Native and methyl-esterified sialylated glycans were analyzed with 2,4,6-trihydroxyacetophenone（THAP）and 2,5-dihydroxybenzoic acid（DHB）as matrix by a matrix-assisted laser desorption/ionization time-of-flight mass...Native and methyl-esterified sialylated glycans were analyzed with 2,4,6-trihydroxyacetophenone（THAP）and 2,5-dihydroxybenzoic acid（DHB）as matrix by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer（MALDI-TOF MS）.High quality negative-ion spectra of commercial sialylated glycan were obtained with THAP as matrix.Detection limit of the glycan was less than 0.1 pmol.After methyl esterification of sialic acid（SA）residue,sialylated glycans were detected sensitively in the positive-ion mode using DHB as matrix.Neutral and sialylated glycans from the mixture of asialofetuin and fetuin were methylesterified and simultaneously recognized in one manipulation.Methyl esterification of SA residue offers a convenient and sensitive way to identify the structure of N-linked glycans for glycan profiling.展开更多
With the size of the biopharmaceutical market exponentially increasing,there is an aligned growth in the importance of data-rich analyses,not only to assess drug product safety but also to assist drug development driv...With the size of the biopharmaceutical market exponentially increasing,there is an aligned growth in the importance of data-rich analyses,not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships.In monoclonal antibodies,many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known.The importance of their function focuses analytical research efforts on the development of robust,accurate and fast methods to support drug development and quality control.Released N-glycan analysis is considered as the gold standard for glycosylation characterisation;however,it is not the only method for quantitative analysis of glycoform heterogeneity.In this study,ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies.While observing good comparability between the quantitative results generated,it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow according to application and the desired depth of data generated.展开更多
BACKGROUND Mass spectrometry-based proteomics and glycomics reveal post-translational modifications providing significant biological insights beyond the scope of genomic sequencing.AIM To characterize the N-linked gly...BACKGROUND Mass spectrometry-based proteomics and glycomics reveal post-translational modifications providing significant biological insights beyond the scope of genomic sequencing.AIM To characterize the N-linked glycoproteomic profile in esophageal squamous cell carcinoma(ESCC)via two complementary approaches.METHODS Using tandem multilectin affinity chromatography for enrichment of N-linked glycoproteins,we performed N-linked glycoproteomic profiling in ESCC tissues by two-dimensional gel electrophoresis(2-DE)-based and isobaric tags for relative and absolute quantification(iTRAQ)labeling-based mass spectrometry quantitation in parallel,followed by validation of candidate glycoprotein biomarkers by Western blot.RESULTS 2-DE-based and iTRAQ labeling-based quantitation identified 24 and 402 differentially expressed N-linked glycoproteins,respectively,with 15 in common,demonstrating the outperformance of iTRAQ labeling-based quantitation over 2-DE and complementarity of these two approaches.Proteomaps showed the distinct compositions of functional categories between proteins and glycoproteins with differential expression associated with ESCC.Western blot analysis validated the up-regulation of total procathepsin D and high-mannose procathepsin D,and the downregulation of total haptoglobin,high-mannose clusterin,and GlcNAc/sialic acid-containing fraction of 14-3-3ζ in ESCC tissues.The serum levels of glycosylated fractions of clusterin,prolinearginine-rich end leucine-rich repeat protein,and haptoglobin in patients with ESCC were remarkably higher than those in healthy controls.CONCLUSION Our study provides insights into the aberrant N-linked glycoproteome associated with ESCC,which will be a valuable resource for future investigations.展开更多
