Acid Glycan Research Articles

Structural Characterization of Human Bufavirus 1: Receptor Binding and Endosomal pH-Induced Changes.

Bufaviruses (BuV) are members of the Parvoviridae of the Protoparvovirus genus. They are non-enveloped, T = 1 icosahedral ssDNA viruses isolated from patients exhibiting acute diarrhea. The lack of treatment options and a limited understanding of their disease mechanisms require studying these viruses on a molecular and structural level. In the present study, we utilize glycan arrays and cell binding assays to demonstrate that BuV1 capsid binds terminal sialic acid (SIA) glycans. Furthermore, using cryo-electron microscopy (cryo-EM), SIA is shown to bind on the 2/5-fold wall of the capsid surface. Interestingly, the capsid residues stabilizing SIA binding are conserved in all human BuVs identified to date. Additionally, biophysical assays illustrate BuV1 capsid stabilization during endo-lysosomal (pH 7.4-pH 4) trafficking and capsid destabilization at pH 3 and less, which correspond to the pH of the stomach. Hence, we determined the cryo-EM structures of BuV1 capsids at pH 7.4, 4.0, and 2.6 to 2.8 Å, 3.2 Å, and 2.7 Å, respectively. These structures reveal capsid structural rearrangements during endo-lysosomal escape and provide a potential mechanism for this process. The structural insights gained from this study will add to the general knowledge of human pathogenic parvoviruses. Furthermore, the identification of the conserved SIA receptor binding site among BuVs provides a possible targetable surface-accessible pocket for the design of small molecules to be developed as anti-virals for these viruses.

The gut microbial differences between pre-released and wild red deer: Firmicutes abundance may affect wild adaptation after release.

Insufficient density of red deer has affected the stability of forest ecosystems and the recovery of large carnivores (represented by Amur tiger). Conservation translocations from captivity to the wild has become an important way to restore the red deer populations. However, the difference in gut microbes between pre-release and wild red deer may affect the feeding adaptability of red deer after release. In this study, we clarified the differences in gut microbes between pre-released and wild red deer and screened the key gut microbes of the red deer involved in feeding by using metagenomic sequencing and feeding analysis. The results showed that the microbial difference between pre-released and wild red deer was mainly related to Firmicutes represented by Eubacteriales and Clostridia, and Firmicutes abundance in pre-released red deer (68.23%) was significantly lower than that of wild red deer (74.91%, p < 0.05). The expression of microbial metabolic pathways in pre-released red deer were significantly lower than those in wild red deer (p < 0.05), including carbohydrate metabolism, amino acid metabolism, glycan biosynthesis and metabolism, etc. The combinations of Firmicutes were significantly positively correlated with the intake of plant fiber and carbohydrate (p < 0.05), and were key microbes to help red deer deal with wild plant resources. Additionally, the combinations of Firmicutes represented by Eubacteriales and Clostridia lacking in pre-released red deer contributed the most to expression of microbial metabolic pathways (importance > 1), showing a significant positive correlation (p < 0.05). This study indicates that high abundance of Firmicutes is an important guarantee for red deer to adapt to the wild feeding environment, which provides critical implications for the recovery of red deer populations and the protection of endangered ungulates.

Fingerprint profiling for quality evaluation and the related biological activity analysis of polysaccharides from Liuweizhiji Gegen-Sangshen beverage.

