IFN-γ, IL-4, and IL-10 were secreted to aid immune responses. Overall, the AAV9-RABVG was a single dose rabies vaccine candidate with great promising by inducing robust, long-term humoral responses and both Th1 and Th2 cell-mediated immune responses in mice and non-human primates.

Zika virus (ZIKV), a family member in the Flavivirus genus, has re-emerged as a global public health concern. The envelope (E) proteins of flaviviruses play a dual role in viral assembly and entry. To identify the key residues of E in virus entry, we generated a ZIKV trans-complemented particle (ZIKVTCP) system, in which a subgenomic reporter replicon was packaged by trans-complementation with expression of CprME. We performed mutagenesis studies of the loop regions that protrude from the surface of the virion in the E ectodomains (DI, DII, DIII). Most mutated ZIKVTCPs exhibited deficient egress. Mutations in DII and in the hinge region of DI and DIII affected prM expression. With a bioorthogonal system, photocrosslinking experiments identified crosslinked intracellular E trimers and demonstrated that egress-deficient mutants in DIII impaired E trimerization. Of these mutants, an E-trimerization-dead mutation D389A that nears the E-E interface between two neighbouring spikes in the immature virion completely abolished viral egress. Several mutations abolished ZIKVTCPs’ entry, without severely affecting viral egress. Further virus binding experiments demonstrated a deficiency of the mutated ZIKVTCPs in virus attachment. Strikingly, synthesized peptide containing residues of two mutants (268-273aa in DII) could bind to host cells and significantly compete for viral attachment and interfere with viral infection, suggesting an important role of these resides in virus entry. Our findings uncovered the requirement for DIII mediated-E trimerization in viral egress, and discovered a key residue group in DII that participates in virus entry.

Rabies is a zoonotic infectious disease with a high fatality rate. It is caused by a virus in the genus Lyssavirus and is a global public health threat. The rabies virus invades and infects cells mainly via a glycoprotein, which may involve multiple receptors. Neutralizing antibodies against the rabies virus function by blocking the binding of the glycoprotein to a receptor or preventing the membrane fusion process. Vaccination combined with anti-rabies virus neutralizing antibodies is essential for postexposure prophylaxis for category III exposure to the rabies virus. In this review, we discussed the neutralizing epitopes of the rabies virus and the neutralization mechanism of monoclonal antibodies. The neutralizing antibodies that have been commercialized or are under development are also summarized. Our review would provide a basis for the further development of safe and effective broad-spectrum neutralizing antibodies to replace the rabies virus immunoglobulin in rabies post-exposure prophylaxis.

Subunit vaccines based on recombinant viral antigens are valuable interventions to fight existing and evolving viruses and can be produced at large-scale in plant-based expression systems. The recombinant viral antigens are often derived from glycosylated envelope proteins of the virus and glycosylation plays an important role for the immunogenicity by shielding protein epitopes. The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a principal target for vaccine development and has been produced in plants, but the yields of recombinant RBD variants were low and the role of the N-glycosylation in RBD from different SARS-CoV-2 variants of concern is less studied. Here, we investigated the expression and glycosylation of six different RBD variants transiently expressed in leaves of Nicotiana benthamiana. All of the purified RBD variants were functional in terms of receptor binding and displayed almost full N-glycan occupancy at both glycosylation sites with predominately complex N-glycans. Despite the high structural sequence conservation of the RBD variants, we detected a variation in yield which can be attributed to lower expression and differences in unintentional proteolytic processing of the C-terminal polyhistidine tag used for purification. Glycoengineering towards a human-type complex N-glycan profile with core α1,6-fucose, showed that the reactivity of the neutralizing antibody S309 differs depending on the N-glycan profile and the RBD variant.