To explore the effects of traditional herbal medicine Ganoderrna tsugae（G, tsugae） on immunomodulatory and antitumor activities, the crude polysaccharides of G. tsugae were purified by filtration, diethylaminoethyl...To explore the effects of traditional herbal medicine Ganoderrna tsugae（G, tsugae） on immunomodulatory and antitumor activities, the crude polysaccharides of G. tsugae were purified by filtration, diethylaminoethyl（DEAE） sepharose-fast flow chromatography and sephadex G-100 size-exclusion chromatography. Two main fractions, pro- tein-containing glyeans CSSLP-1 and CSSLP-2, were obtained via the gradient elution. The protein content, molecu- lar weight, and monosaccharide composition of the two fractions were analyzed. Furthermore, the influence of the protein-containing glycans from G. tsugae on the activation of human acute monocytic leukemia cell line（THP-l） and their antitumor activities to the human hepatocellular liver carcinoma cell（HepG-2） in vitro were evaluated. The re- sults indicate that CSSLP-1 and CSSLP-2 could increase the pinocytie activity of THP-1 cells and induce THP-1 cells to produce the eytokines of TNFa and IL-2, significantly. CSSLP-1 and CSSLP-2 also played an inhibiting effect on the cancer cell（HepG-2）. Moreover, the anti-proliferation activity of CSSLP-1 and CSSLP-2 increased with the par- ticipation of TNFa and IL-2 or other antitumor factors induced from THP-1 cells by G. tsugae protein-containing glycan fractions.展开更多
Background Optimal gut health is important to maximize growth performance and feed efficiency in broiler chickens.A total of 1,365 one-day-old male Ross 308 broiler chickens were randomly divided into 5 treatments gro...Background Optimal gut health is important to maximize growth performance and feed efficiency in broiler chickens.A total of 1,365 one-day-old male Ross 308 broiler chickens were randomly divided into 5 treatments groups with 21 replicates,13 birds per replicate.The present research investigated effects of microbial muramidase or a precision glycan alone or in combination on growth performance,apparent total tract digestibility,total blood carotenoid content,intestinal villus length,meat quality and gut microbiota in broiler chickens.Treatments included:NC:negative control(basal diet group);PC:positive control(basal diet+0.02%probiotics);MR:basal diet+0.035%microbial muramidase;PG:basal diet+0.1%precision glycan;and MRPG:basal diet+0.025%MR+0.1%PG,respectively.Results MRPG group increased the body weight gain and feed intake(P<0.05)compared with NC group.Moreover,it significantly increased total serum carotenoid(P<0.05)and MRPG altered the microbial diversity in ileum contents.The MRPG treatment group increased the abundance of the phylum Firmicutes,and family Lachnospiraceae,Ruminococcaceae,Oscillospiraceae,Lactobacillaceae,Peptostreptococcaceae and decreased the abundance of the phylum Campilobacterota,Bacteroidota and family Bacteroidaceae.Compared with the NC group,the chickens fed MRPG showed significantly increased in duodenum villus length at end the trial.Conclusion In this study,overall results showed that the synergetic effects of MR and PG showed enhancing growth performance,total serum carotenoid level and altering gut microbiota composition of broilers.The current research indicates that co-supplementation of MR and PG in broiler diets enhances intestinal health,consequently leading to an increased broiler production.展开更多
The glycosylation of proteins is responsible for their structural and functional roles in many cellular activities.This work describes a strategy that combines an efficient release, labeling and liquid chromatography...The glycosylation of proteins is responsible for their structural and functional roles in many cellular activities.This work describes a strategy that combines an efficient release, labeling and liquid chromatography–mass spectral analysis with the use of a comprehensive database to analyze N-glycans. The analytical method described relies on a recently commercialized kit in which quick deglycosylation is followed by rapid labeling and cleanup of labeled glycans. This greatly improves the separation, mass spectrometry（MS） analysis and fluorescence detection of N-glycans. A hypothetical database, constructed using Glyc Resoft, provides all compositional possibilities of N-glycans based on the common sugar residues found in N-glycans. In the initial version this database contains ＆gt; 8,700 N-glycans, and is compatible with MS instrument software and expandable. N-glycans from four different well-studied glycoproteins were analyzed by this strategy. The results provided much more accurate and comprehensive data than that had been previously reported. This strategy was then used to analyze the N-glycans present on the membrane glycoproteins of gastric carcinoma cells with different degrees of differentiation. Accurate and comprehensive N-glycan data from those cells was obtained efficiently and their differences were compared corresponding to their differentiation states. Thus, the novel strategy developed greatly improves accuracy, efficiency and comprehensiveness of N-glycan analysis.展开更多