Liuweizhiji Gegen-Sangshen beverage (LGS) is popular in China, which has been used for alleviating alcohol-mediated discomfort and preventing alcoholic liver disease (ALD). This beverage is consisted of six herbal components that are known as functional foods and fruits. LGS is rich in polysaccharides, however, the activity and quality evaluation of LGS-derived polysaccharides remain unexplored. The purpose of this study is thus to establish a comprehensive quality control methodology for the assessment of LGS polysaccharides (LGSP) and to further explore the anti-oxidant, anti-inflammatory as well as prebiotic effect of LGSP. LGSP was extracted, followed by analysis of molecular weight distribution, monosaccharide content and structural characterization via integrating the application of high-performance size exclusion chromatography (HPSEC), 1-phenyl-3-methyl-5-pyrazolone-HPLC (PMP-HPLC), fourier transform infrared spectroscopy (FT-IR) as well as nuclear magnetic resonance spectroscopy (NMR) techniques. The anti-oxidation activity of LGSP was determined by DPPH, ABTS, hydroxyl radical scavenging capacity and total antioxidant capacity. The anti-inflammation of LGSP were assessed on the RAW 264.7 cells. The effect of LGSP on growth of Lactobacillus, Bifidobacterium bifidum and Bifidobacterium adolescentis was evaluated. The results demonstrated that LGSP had two molecular weight distribution peaks, with the average molecular weights of (6.569 ± 0.12) × 104 Da and (4.641 ± 0.30) × 104 Da. LGSP was composed of 8 monosaccharides, with galacturonic acid, glucose rhamnose and galactose representing the highest molar ratios. Homogalacturonic acid (HG) type and rhamnosegalacturonic acid glycans I (RG-I) type and α-1,4-glucan were present in LGSP. LGSP concentration in LGS was 17.94 ± 0.28 mg/mL. Furthermore, fingerprint analysis combined with composition quantification of 10 batches of LGSP demonstrated that there was a high similarity among batches. Notably, LGSP exhibited anti-oxidant effect and inhibited expressions of pro-inflammatory factors (TNF-α and IL-6) in LPS-stimulated RAW 264.7 cells. In addition, LGSP remarkably promoted the proliferation of probiotics Lactobacillus, Bifidobacterium bifidum and Bifidobacterium adolescentis, showing good prebiotic activity. The results of present study would be of help to gain the understanding of structure-activity relationship of LGSP, provide a reference for quality evaluation of bioactive LGSP, and facilitate development of unique health and functional products in the future.

Purification of an Acidic Polysaccharide with Anticoagulant Activity from the Marine Sponge Sarcotragus spinosulus.

Thromboembolic conditions are the most common cause of death in developed countries. Anticoagulant therapy is the treatment of choice, and heparinoids and warfarin are the most adopted drugs. Sulphated polysaccharides extracted from marine organisms have been demonstrated to be effective alternatives, blocking thrombus formation by inhibiting some factors involved in the coagulation cascade. In this study, four acidic glycan fractions from the marine sponge Sarcotragus spinosulus were purified by anion-exchange chromatography, and their anticoagulant properties were investigated through APTT and PT assays and compared with both standard glycosaminoglycans and holothurian sulphated polysaccharides. Moreover, their topographic localization was assessed through histological analysis, and their cytocompatibility was tested on a human fibroblast cell line. A positive correlation between the amount of acid glycans and the inhibitory effect towards both the intrinsic and extrinsic coagulation pathways was observed. The most effective anticoagulant activity was shown by a highly charged fraction, which accounted for almost half (about 40%) of the total hexuronate-containing polysaccharides. Its preliminary structural characterization, performed through infrared spectroscopy and nuclear magnetic resonance, suggested that it may consist of a fucosylated chondroitin sulphate, whose unique structure may be responsible for the anticoagulant activity reported herein for the first time.

Aluminum exposure alters the pedal mucous secretions of the chocolate-band snail, Eobania vermiculata (Gastropoda: Helicidae).

Aluminum (Al) is used in everyday life and present in food drugs, packaging, industry, and agriculture. Although it is the most common metal in the Earth crust, a correlation has been demonstrated between its presence and various pathologies, even serious ones, especially of a neurological type. However, there is a histological gap regarding the role Al can have in contact with the covering and secreting epithelia. The alterations of the ventral and dorsal foot mucocytes and their secretions of the snail Eobania vermiculata caused by Al were investigated in situ by histochemical and lectin-histochemical techniques. Administration to different experimental groups took place for 3 and 9 days with 50 and 200 μM of AlCl3. Several types of mucocytes were detected with a prevalent secretion of acid glycans in the foot of E. vermiculata. Sulfated glycans prevail in the dorsal region, with one type showing only fucosylated residues and another also having galactosaminylated and glycosaminylated residues. Carboxylated glycans prevail in the ventral region, with presence of galactosaminylated, glycosaminylated, and fucosylated residuals in both cells. Snails treated presented a general decrease of mucin amount in the secreting cells and affected the mucus composition. These changes could alter the rheological and functional properties of the mucus with possible implications for the health of the treated animals. RESEARCH HIGHLIGHTS: Snails were fed with Al-contaminated lettuce at different concentrations. In the foot mucocytes produced mucus with prevailing acidic glycans. In the treated resulted a reduction in the amount of mucus and an alteration of glycan composition.

