Research progress of SMIM1 in Vel blood type, tumor and plasmodium infection / 中国输血杂志

Small integral membrane protein 1(SMIM1) gene encodes Vel blood group antigen, and homozygous deletion of 17 bases pairs in its exon 3 leads to Vel negative phenotype. As a rare phenotype, accurate identification and prevention of transfusion reaction for Vel negative phenotype is particularly important. In addition to serving as a Vel blood group antigen, recent bioinformatics analysis suggests that SMIM1 protein may be a new tumor biomarker involved in the occurrence and development of numerous tumors. Due to the similarity in structure between SMIM1 protein and glycoprotein, and its phosphorylation in red blood cells infected with Plasmodium falciparum, it is speculated that SMIM1 protein may be involved in the development of malaria. Therefore, this article provides a review of the related research on SMIM1 and its coding protein in Vel blood type, tumor and plasmodium infection.

Progress on FUT2 gene and gut microbial composition in relation to rotavirus susceptibility / 国际儿科学杂志

Rotavirus(RV) is currently the leading cause of severe diarrhea in infants and young children worldwide, and RV has been found to recognize and bind to histo-blood group antigens(HBGAs) through its structural protein VP8, and the FUT2 gene determines the expression of HBGAs in epithelial tissues and secretion in body fluids.Individuals with loss of functional enzyme activity due to mutations in the FUT2 gene, called non-secretors, are unable to express and secrete HBGAs in the mucosa and body fluids, and non-secretors have been found to be resistant to diarrhea caused by RV.Studies have shown that microbial composition is genetically regulated by the host, and hundreds of genetic loci are involved in regulating the composition of human gut microbes, including FUT2.Sterile animal models reduce the rate of RV infection, suggesting that intestinal bacteria are associated with the process of RV infection.These studies reveal that secretory status directly influences individual susceptibility to RV, and its effect on gut microbial composition indirectly modulates human susceptibility to RV.This article reviews the correlation between FUT2 and gut microbial composition with RV susceptibility, with the aim of opening new avenues for personalized prevention and treatment of RV infection.

Preliminary study of ssDNA aptamer cytotoxicity on RhD+ red blood cells / 中国输血杂志

【Objective】 To investigate the effect of ssDNA aptamer of RhD blood group antigen on erythrocyte toxicity. 【Methods】 Two full-length ssDNA aptamers(82 bp) of RhD blood group antigen were obtained by gene synthesis.Five samples of whole blood with EDTA anticoagulant were collected to prepare red blood cell suspensions (4×107/mL), which were split into 10 tubes(100 μL/tube), corresponding to 5 experimental groups and 5 controls.Two monospecific full-length ssDNA sequences (100 pmol/μL, 5μL each) were added into the experimental group, while the same amount of normal saline into the control.After treatment, the experimental group and the control were incubated for 60 min at 37℃.After washing, they were suspended in LISS solution and stored at 4℃.The experimental group and the control were set according to different time point during storage (1 h, 1 d, 3 d, 10 d and 17 d), with 5 tubes in each group.For erythrocytes in LISS suspension at different storage time, Annexin V labeled with FITC was used as a probe to label the phosphatidylserine (PS) content and Fluo-4 to label Ca2+ .The eversion of PS and the change of Ca2+ concentration in red blood cells in LISS suspensions were determined by flow cytometry. 【Results】 After incubation, all groups were examined under the light microscope.No agglutination occurred in the experimental group, while agglutination occurred in the control.Flow cytometry showed that the number of Annexin V-FITC staining cells of suspended erythrocytes at the same storage time-point was similar between the experimental group and the control, with no significant differences.In the experimental group, apoptosis rate of Annexin V cells at 10-day storage(6.06±1.38) was significantly higher than that at 1-hour storage(P<0.05), so as at 17-day storage(7.77±1.23) than 1-hour, 1-day and 3-day storage(P<0.05). The apoptosis rate of Fluo-4 AM cell in suspended RBCs at the same storage time-point was similar between the two groups(P>0.05). In the experimental group, the apoptosis rate of Fluo-4 AM cell at the 3-day, 10-day and 17-day storage was 20.84±4.16, 22.35±3.37 and 27.06±2.81, respectively(P<0.05). 【Conclusion】 ssDNA aptamer was not found to have any cytotoxic effects on red blood cells, and RhD ssDNA aptamer may be used as a material for the detection and preparation of universal blood.