Malignant tumors are complex structures composed of cancer cells and tumor microenvironmental cells.In this complex structure,cells cross-talk and interact,thus jointly promoting cancer development and metastasis.Rece...Malignant tumors are complex structures composed of cancer cells and tumor microenvironmental cells.In this complex structure,cells cross-talk and interact,thus jointly promoting cancer development and metastasis.Recently,immunoregulatory molecule-based cancer immunotherapy has greatly improved treatment efficacy for solid cancers,thus enabling some patients to achieve persistent responses or cure.However,owing to the development of drug-resistance and the low response rate,immunotherapy against the available targets PD-1/PD-L1 or CTLA-4 has limited benefits.Although combination therapies have been proposed to enhance the response rate,severe adverse effects are observed.Thus,alternative immune checkpoints must be identified.The SIGLECs are a family of immunoregulatory receptors(known as glyco-immune checkpoints)discovered in recent years.This review systematically describes the molecular characteristics of the SIGLECs,and discusses recent progress in areas including synthetic ligands,monoclonal antibody inhibitors,and Chimeric antigen receptor T(CAR-T)cells,with a focus on available strategies for blocking the sialylated glycan-SIGLEC axis.Targeting glyco-immune checkpoints can expand the scope of immune checkpoints and provide multiple options for new drug development.展开更多
BACKGROUND Hepatitis B virus (HBV) infection is the primary cause of hepatitis with chronic HBV infection,which may develop into liver fibrosis,cirrhosis and hepatocellular carcinoma.Detection of early-stage fibrosis ...BACKGROUND Hepatitis B virus (HBV) infection is the primary cause of hepatitis with chronic HBV infection,which may develop into liver fibrosis,cirrhosis and hepatocellular carcinoma.Detection of early-stage fibrosis related to HBV infection is of great clinical significance to block the progression of liver lesion.Direct liver biopsy is regarded as the gold standard to detect and assess fibrosis;however,this method is invasive and prone to clinical sampling error.In order to address these issues,we attempted to find more convenient and effective serum markers for detecting HBV-induced early-stage liver fibrosis.AIM To investigate serum N-glycan profiling related to HBV-induced liver fibrosis and verify multiparameter diagnostic models related to serum N-glycan changes.METHODS N-glycan profiles from the sera of 432 HBV-infected patients with liver fibrosis were analyzed.Significant changed N-glycan levels (peaks)(P <0.05) in differentfibrosis stages were selected in the modeling group,and multiparameter diagnostic models were established based on changed N-glycan levels by logistic regression analysis.The receiver operating characteristic (ROC) curve analysis was performed to evaluate diagnostic efficacy of N-glycans models.These models were then compared with the aspartate aminotransferase to platelet ratio index (APRI),fibrosis index based on the four factors (FIB-4),glutamyltranspeptidase platelet albumin index (S index),GlycoCirrho-test,and GlycoFibro-test.Furthermore,we combined multiparameter diagnostic models with alanine aminotransferase (ALT) and platelet (PLT) tests and compared their diagnostic power.In addition,the diagnostic accuracy of N-glycan models was also verified in the validation group of patients.RESULTS Multiparameter diagnostic models constructed based on N-glycan peak 1,3,4and 8 could distinguish between different stages of liver fibrosis.The area under ROC curves (AUROCs) of Model A and Model B were 0.890 and 0.752,respectively differentiating fibrosis F0-F1 from F2-F4,and F0-F2 from F3-F4,and surpassing other serum panels.However,AUROC (0.747) in Model C used for the diagnosis of F4 from F0-F3 was lower than AUROC (0.795) in FIB-4.In combination with ALT and PLT,the multiparameter models showed better diagnostic power (AUROC=0.912,0.829,0.885,respectively) when compared with other models.In the validation group,the AUROCs of the three combined models (0.929,0.858,and 0.867,respectively) were still satisfactory.We also applied the combined models to distinguish adjacent fibrosis stages of 432patients (F0-F1/F2/F3/F4),and the AUROCs were 0.917,0.720 and 0.785.CONCLUSION Multiparameter models based on serum N-glycans are effective supplementary markers to distinguish between adjacent fibrosis stages of patients caused by HBV,especially in combination with ALT and PLT.展开更多