Succinylation-Alcian Blue Staining of Mucins on Polyvinylidene Difluoride Membrane.

Mucins are often stained with the basic dye Alcian blue, but mucins with a low acidic glycan content cannot be stained with it. Succinylation-Alcian blue staining is a method that temporarily modifies glycans with succinic acid to visualize mucins with low acidic glycan content. This method can be used to stain mucins on polyvinylidene difluoride (PVDF) membranes separated via supported molecular matrix electrophoresis (SMME) and mucins blotted onto PVDF membranes from gel electrophoreses. The succinyl groups of the modified glycans can be easily and completely removed by releasing O-glycan from the stained mucin bands. Therefore, the glycans can be analyzed using the same methods as those used for mucins with a high acidic glycan content.

Novel Selective Quantification of Zinpentraxin Alfa Biotherapeutic in the Presence of Endogenous Isomer in Plasma Samples of Idiopathic Pulmonary Fibrosis Patients Using Immunoaffinity LC–MS

Idiopathic pulmonary fibrosis (IPF) is a progressive fatal interstitial lung disease that affects three million patients worldwide and currently without an effective cure. Zinpentraxin alfa, a recombinant human pentraxin-2 (rhPTX-2) protein, has been evaluated as a potential drug candidate for the treatment of IPF. Clinical pharmacokinetic analysis of zinpentraxin alfa has been challenging historically due to interference from serum amyloid P component (SAP), an endogenous human pentraxin-2 protein. These molecules share an identical primary amino acid sequence and glycan composition; however, zinpentraxin alfa possesses α2,3-linked terminal sialic acid residues while SAP is an α2,6-linked isomer. By taking advantage of this only structural difference, we developed a novel assay strategy where α2,3-sialidase was used to selectively hydrolyze α2,3-linked sialic acid residues, resulting in desialylated zinpentraxin alfa versus unchanged sialylated SAP, following an immunoaffinity capture step. Subsequent tryptic digestion produced a unique surrogate asialo-glycopeptide from zinpentraxin alfa and allowed specific quantification of the biotherapeutic in human plasma. In addition, a common peptide shared by both molecules was selected as a surrogate to determine total hPTX-2 concentrations, i.e., sum of zinpentraxin alfa and SAP. The quantification methods for both zinpentraxin alfa and total hPTX-2 were validated and used in pharmacokinetic assessment in IPF patients. The preliminary results suggest that endogenous SAP levels remained largely constant in IPF patients throughout the treatment with zinpentraxin alfa. Our novel approach provides a general bioanalytical strategy to selectively quantify α2,3-sialylated glycoproteins in the presence of their corresponding α2,6-linked isomers.Graphical

Metagenomics-based exploration of key soil microorganisms contributing to continuously planted Casuarina equisetifolia growth inhibition and their interactions with soil nutrient transformation.

Casuarina equisetifolia (C. equisetifolia) is an economically important forest tree species, often cultivated in continuous monoculture as a coastal protection forest. Continuous planting has gradually affected growth and severely restricted the sustainable development of the C. equisetifolia industry. In this study, we analyzed the effects of continuous planting on C. equisetifolia growth and explored the rhizosphere soil microecological mechanism from a metagenomic perspective. The results showed that continuous planting resulted in dwarfing, shorter root length, and reduced C. equisetifolia seedling root system. Metagenomics analysis showed that 10 key characteristic microorganisms, mainly Actinoallomurus, Actinomadura, and Mycobacterium, were responsible for continuously planted C. equisetifolia trees. Quantitative analysis showed that the number of microorganisms in these three genera decreased significantly with the increase of continuous planting. Gene function analysis showed that continuous planting led to the weakening of the environmental information processing-signal transduction ability of soil characteristic microorganisms, and the decrease of C. equisetifolia trees against stress. Reduced capacity for metabolism, genetic information processing-replication and repair resulted in reduced microbial propagation and reduced microbial quantity in the rhizosphere soil of C. equisetifolia trees. Secondly, amino acid metabolism, carbohydrate metabolism, glycan biosynthesis and metabolism, lipid metabolism, metabolism of cofactors and vitamins were all significantly reduced, resulting in a decrease in the ability of the soil to synthesize and metabolize carbon and nitrogen. These reduced capacities further led to reduced soil microbial quantity, microbial carbon and nitrogen, microbial respiration intensity, reduced soil enzyme nutrient cycling and resistance-related enzyme activities, a significant reduction in available nutrient content of rhizosphere soils, a reduction in the ion exchange capacity, and an impediment to C. equisetifolia growth. This study provides an important basis for the management of continuously planted C. equisetifolia plantations.