Establishment and identification of a human megakaryocytic cell line with low Ley expression / 中国输血杂志

【Objective】 To establish a stable human megakaryocytic cell line with low expression of Ley antigen to further study the role of Ley on activation of platelets. 【Methods】 The expression level of the Ley antigen in a human megakaryocytic cell line, DAMI, was determined using Western Blot and flow cytometry. The expression level of genes that encode fucosyltransferase (FUTs), which was involved in the biosynthesis of Ley antigen, was also determined to identify the candidate genes to be knocked out. The candidate FUT gene was knocked out via a CRISPR/Cas 9 gene knockout system and cells with low Ley antigen expression were sorted by flow cytometry. The sorted cell line was cultured and characterized. 【Results】 The Ley was expressed intensively on DAMI cell. FUT1 and FUT4 mRNA was expressed relatively higher, both may be key enzymes for the biosynthesis of the Ley antigen. In the DAMI cell line with the knockout of FUT1 gene, the expression of the Ley adntigen was remarkedly reduced, while cell proliferation was not affected compared to the wildtype control cells. 【Conclusions】 Since various FUTs contributes to the biosynthesis of the Ley antigen, the knockout of the primary one of them cannot totally block its biosynthesis, but only reduce its expression. In this study, a stable FUT gene knockout human megakaryocyticcell line is established using CRISPR/Cas 9 technology, which provides basis for the study of the impact of the Ley antigen on platelet functions.

Effects of GYP mRNA alternative splicing on cell surface localization of MNS blood group glycoprotein GPA and GPB / 中国输血杂志

【Objective】 To analyze the polymorphisms of GYPA and GYPB mRNA spliceosomes associated with MNS blood group, and to explore the mechanism of subcellular localization of GPA and GPB protein isomerism encoded by various spliceosomes as well as the expression of MNS blood group antigen. 【Methods】 Ten blood samples of voluntary blood donors were randomly selected. The total mRNA of peripheral blood was extracted and reversed into cDNA. Nested PCR was used to amplify reading open frame of GYPA and GYPB gene, and sequencing was performed by Sanger. The base sequence obtained was compared with GYPA(NCBI: NM_002099) and GYPB(NCBI: Nm_002100.5). After the wild type and various splicing isomer of the open reading frame of GYPA and GYPB had been obtained, they were fused with the encoding gene of green fluorescent protein (GFP) by fusion PCR technology, then cloned and transfected into HEK293 cells for over expression. The subcellular localization of GPA-GFP and GPB-GFP fused fluorescent proteins was monitored by focusing laser scanning microscope. 【Results】 Exon-1 and Exon-2 were missing in GYPA mRNA of the 2 samples, and 2~26 amino acids were missing in the predicted GPA isomer, and the full length sequence of GYPB mRNA was complete. GYPA mRNA was intact in 6 samples, exon-2 was missing in GYPB mRNA, 13~45 amino acids were missing in the predicted GPB protein isomer, and other exon sequences were intact. One sample had intact GYPA mRNA, and 364~385 bases in exon-5 of GYPB mRNA were replaced by AG, indicating truncation of amino acid signal peptide. The GYP mRNA sequences of other samples were complete. The fluorescence signal of GP-GFP fusion protein showed that all GPA and GPB glycoprotein isomers, cloned according to various RNA splicing, could demonstrate the orientation distribution on the cell membrane surface, while some alternative splicing leaded to different degrees of protein dispersion in the cell, and affected the distribution speed and proportion of protein on the cell surface, which might be one of the reasons for the strength variation of MNS antigen. 【Conclusion】 The GYP mRNA spliceosome is obviously polymorphic, but the partial deletion of GYP mRNA fragment does not affect the localization and distribution of the protein isomers encoded by GYP mRNA on the cell surface, which can ensure the expression of MNS antigen characteristics.