Lassa virus(LASV)belongs to the Mammarenavirus genus(family Arenaviridae)and causes severe hemorrhagic fever in humans.The glycoprotein complex(GPC)contains eleven N-linked glycans that play essential roles in GPC fun...Lassa virus(LASV)belongs to the Mammarenavirus genus(family Arenaviridae)and causes severe hemorrhagic fever in humans.The glycoprotein complex(GPC)contains eleven N-linked glycans that play essential roles in GPC functionalities such as cleavage,transport,receptor recognition,epitope shielding,and immune response.We used three mutagenesis strategies(asparagine to glutamine,asparagine to alanine,and serine/tyrosine to alanine mutants)to abolish individual glycan chain on GPC and found that all the three strategies led to cleavage inefficiency on the 2nd(N89),5th(N119),or 8th(N365)glycosylation motif.To evaluate N to Q mutagenesis for further research,it was found that deletion of the 2nd(N89Q)or 8th(N365Q)glycan completely inhibited the transduction efficiency of pseudotyped particles.We further investigated the role of individual glycan on GPC-mediated immune response by DNA immunization of mice.Deletion of the individual 1st(N79Q),3rd(N99Q),5th(N119Q),or 6th(N167Q)glycan significantly enhanced the proportion of effector CD4+cells,whereas deletion of the 1st(N79Q),2nd(N89Q),3rd(N99Q),4th(N109Q),5th(N119Q),6th(N167Q),or 9th(N373Q)glycan enhanced the proportion of CD8+effector T cells.Deletion of specific glycan improves the Th1-type immune response,and abolishment of glycan on GPC generally increases the antibody titer to the glycan-deficient GPC.However,the antibodies from either the mutant or WT GPC-immunized mice show little neutralization effect on wild-type LASV.The glycan residues on GPC provide an immune shield for the virus,and thus represent a target for the design and development of a vaccine.展开更多
基金Supported by the National Natural Science Foundation of China(30800193)Grant from Centre for International Mobility(CIMO),Finland
文摘Native and methyl-esterified sialylated glycans were analyzed with 2,4,6-trihydroxyacetophenone（THAP）and 2,5-dihydroxybenzoic acid（DHB）as matrix by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer（MALDI-TOF MS）.High quality negative-ion spectra of commercial sialylated glycan were obtained with THAP as matrix.Detection limit of the glycan was less than 0.1 pmol.After methyl esterification of sialic acid（SA）residue,sialylated glycans were detected sensitively in the positive-ion mode using DHB as matrix.Neutral and sialylated glycans from the mixture of asialofetuin and fetuin were methylesterified and simultaneously recognized in one manipulation.Methyl esterification of SA residue offers a convenient and sensitive way to identify the structure of N-linked glycans for glycan profiling.
文摘With the size of the biopharmaceutical market exponentially increasing,there is an aligned growth in the importance of data-rich analyses,not only to assess drug product safety but also to assist drug development driven by the deeper understanding of structure/function relationships.In monoclonal antibodies,many functions are regulated by N-glycans present in the constant region of the heavy chains and their mechanisms of action are not completely known.The importance of their function focuses analytical research efforts on the development of robust,accurate and fast methods to support drug development and quality control.Released N-glycan analysis is considered as the gold standard for glycosylation characterisation;however,it is not the only method for quantitative analysis of glycoform heterogeneity.In this study,ten different analytical workflows for N-glycan analysis were compared using four monoclonal antibodies.While observing good comparability between the quantitative results generated,it was possible to appreciate the advantages and disadvantages of each technique and to summarise all the observations to guide the choice of the most appropriate analytical workflow according to application and the desired depth of data generated.
基金Supported by National Natural Science Foundation of China,No.81072039 and No.81872037.