Dissecting the Heterogeneous Glycan Profiles of Recombinant Coronavirus Spike Proteins with Individual Ion Mass Spectrometry.

Surface-embedded glycoproteins, such as the spike protein trimers of coronaviruses MERS, SARS-CoV, and SARS-CoV-2, play a key role in viral function and are the target antigen for many vaccines. However, their significant glycan heterogeneity poses an analytical challenge. Here, we utilized individual ion mass spectrometry (I2MS), a multiplexed charge detection measurement with similarities to charge detection mass spectrometry (CDMS), in which a commercially available Orbitrap analyzer is used to directly produce mass profiles of these heterogeneous coronavirus spike protein trimers under native-like conditions. Analysis by I2MS shows that glycosylation contributes to the molecular mass of each protein trimer more significantly than expected by bottom-up techniques, highlighting the importance of obtaining complementary intact mass information when characterizing glycosylation of such heterogeneous proteins. Enzymatic dissection to remove sialic acid or N-linked glycans demonstrates that I2MS can be used to better understand the glycan profile from a native viewpoint. Deglycosylation of N-glycans followed by I2MS analysis indicates that the SARS-CoV-2 spike protein trimer contains glycans that are more difficult to remove than its MERS and SARS-CoV counterparts, and these differences are correlated with solvent accessibility. I2MS technology enables characterization of protein mass and intact glycan profile and is orthogonal to traditional mass analysis methods such as size exclusion chromatography-multiangle light scattering (SEC-MALS) and field flow fractionation-multiangle light scattering (FFF-MALS). An added advantage of I2MS is low sample use, requiring 100-fold less than other methodologies. This work highlights how I2MS technology can enable efficient development of vaccines and therapeutics for pharmaceutical development.

The pan-cancer multi-omics landscape of key genes of sialylation combined with RNA-sequencing validation

BackgroundSialylation, the process of salivary acid glycan synthesis, plays a pivotal function in tumor growth, immune escape, tumor metastasis, and resistance to drugs. However, the association between sialylation and prognosis, tumor microenvironment (TME), and treatment response in a variety of cancers remains unclear. MethodsA comprehensive survey of the expression profile, prognostic value, and genetic and epigenetic alterations of sialylation-related genes was performed in pan-cancer. Subsequently, the single-sample gene set enrichment analysis (ssGSEA) algorithm was used to compute sialylation pathway scores in pan-cancer. Correlations of sialylation pathway scores with clinical features, prognosis, and TME were evaluated using multiple algorithms. Finally, the efficacy of the sialylation pathway score in determining the effect of immunotherapy was evaluated. The expression of sialylation-related genes were verified by RNA-sequencing. ResultsSignificant differences were observed in sialylation-related genes expression between tumors and adjacent normal tissues for most cancer types. Sialylation pathway scores differed according to the type of tumor, where the poor prognosis was correlated with high sialylation pathway scores in uveal melanoma (UVM) and pancreatic adenocarcinoma (PAAD). In addition, sialylation pathway scores were positively associated with the ImmuneScore, StromalScore and immune-related pathways. Moreover, the level of immune cells infiltration was higher in tumors with higher sialylation pathway scores. Finally, patients with high sialylation pathway scores were more sensitive to immunotherapy. ConclusionSialylation-related genes are essential in pan-cancer. The sialylation pathway score may be used as a biomarker in oncology patients.

Changes in the structure of polysaccharides under different extraction methods

Changes in the structure of polysaccharides under different extraction methods

Site-Specific Antibody Conjugation with Payloads beyond Cytotoxins.