Comparison of tube method versus gel method for the results of blood group IgM antibody titration in ABO-incompatible kidney transplant recipients / 中华器官移植杂志

Objective:To explore the critical value of different blood group antibody titration in ABO blood group incompatible kidney transplant(ABOi-KT)recipients by tube and gel methods to provide rationales for selecting the threshold value of antibody titration before ABOi-KT.Methods:From January 2019 to April 2021, 681 blood group antibody titrations were performed for 214 ABOi-KT recipients.There were type A( n=135), type B( n=168)and type O( n=378). The difference, correlation and consistency of two methods were statistically analyzed. Results:Tube method was 2 gradients lower than gel method(4-fold dilution)and the results were significantly different( P<0.000 1). Spearman's test indicated that the results of two methods were significantly correlated( P<0.000 1). The results of intraclass correlation coefficient showed that the consistency of two methods was general for type A recipients(ICC=0.640), decent for type B recipients(ICC=0.751)and poor for type O recipients(ICC<0.4). When the critical value of tube method was set, titration of type A anti-B was 16, titration of type B anti-A 8 and titration of type O anti-A/B 8.And the corresponding critical values of gel was type A anti-B 32, type B anti-A 16 and type O anti-A/B 16. Conclusions:The results of ABO blood group IgM antibody titration by gel and tube methods are correlative.And gel method is recommended for more stable and reproducible results.

Research progress in the application of identification technology for infectious Norovirus / 中华实验和临床病毒学杂志

Norovirus, as one of the main pathogens causing non-bacterial gastroenteritis, can cause serious public health problems and economic losses around the world. In recent years, the outbreaks caused by the virus in China are on the rise. Human Norovirus (HuNoV) can hardly be cultivated in-vitro. The nucleic acid detection method (such as RT-qPCR) has the highest sensitivity and specificity, but it was not established that the correlation between the detected viral genome and viral infectivity, which leads to inaccurate judgment of safety risks. Here, the in-vitro and in-vivo culture models, viral genome integrity and capsid protein integrity were cut into three aspects. The research progress and characteristics of infectious Norovirus identification technology in recent years were reviewed and discussed, and the future development trend of this technology was prospected. The aim is to further improve the accuracy of Norovirus quantitative detection and provide a theoretical basis for its application in the field of food safety testing.

Protein expression and receptor binding specificity of porcine P19 rotavirus VP8* / 中华实验和临床病毒学杂志

Objective@#To explore the receptor binding specificity of VP8* protein of porcine P[19] rotaviruses (RVs) with oligosaccharides.@*Methods@#The porcine P[19] VP8* protein was expressed and purified. The receptor binding specificity of P[19] VP8* was analyzed by oligosaccharide binding and saliva binding assay.@*Results@#The P[19] VP8* protein showed significant binding to mucin cores, especially mucin core 2.@*Conclusions@#Mucin core 2 may be a potential receptor for the porcine P[19] RV, which provides certain basis for the study of virus infection mechanism and RV surveillance.

Evaluation of immune effects of virus-like particles of recombinant GⅡ. 17 norovirus / 中华微生物学和免疫学杂志

Objective To evaluate the immune effects of virus-like particles ( VLPs) assembled from the capsid protein VP1 of a recombinant norovirus ( NoV) GⅡ. 17 genotype. Methods The recombi-nant NoV GⅡ. 17 VP1 VLPs were purified, and then tested by SDS-PAGE and Western blot to analyze the purity. The size, morphology and diameter distribution of the recombinant VLPs were detected by transmis-sion electron microscopy ( TEM) and dynamic light scattering ( DLS) analyzer. The recombinant VP1 VLPs adsorbed by aluminium adjuvant were used to immunize BALB/c mice. Serum samples were collected after immunization. Specific antibody level and neutralizing antibody activity were evaluated with enzyme linked immunosorbent assay ( ELISA) and histo-blood group antigen ( HBGA)-VLP blocking test. Cross-reactivity of serum samples with GⅠ. 1 and GⅡ. 4 VP1 VLPs were detected. Moreover, cross-protection against GⅠ. 1 and GⅡ. 4 VP1 VLPs was analyzed. Results The purity of the recombinant NoV GⅡ. 17 VP1 VLP was greater than 90％ and specific bands were detected by Western blot. TEM images and DLS experiments showed that VLPs were 30-50 nm in size with good morphology and uniformity, indicating that the recombi-nant VLPs were similar to the wildtype virus. High titers of specific antibodies were detected in serum sam-ples of the immunized mice. A certain degree of cross-reactions between serum samples and VP1 VLPs of NoV GⅠ. 1 and GⅡ. 4 were observed, but no cross-protection was detected. Conclusion The recombinant GⅡ. 17 VP1 VLPs in combination with aluminum adjuvant can induce higher titers of HBGA blocking anti-bodies in mice, suggesting that it could be used as a candidate target antigen for norovirus vaccine.