文摘BACKGROUND Mass spectrometry-based proteomics and glycomics reveal post-translational modifications providing significant biological insights beyond the scope of genomic sequencing.AIM To characterize the N-linked glycoproteomic profile in esophageal squamous cell carcinoma(ESCC)via two complementary approaches.METHODS Using tandem multilectin affinity chromatography for enrichment of N-linked glycoproteins,we performed N-linked glycoproteomic profiling in ESCC tissues by two-dimensional gel electrophoresis(2-DE)-based and isobaric tags for relative and absolute quantification(iTRAQ)labeling-based mass spectrometry quantitation in parallel,followed by validation of candidate glycoprotein biomarkers by Western blot.RESULTS 2-DE-based and iTRAQ labeling-based quantitation identified 24 and 402 differentially expressed N-linked glycoproteins,respectively,with 15 in common,demonstrating the outperformance of iTRAQ labeling-based quantitation over 2-DE and complementarity of these two approaches.Proteomaps showed the distinct compositions of functional categories between proteins and glycoproteins with differential expression associated with ESCC.Western blot analysis validated the up-regulation of total procathepsin D and high-mannose procathepsin D,and the downregulation of total haptoglobin,high-mannose clusterin,and GlcNAc/sialic acid-containing fraction of 14-3-3ζ in ESCC tissues.The serum levels of glycosylated fractions of clusterin,prolinearginine-rich end leucine-rich repeat protein,and haptoglobin in patients with ESCC were remarkably higher than those in healthy controls.CONCLUSION Our study provides insights into the aberrant N-linked glycoproteome associated with ESCC,which will be a valuable resource for future investigations.
基金Supported by the National Natural Science Foundation of China(No.30870552)
文摘To explore the effects of traditional herbal medicine Ganoderrna tsugae（G, tsugae） on immunomodulatory and antitumor activities, the crude polysaccharides of G. tsugae were purified by filtration, diethylaminoethyl（DEAE） sepharose-fast flow chromatography and sephadex G-100 size-exclusion chromatography. Two main fractions, pro- tein-containing glyeans CSSLP-1 and CSSLP-2, were obtained via the gradient elution. The protein content, molecu- lar weight, and monosaccharide composition of the two fractions were analyzed. Furthermore, the influence of the protein-containing glycans from G. tsugae on the activation of human acute monocytic leukemia cell line（THP-l） and their antitumor activities to the human hepatocellular liver carcinoma cell（HepG-2） in vitro were evaluated. The re- sults indicate that CSSLP-1 and CSSLP-2 could increase the pinocytie activity of THP-1 cells and induce THP-1 cells to produce the eytokines of TNFa and IL-2, significantly. CSSLP-1 and CSSLP-2 also played an inhibiting effect on the cancer cell（HepG-2）. Moreover, the anti-proliferation activity of CSSLP-1 and CSSLP-2 increased with the par- ticipation of TNFa and IL-2 or other antitumor factors induced from THP-1 cells by G. tsugae protein-containing glycan fractions.
基金supported by Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the Ministry of Education(NRF-RS-2023-00275307)。
文摘Background Optimal gut health is important to maximize growth performance and feed efficiency in broiler chickens.A total of 1,365 one-day-old male Ross 308 broiler chickens were randomly divided into 5 treatments groups with 21 replicates,13 birds per replicate.The present research investigated effects of microbial muramidase or a precision glycan alone or in combination on growth performance,apparent total tract digestibility,total blood carotenoid content,intestinal villus length,meat quality and gut microbiota in broiler chickens.Treatments included:NC:negative control(basal diet group);PC:positive control(basal diet+0.02%probiotics);MR:basal diet+0.035%microbial muramidase;PG:basal diet+0.1%precision glycan;and MRPG:basal diet+0.025%MR+0.1%PG,respectively.Results MRPG group increased the body weight gain and feed intake(P<0.05)compared with NC group.Moreover,it significantly increased total serum carotenoid(P<0.05)and MRPG altered the microbial diversity in ileum contents.The MRPG treatment group increased the abundance of the phylum Firmicutes,and family Lachnospiraceae,Ruminococcaceae,Oscillospiraceae,Lactobacillaceae,Peptostreptococcaceae and decreased the abundance of the phylum Campilobacterota,Bacteroidota and family Bacteroidaceae.Compared with the NC group,the chickens fed MRPG showed significantly increased in duodenum villus length at end the trial.Conclusion In this study,overall results showed that the synergetic effects of MR and PG showed enhancing growth performance,total serum carotenoid level and altering gut microbiota composition of broilers.The current research indicates that co-supplementation of MR and PG in broiler diets enhances intestinal health,consequently leading to an increased broiler production.