As antibody-drug conjugates have become a very important modality for cancer therapy, many site-specific conjugation approaches have been developed for generating homogenous molecules. The selective antibody coupling is achieved through antibody engineering by introducing specific amino acid or unnatural amino acid residues, peptides, and glycans. In addition to the use of synthetic cytotoxins, these novel methods have been applied for the conjugation of other payloads, including non-cytotoxic compounds, proteins/peptides, glycans, lipids, and nucleic acids. The non-cytotoxic compounds include polyethylene glycol, antibiotics, protein degraders (PROTAC and LYTAC), immunomodulating agents, enzyme inhibitors and protein ligands. Different small proteins or peptides have been selectively conjugated through unnatural amino acid using click chemistry, engineered C-terminal formylglycine for oxime or click chemistry, or specific ligation or transpeptidation with or without enzymes. Although the antibody protamine peptide fusions have been extensively used for siRNA coupling during early studies, direct conjugations through engineered cysteine or lysine residues have been demonstrated later. These site-specific antibody conjugates containing these payloads other than cytotoxic compounds can be used in proof-of-concept studies and in developing new therapeutics for unmet medical needs.

3D bioprinted colorectal cancer models based on hyaluronic acid and signalling glycans

In cancer microenvironment, aberrant glycosylation events of ECM proteins and cell surface receptors occur. We developed a protocol to generate 3D bioprinted models of colorectal cancer (CRC) crosslinking hyaluronic acid and gelatin functionalized with three signalling glycans characterized in CRC, 3′-Sialylgalactose, 6′-Sialylgalactose and 2′-Fucosylgalactose. The crosslinking, performed exploiting azide functionalized gelatin and hyaluronic acid and 4arm-PEG-dibenzocyclooctyne, resulted in biocompatible hydrogels that were 3D bioprinted with commercial CRC cells HT-29 and patient derived CRC tumoroids. The glycosylated hydrogels showed good 3D printability, biocompatibility and stability over the time. SEM and synchrotron radiation SAXS/WAXS analysis revealed the influence of glycosylation in the construct morphology, whereas MALDI-MS imaging showed that protein profiles of tumoroid cells vary with glycosylation, indicating that sialylation and fucosylation of ECM proteins induce diverse alterations to the proteome of the tumoroid and surrounding cells.

Applying imaging mass spectrometry to define the N-glycan profiles of co-localized virus and immune cell infiltrates in post-COVID-19 infected lung autopsy tissues

The current COVID-19 pandemic is characterized by a broad range of disease severity in patients. This diversity in clinical manifestations has complicated our understanding of the SARS-CoV-2 pathogenesis and highlights the significance of an individual’s ability to mount an effective viral immune response. Glycosylation is a common post-translational modification occurring in complex organisms and is imperative for immune cell function. In this study, a combination approach with immunohistochemistry (IHC) and matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was utilized to determine the spatial distribution of N-glycans and immune cell populations in COVID-19 lung tissues. Tissues from seven SARS-CoV-2, PCR + donors were analyzed. Tissues represented a spectrum of time spent on ventilators which was reflected in their respective viral infection status and lung pathologies. N-glycan distributions in the MALDI-IMS images were then correlated with H&amp;amp;E staining and IHC of SARS-CoV-2 spike protein, CD4, CD8, CD163 and CD11b. Distinct and shared N-glycan signatures were identified in association with specific immune cell types, and their co-localization with the viral spike protein. Additionally, we observed unique patterns of α2,3-linked and α2,6-linked sialic acid glycans that associated with both immune cell populations and fibrotic regions within the tissue architecture. N-glycan MALDI-IMS is an effective tool to further understand tissue-localized immune cell populations in response to emerging viral pathogens such as SARS-CoV-2.

Microbiome-metabolome analysis reveals alterations in the composition and metabolism of caecal microbiota and metabolites with dietary Enteromorpha polysaccharide and Yeast glycoprotein in chickens.