Physicochemical and immunological characterization of two forms of recombinant norovirus GⅡ.4 virus-like particles assembled in Hansenula polymorpha / 中华微生物学和免疫学杂志

Objective To investigate the physicochemical properties and immunogenicity of virus like particles(VLPs) in two different conformations assembled from the essential capsid protein VP1 of GⅡ.4 norovirus(NoV) in Hansenula polymorpha. Methods NoV GⅡ.4 VLPs in two different conforma-tions were prepared from high-density fermentation of recombinant engineered strains and VLPs purification. Physicochemical properties of the two forms of VLPs were identified by Western blot,size-exclusion high per-formance liquid chromatography (SEC-HPLC), dynamic light scattering(DLS) and transmission electron microscopy. Serum VLPs binding activities and blocking activities against VLPs binding to histo-blood group antigen(HBGA-VLPs) were evaluated after immunization of BALB/c mice with the two forms of VLPs. Re-sults VLPs of two different diameters with high homogeneity were obtained after purification. DLS results showed that particle sizes of two VLPs were 53.98 nm and 45.18 nm,respectively. The two VLPs were sim-ilar in binding abilities to HBGA receptors. Serum VLPs binding activities and blocking activities against HBGA-VLPs were found higher in NoV-VLP-L than NoV-VLP-S,but the difference was not statistically sig-nificant (P>0.05). Conclusion VLPs in two different conformations were obtained by expressing NoV GⅡ.4 VP1 proteins in Hansenula polymorpha. Though they were similar in physicochemical properties and immunogenicity,the NoV-VLP-L might be potential antigen candidates for the development of recombinant human norovirus vaccine.

Evaluation of immune effects of recombinant norovirus GⅠ. 1 and GⅡ. 4 virus-like particles / 中华微生物学和免疫学杂志

Objective To evaluate the immune effects of virus-like particles ( VLPs) of VP1 pro-teins derived from norovirus GⅠ. 1 and GⅡ. 4 genotypes expressed in Hansenula polymorpha expression sys-tem. Methods SDS-PAGE and Western blot assay were performed to detect the purity of GⅠ. 1 and GⅡ. 4 VP1 proteins after purification. Morphologies of the recombinant VLPs were observed under transmission electron microscopy ( TEM) . Sizes and distributions of the VLPs were analyzed by dynamic light scattering analyzer. BT50(50% of blocking titer) was detected by HBGA (histo-blood group antigen) blocking assay in BALB/c mice immunized with different regimens. Results SDS-PAGE analysis of the purified recombinant GⅠ. 1 and GⅡ. 4 VP1 proteins showed that their purity were greater than 90%. Western blot assay con-firmed the specific bands of VLPs. TEM images showed that the sizes of purified GⅠ. 1 and GⅡ. 4 VP1 VLPs were at a mean diameter of 30-50 nm with clear border and high homogeneity, which was similar to that of wild virus. BT50 significantly increased in the groups, in which Al( OH) 3 was used as adjuvant. Con-clusion Animal studies have shown that administration of GⅠ. 1 and GⅡ. 4 VP1 VLPs in the presence of Al( OH) 3 induces detectable HBGA-blocking antibody, indicating that GⅠ. 1 and GⅡ. 4 VP1 VLPs are promising candidates for norovirus vaccine.