基金the National Natural Science Foundation of China (81473179 and 81673388)Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, YX13200111)the funding for Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-PsychoDiseases (BM2013003)
文摘The glycosylation of proteins is responsible for their structural and functional roles in many cellular activities.This work describes a strategy that combines an efficient release, labeling and liquid chromatography–mass spectral analysis with the use of a comprehensive database to analyze N-glycans. The analytical method described relies on a recently commercialized kit in which quick deglycosylation is followed by rapid labeling and cleanup of labeled glycans. This greatly improves the separation, mass spectrometry（MS） analysis and fluorescence detection of N-glycans. A hypothetical database, constructed using Glyc Resoft, provides all compositional possibilities of N-glycans based on the common sugar residues found in N-glycans. In the initial version this database contains ＆gt; 8,700 N-glycans, and is compatible with MS instrument software and expandable. N-glycans from four different well-studied glycoproteins were analyzed by this strategy. The results provided much more accurate and comprehensive data than that had been previously reported. This strategy was then used to analyze the N-glycans present on the membrane glycoproteins of gastric carcinoma cells with different degrees of differentiation. Accurate and comprehensive N-glycan data from those cells was obtained efficiently and their differences were compared corresponding to their differentiation states. Thus, the novel strategy developed greatly improves accuracy, efficiency and comprehensiveness of N-glycan analysis.
基金supported by the Shanghai Science and Technology Committee (Grant Nos. 20DZ2201900 to Y.Y. and 23ZR1432500 to W.P.)National Natural Science Foundation of China (Grant Nos. 82072602 to Y.Y.+4 种基金91853121, 21977066, and 22177069 to W.P.)Innovation Foundation of Translational Medicine of Shanghai Jiao Tong University School of Medicine(Grant No. TM202001 to Y.Y.)Collaborative Innovation Center for Clinical and Translational Science by Chinese Ministry of Education&Shanghai (Grant No. CCTS-2022202 to Y.Y.)Shanghai Pilot Program for Basic Research-Shanghai Jiao Tong University (Grant No. 21TQ1400210 to W.P.)Medical-Engineering Interdisciplinary Research Foundation of Shanghai Jiao Tong University (Grant No. YG2022ZD001 to W.P.)
文摘Malignant tumors are complex structures composed of cancer cells and tumor microenvironmental cells.In this complex structure,cells cross-talk and interact,thus jointly promoting cancer development and metastasis.Recently,immunoregulatory molecule-based cancer immunotherapy has greatly improved treatment efficacy for solid cancers,thus enabling some patients to achieve persistent responses or cure.However,owing to the development of drug-resistance and the low response rate,immunotherapy against the available targets PD-1/PD-L1 or CTLA-4 has limited benefits.Although combination therapies have been proposed to enhance the response rate,severe adverse effects are observed.Thus,alternative immune checkpoints must be identified.The SIGLECs are a family of immunoregulatory receptors(known as glyco-immune checkpoints)discovered in recent years.This review systematically describes the molecular characteristics of the SIGLECs,and discusses recent progress in areas including synthetic ligands,monoclonal antibody inhibitors,and Chimeric antigen receptor T(CAR-T)cells,with a focus on available strategies for blocking the sialylated glycan-SIGLEC axis.Targeting glyco-immune checkpoints can expand the scope of immune checkpoints and provide multiple options for new drug development.