The intestinal microbiome is responsible for the fermentation of complex carbohydrates and orchestrates the immune system through gut microbiota-derived metabolites. In our previous study, we reported that supplementation of Enteromorpha polysaccharide (EP) and yeast glycoprotein (YG) in combination synergistically improved antioxidant activities, serum lipid profile, and fatty acid metabolism in chicken. However, the mechanism of action of these polysaccharides remains elusive. The present study used an integrated 16S-rRNA sequencing technology and untargeted metabolomics technique to reveal the mechanism of action of EP+YG supplementation in broiler chickens fed basal diet or diets supplemented with EP+YG (200mg/kg EP + 200mg/kg YG). The results showed that EP+YG supplementation altered the overall structure of caecal microbiota as evidenced by β diversities analysis. Besides, EP+YG supplementation changed the microbiota composition by altering the community profile at the phylum and genus levels. Furthermore, Spearman correlation analysis indicated a significant correlation between altered microbiota genera vs serum cytokine levels and microbiota genera vs volatile fatty acids production. Predicted functional analysis showed that EP+YG supplementation significantly enriched amino acid metabolism, nucleotide metabolism, glycan biosynthesis and metabolism, energy metabolism, and carbohydrate metabolism. Metabolomics analysis confirmed that EP+YG supplementation modulates a myriad of caecal metabolites by increasing some metabolites, including pyruvic acid, pyridoxine, spermidine, spermine, and dopamine, and decreasing metabolites related to lipid metabolisms such as malonic acid, oleic acid, and docosahexaenoic acid. The quantitative enrichment analysis results further showed that glycolysis/gluconeogenesis, citric acid cycle, tyrosine metabolism, glycine, serine, and threonine metabolism, and cysteine and methionine metabolism were the most important enriched pathways identified with enrichment ratio >11, whereas, fatty acid biosynthesis and biosynthesis of unsaturated fatty acids pathways were suppressed. Together, the 16S-rRNA and untargeted metabolomics results uncovered that EP+YG supplementation modulates intestinal microbiota and their metabolites, thereby influencing the important metabolism pathways, suggesting a potential feed additive.

Aeromonas salmonicida binds α2-6 linked sialic acid, which is absent among the glycosphingolipid repertoires from skin, gill, stomach, pyloric caecum, and intestine

ABSTRACT Carbohydrates can both protect against infection and act as targets promoting infection. Mucins are major components of the slimy mucus layer covering the fish epithelia. Mucins can act as decoys for intimate pathogen interaction with the host afforded by binding to glycosphingolipids in the host cell membrane. We isolated and characterized glycosphingolipids from Atlantic salmon skin, gill, stomach, pyloric caeca, and intestine. We characterized the glycosphingolipids using liquid chromatography – mass spectrometry and tandem mass spectrometry and the glycan repertoire was compared with the glycan repertoire of mucins from the same epithelia. We also investigated Aeromonas salmonicida binding using chromatogram and microtiter well based binding assays. We identified 29 glycosphingolipids. All detected acid glycans were of the ganglio-series (unless shorter) and showed a high degree of polysialylation. The non-acid glycans were mostly composed of the neolacto, globo, and ganglio core structures. The glycosphingolipid repertoire differed between epithelia and the proportion of the terminal moieties of the glycosphingolipids did not reflect the terminal moieties on the mucins from the same epithelia. A. salmonicida did not bind the Atlantic salmon glycosphingolipids. Instead, we identified that A. salmonicida binding to sialic acid occurred to α2–6 Neu5Ac but not to α2–3 Neu5Ac. α2–6 Neu5Ac was present on mucins whereas mainly α2–3 Neu5Ac was found on the glycosphingolipids, explaining the difference in A. salmonicida binding ability between these host glycoconjugates. A. salmonicida´s ability to bind to Atlantic salmon mucins, but not the glycosphingolipids, is likely part of the host defence against this pathogen.

Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping

The evaluation of the protein glycosylation states of samples of aflibercept obtained from three different regions was conducted by site-specific N-linked glycan microheterogeneity profiling. Glycopeptide-based nano-LC MSMS mapping of tryptic-digested samples of each aflibercept lot provided site-specific information about glycan microheterogeneity on each of the five N-glycosylation sites (two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region). Next, the glycopeptide-mapping results obtained from the three different aflibercept lots were compared to evaluate the similarity between the samples. Three aflibercept lots showed a high degree of similarity in glycan composition, fucosylation level, sialylation level, and branching, when all five N-glycosylation sites were assessed together as a group. On the other hand, noticeable variations between lots in the glycan types and sialylation levels on the two sites of the VEGFR-2 domain were observed when each of the five N-glycosylation sites were assessed using the glycopeptide-based method. The presence of N-glycolylneuraminic acid (NeuGc) glycans, which may mediate adverse immune reactions in antibody therapeutics, were also detected on the sites of VEGFR1 and VEGFR2 domains, but not on the IgG Fc domain site. These results imply that analyses of the glycosylation profiles of fusion proteins containing multiple N-glycosylation sites, such as aflibercept, being done as a part of quality control for the therapeutics manufacturing process or for biosimilar development, can be done with a more applicable outcome by assessing each site separately.

Comparison of Bacterial and Fungal Community Structure and Potential Function Analysis of Yak Feces before and after Weaning.