ABSTRACT Increased viral surveillance has led to the isolation and identification of numerous uncharacterized paramyxoviruses, rapidly expanding our understanding of paramyxoviral diversity beyond the bounds of known genera. Despite this diversity, a key feature that unites paramyxoviruses is the presence of a receptor-binding protein (RBP), which facilitates host-cell attachment and plays a fundamental role in determining host range. Here, we study the RBP presented on the surface of rodent-borne paramyxoviruses Mossman and Nariva (MosV and NarV, respectively), viruses that constitute founding members of the recently defined Narmovirus genus within the Paramyxoviridae family. Crystallographic analysis of the C-terminal head region of the dimeric MosV and NarV RBPs demonstrates that while these glycoproteins retain the canonical six-bladed β-propeller fold found in other paramyxoviral RBPs, they lack the structural motifs associated with established paramyxovirus host-cell receptor entry pathways. Consistent with MosV-RBP and NarV-RBP undergoing a distinct entry pathway from other characterized paramyxoviruses, structure-based phylogenetic analysis demonstrates that these six-bladed β-propeller head domains form a singular structural class that is distinct from other paramyxoviral RBPs. Additionally, using an integrated crystallographic and small-angle X-ray scattering analysis, we confirm that MosV-RBP and NarV-RBP form homodimeric arrangements that are distinct from those adopted by other paramyxovirus RBPs. Altogether, this investigation provides a molecular-level blueprint of the narmovirus RBP that broadens our understanding of the structural space and functional diversity available to paramyxovirus RBPs. IMPORTANCE Genetically diverse paramyxoviruses are united in their presentation of a receptor-binding protein (RBP), which works in concert with the fusion protein to facilitate host-cell entry. The C-terminal head region of the paramyxoviral RBP, a primary determinant of host-cell tropism and inter-species transmission potential, forms structurally distinct classes dependent upon protein and glycan receptor specificity. Here, we reveal the architecture of the C-terminal head region of the RBPs from Nariva virus (NarV) and Mossman virus (MosV), two archetypal rodent-borne paramyxoviruses within the recently established genus Narmovirus, family Paramyxoviridae. Our analysis reveals that while narmoviruses retain the general architectural features associated with paramyxoviral RBPs, namely, a six-bladed β-propeller fold, they lack the structural motifs associated with known receptor-mediated host-cell entry pathways. This investigation indicates that the RBPs of narmoviruses exhibit pathobiological features that are distinct from those of other paramyxoviruses.

ABSTRACT In order to produce therapeutic glycoproteins modified with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. In this study, a Pichia pastoris mutant Glyco4, which has a humanized N-glycosylation pathway and could successfully generate the human-like N-glycans, was carefully characterized. Glyco4 displays a significant growth delay and cell wall defects. Comparative transcriptomics reveals that manipulating the N-glycosylation pathway could notably affect the expression pattern of numerous biological pathways in Glyco4. Among the differentially expressed genes, the down-regulation of PpSPI1 was proven to be the main cause of the cell wall defects in Glyco4. Deletion of PpSPI1 in P. pastoris GS115 strain presented growth delay and weaker resistance to cell wall/membrane perturbing agents. PpSpi1 was shown to be a glycosylphosphatidylinositol-anchored cell wall glycoprotein and involved in the formation of the mannoprotein layer on the outer surface of cell wall. Overexpressing PpSPI1 in Glyco4 could partially recover the cell wall defects and also improve its resistance to cell wall perturbing agents and osmotic stress. Thus, overexpression of PpSpi1 is a useful strategy to facilitate P. pastoris industrial applications in the manufacture of human glycoproteins. IMPORTANCE Engineering of biological pathways in various microorganisms is a promising direction for biotechnology. Since the existing microbial cells have evolved over a long period of time, any artificial engineering may cause some unexpected and harmful effects on them. Systematically studying and evaluating these engineered strains are very important and necessary. In order to produce therapeutic proteins with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. The properties of a P. pastoris strain with humanized N-glycosylation machinery were carefully evaluated in this study. Our work has identified a key glycoprotein (PpSpi1) responsible for the poor growth and morphological defects of this glycoengineered strain. Overexpression of PpSpi1 could significantly rescue the growth defect of the glycoengineered P. pastoris and facilitate its future industrial applications.