基金Supported by Major Science and Technology Special Project of China Thirteenth Five-Year Plan,No.2018ZX10732401-003-015；Guangxi Key Laboratory for the Prevention and Control of Viral Hepatitis,No.GXCDCKL201901
文摘BACKGROUND Hepatitis B virus (HBV) infection is the primary cause of hepatitis with chronic HBV infection,which may develop into liver fibrosis,cirrhosis and hepatocellular carcinoma.Detection of early-stage fibrosis related to HBV infection is of great clinical significance to block the progression of liver lesion.Direct liver biopsy is regarded as the gold standard to detect and assess fibrosis;however,this method is invasive and prone to clinical sampling error.In order to address these issues,we attempted to find more convenient and effective serum markers for detecting HBV-induced early-stage liver fibrosis.AIM To investigate serum N-glycan profiling related to HBV-induced liver fibrosis and verify multiparameter diagnostic models related to serum N-glycan changes.METHODS N-glycan profiles from the sera of 432 HBV-infected patients with liver fibrosis were analyzed.Significant changed N-glycan levels (peaks)(P <0.05) in differentfibrosis stages were selected in the modeling group,and multiparameter diagnostic models were established based on changed N-glycan levels by logistic regression analysis.The receiver operating characteristic (ROC) curve analysis was performed to evaluate diagnostic efficacy of N-glycans models.These models were then compared with the aspartate aminotransferase to platelet ratio index (APRI),fibrosis index based on the four factors (FIB-4),glutamyltranspeptidase platelet albumin index (S index),GlycoCirrho-test,and GlycoFibro-test.Furthermore,we combined multiparameter diagnostic models with alanine aminotransferase (ALT) and platelet (PLT) tests and compared their diagnostic power.In addition,the diagnostic accuracy of N-glycan models was also verified in the validation group of patients.RESULTS Multiparameter diagnostic models constructed based on N-glycan peak 1,3,4and 8 could distinguish between different stages of liver fibrosis.The area under ROC curves (AUROCs) of Model A and Model B were 0.890 and 0.752,respectively differentiating fibrosis F0-F1 from F2-F4,and F0-F2 from F3-F4,and surpassing other serum panels.However,AUROC (0.747) in Model C used for the diagnosis of F4 from F0-F3 was lower than AUROC (0.795) in FIB-4.In combination with ALT and PLT,the multiparameter models showed better diagnostic power (AUROC=0.912,0.829,0.885,respectively) when compared with other models.In the validation group,the AUROCs of the three combined models (0.929,0.858,and 0.867,respectively) were still satisfactory.We also applied the combined models to distinguish adjacent fibrosis stages of 432patients (F0-F1/F2/F3/F4),and the AUROCs were 0.917,0.720 and 0.785.CONCLUSION Multiparameter models based on serum N-glycans are effective supplementary markers to distinguish between adjacent fibrosis stages of patients caused by HBV,especially in combination with ALT and PLT.
基金This work was supported by the National Key Research and Development Program of China(2018YFA0507204)the National Natural Sciences Foundation of China(31670165)+1 种基金Wuhan National Biosafety Laboratory,Chinese Academy of Sciences Advanced Customer Cultivation Project(2019ACCP-MS03)the Open Research Fund Program of the State Key Laboratory of Virology of China(2018IOV001).Author Contributions。
文摘Lassa virus(LASV)belongs to the Mammarenavirus genus(family Arenaviridae)and causes severe hemorrhagic fever in humans.The glycoprotein complex(GPC)contains eleven N-linked glycans that play essential roles in GPC functionalities such as cleavage,transport,receptor recognition,epitope shielding,and immune response.We used three mutagenesis strategies(asparagine to glutamine,asparagine to alanine,and serine/tyrosine to alanine mutants)to abolish individual glycan chain on GPC and found that all the three strategies led to cleavage inefficiency on the 2nd(N89),5th(N119),or 8th(N365)glycosylation motif.To evaluate N to Q mutagenesis for further research,it was found that deletion of the 2nd(N89Q)or 8th(N365Q)glycan completely inhibited the transduction efficiency of pseudotyped particles.We further investigated the role of individual glycan on GPC-mediated immune response by DNA immunization of mice.Deletion of the individual 1st(N79Q),3rd(N99Q),5th(N119Q),or 6th(N167Q)glycan significantly enhanced the proportion of effector CD4+cells,whereas deletion of the 1st(N79Q),2nd(N89Q),3rd(N99Q),4th(N109Q),5th(N119Q),6th(N167Q),or 9th(N373Q)glycan enhanced the proportion of CD8+effector T cells.Deletion of specific glycan improves the Th1-type immune response,and abolishment of glycan on GPC generally increases the antibody titer to the glycan-deficient GPC.However,the antibodies from either the mutant or WT GPC-immunized mice show little neutralization effect on wild-type LASV.The glycan residues on GPC provide an immune shield for the virus,and thus represent a target for the design and development of a vaccine.