Weaning is one of the most stressful periods in yak growth. However, the impact of weaning on microbial diversity, structure, and potential function of yak feces is not clear. In this study, 12 Xinjiang yaks aged 3, 4, 5, and 6 months old were selected to collect fresh feces before and after weaning. Through 16S rRNA and ITS high-throughput sequencing, the dynamic distribution and potential function of yak fecal, bacterial, and fungal communities in each month were revealed. The study found that the richness of fungi had a significant impact on weaning. At the phylum level, Firmicutes, Bacteroidetes, Ascomycota, and Basidiomycota, and at the genus level, 5-7N15, Oscillospira, Roseburia, Dorea, Preussia, Neoascochyta, Naganishia, and Sporormiella were enriched in yak feces of different months old. The abundance and proportion of bacteria Firmicutes, Bacteroidetes, 5-7N15, and fungi Mucoromyceta changed significantly before and after weaning. With the increase of months, Verrucomicrobia and Akkermansia have shown a downward trend. Through the prediction and analysis of fecal microbial function, it was found that at the level of primary pathways, weaning has a significant impact on cellular processes, environmental information processing, genetic information processing, metabolism, and organismal systems. At the level of secondary metabolic pathways, weaning has a significant impact on cell motility, signal transduction, folding, sorting and degradation, translation, amino acid metabolism, glycan biosynthesis and metabolism, metabolism of terpenoids and polyketides, and xenobiotics biodegradation and metabolism. In addition, by analyzing the differences in functional pathways and microbial composition between sample groups of different months, it was found that the differences in functional pathways were related to the abundance differences of some microorganisms. In general, the changes in the composition and structure of yak fecal microflora may reflect the adaptability of the intestinal microbiota.

Systematic Optimisation of Microtiter Plate Lectin Assay to Improve Sialic Acid Linkage Detection.

We aimed to develop a high-throughput lectin assay with minimized background signals to investigate the interactions of lectins and sialic acid glycans, focusing on Prostate- Specific Antigen (PSA). High background signals resulting from nonspecific binding are a significant concern for microtiter plate-based Enzyme-Linked Lectin Sorbent Assays (ELISAs), as they can mask specific binding signals and cause false-positive results. In this study, we constructed an ELISA based on different washing step parameters, including the number of washing cycles, NaCl and Tween-20 concentrations, and the type of blocking agent and evaluated the effects on both specific and nonspecific binding signals. Furthermore, we performed a PSA binding assay using the optimized ELISA. The optimal washing parameters based on the highest specific binding signal proposed four cycles of washing steps using a washing buffer containing a high salt concentration (0.5 M NaCl) and mild detergent (0.05% Tween-20). The utilization of the optimized washing parameters in this assay was shown to be sufficient to obtain the optimal binding signals without the use of any blocking agent. Binding assays performed using the optimized ELISA revealed that the glycan of the PSA sample used in this study mainly consists of terminal α2,6-linked sialic acid, as strongly recognized by Sambucus nigra agglutinin (SNA) with a KD value of 12.38 nM. The ELISA reported in this study provides a simple yet sensitive assay for sialic acid linkage recognition.

MicroRNA expression signature in the striated and smooth adductor muscles of Yesso scallop Patinopecten yessoensis

Increasing evidences point to the potential role of microRNAs (miRNAs) in muscle growth and development in animals. However, knowledge on the identity of miRNAs and their targets in molluscs remains largely unknown. Scallops have one large adductor muscle, composed of fast (striated) and slow (smooth) muscle types, which display great differences in muscle fibers, meat quality, cell types and molecular components. In the present study, we performed a comprehensive investigation of miRNA transcriptomes in fast and slow adductor muscles of Yesso scallop Patinopecten yessoensis. As a result, 47 differentially expressed miRNAs representing ten miRNA families were identified between the striated and smooth adductor muscles. The KEGG enrichment analysis of their target genes were mainly associated with amino acid metabolism, energy metabolism and glycan biosynthesis. The target genes of miR-133 and miR-71 were validated by the dual-luciferase reporter assays and miRNA antagomir treatment in vivo. The identification and functional validation of these different miRNAs in scallops will greatly help our understanding of miRNA regulatory mechanism that achieves the unique muscle phenotypes in scallops. The present findings provide the direct evidences for muscle-specific miRNAs involved in muscle growth and differentiation in molluscs.