Histo-blood group antigen and rotavirus infection / 临床儿科杂志

Rotavirus (RV) is one of the major pathogens responsible for acute viral gastro-enteritis in children. The infec-tion of RV is dependent upon the recognition of the host cell speciifc receptors and attachment. Thus, receptors are the important factors of infection. Recent studies have suggested that a genetic factor might play a role in the susceptibility of hosts to RV infec-tion. Histo-blood group antigens have recently been discovered as receptors binding to RV, which are important for the study of evolution. Thus it will be also crucial for the pathogenesis and epidemiology and prevention and treatment for RV. In this article, we will review the correlation of the RV infection and histo-blood group antigens and further discuss the development of optimal vaccine.

Biological Meaning of the Histo-Blood Group Antigens Composed of Sugar Chains / 대한수혈학회지

All living creatures on this planet, from bacteria to human, produce sugar chains (glycans). This means that sugar chains are essential for living a life. Abundant, diverse, and highly regulated repertoire of glycans are synthesized by glycosylation process in cells. Located in proteins (N-glycans and O-glycans) and lipids (glycosphingolipids), glycans participate in many vital biological processes including molecular recognition, cell adhesion, molecular trafficking and clearance, receptor activation, and signal transduction. Histo-blood group antigens that are composed of sugar chains are expressed under the control of the Secretor, Lewis and ABO glycosyltransferases. They play important roles in microbial infections and cancers. Many of sugar chains associated with histo-blood group antigens are exploited as receptors for microorganisms. Aberrant glycosylation of proteins and lipids occurs commonly during malignant transformation and leads to the expression of tumor-associated glycans. In this review, over the scope of transfusion medicine, we discussed deep down the biologic meaning of sugar chains, through exploring how the sugar chains are synthesized, structured, and functioning.

Loss of Red Cell A Antigen in a Patient with Acute Myeloid Leukemia: A Case Report and Review of the Literature / 대한수혈학회지

Red cell antigens, A, B, and H can be weakened or lost especially in patients with hematologic malignancies. We report a 42-year-old female patient with acute myeloid leukemia, who showed loss of A antigen on her red cells. She showed the persistence of leukemia in spite of three cycles of induction chemotherapy. Her ABO blood group showed a discrepancy: the cell type was O and the serum type was A. Adsorption/elution test could not identify the presence of A antigen on her red cells, and the test for A and B transferases was negative. ABO genotyping using PCR/restriction fragment length polymorphism and sequencing of exons 6 and 7 of the ABO gene demonstrated 467 C>T substitution in exon 7 and confirmed the genotype of A102/O01. She was transfused with leukapheresis products collected from donors with blood group A, but expired of severe sepsis. This is the first Korean case, in which red cell A antigen loss was genetically proven using sequencing, and underscores the necessity of ABO genotyping to solve the ABO discrepancy and to transfuse effectively.

Detection of Helicobacter pylori and BabA (Blood-group Antigen Binding Adhesin) in Saliva and Gastric Tissue by Polymerase Chain Reaction / 임상검사와정도관리

BACKGROUND: Saliva is considered an important vector for the Helicobacter pylori infection. The presence of the babA2 gene, encoding for BabA (blood-group antigen binding adhesin), in the H. pylori genome is crucial for H. pylori-related pathogenesis. METHODS: The study was performed in the group of 215 patients. The detection of H. pylori and babA2 in saliva and gastric tissue was done by PCR (polymerase chain reaction). Moreover, gastric tissues were stained with hematoxylin-eosin as well as with modified Giemsa methods for the analysis of Helicobacter pylori density. RESULTS: The positive rate of H. pylori by nested PCR was 78.6% in gastric tissue and 72.7% in saliva. In addition, the positive rate of H. pylori was 55.5% by the histological analysis of Helicobacter pylori density in gastric tissue. The positive rate of babA2 by PCR was 33.9% in gastric tissue, and 8.2% in saliva. CONCLUSION: We revealed that the H. pylori PCR results obtained in gastric tissue correlated well with those obtained in saliva. As saliva is more available specimen, it is more suitable for clinical application of H. pylori detection by PCR. However, clinical use of - BabA PCR seems to be limited because of its low-sensitivity.