Objective To investigate the differential expression of proteins in the serum of diabetic patients quantitative proteomics technologies, and to identify specific protein biomarkers. Methods Serum samples from three diabetic patients and three healthy controls in Shenzhen in 2023 were collected. Immunoprecipitation and nano liquid chromatography-quadrupole-electrostatic orbitrap-linear ion trap mass spectrometry (easy-nanoLC-Q-orbitrap-fusion) were employed to perform fraction and no-fraction-based quantitative analysis of the serum proteome in diabetic patients and healthy controls. The mass spectrometry data were analyzed by MaxQuant1.6.1.0, and the differential proteins identified by the fraction-based method were subjected to gene ontology (GO) functional clustering and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Results A total of 251 reliable proteins were identified by the no-fraction-based method, among which 17 differentially expressed proteins were selected. In contrast, 494 reliable proteins were identified by the fraction-based method, and 74 differentially expressed proteins were selected. Among them, seven proteins showed consistent changes in both methods. Five proteins were downregulated, including pregnancy zone protein (PZP), carbonic anhydrase 1 (CAH1), mannose-binding protein C (MBL2), fibrinogen alpha chain (FIBA), and haptoglobin (HPT). Two proteins were upregulated, including apolipoprotein(a) (LPA) and complement factor H-related protein 4 (CFH4). GO analysis revealed that the differentially expressed proteins were mainly involved in molecular functions such as glycosaminoglycan binding and heparin binding. KEGG pathway analysis showed that the differentially expressed proteins were mainly associated with complement and coagulation cascades, cell adhesion, PI3K-Akt signaling pathway, and other pathways. Conclusions Fraction-based no-fractional quantitative proteomics analysis provides an effective approach for analyzing differential protein expression in the serum of diabetic patients, and offers a valuable technique for screening specific protein biomarkers of serum samples in large-scale cohorts of disease.

Objective:To investigate the proteins interacting with Mycobacterium tuberculosis Rv1705c in human body. Methods:Rv1705c was prokaryotically expressed and inclusion bodies were collected for further lysis and the purification of Rv1705c. ELISA assay was used to detect the secretion of IFN-γ after stimulating macrophages with Rv1705c protein. Purified and biotin-labeled Rv1705c sample was incubated on the HuProt? human proteome microarray to screen the interacting proteins. GenePix Pro 6.0 software was used to extract all features of the data obtained from the scanned images and further analysis was performed based on bioinformatics databases such as GO and KEGG. GST pull-down was performed to verify the interaction of Rv1705c with PSMA3 and RSAD2.Results:The purification results showed that Rv1705c was expressed in endosomes. The secretion of IFN-γ increased significantly after stimulating macrophages with Rv1705c. A total of 29 potential Rv1705c-interacting proteins were screened, and nine of them showed signal-to-noise ratio (SNR)>1.6, namely PSMA3, NLN, THOP1, UPF3A, RSAD2, OMG, PNKD, STEAP3 and MED8. Further bioinformatics analysis revealed that PSMA3, RSAD2 and C1QBP were involved in innate immune signaling pathway, and there were interactions of PSMA3 and RSAD2 with IFN. GST pull-down assay validated that PSMA3 and RSAD2 interacted with Rv1705c.Conclusions:This study showed that PSMA3 and RSAD2 interacted with Rv1705c, providing reference for further investigation on the mechanism of Mycobacterium tuberculosis infection.

OBJECTIVE: To explore the genetic basis of three children with unexplained developmental delay/intellectual disability (DD/ID). METHODS: Peripheral blood samples were collected from the patients and subjected to chromosomal microarray analysis (CMA). RESULTS: Patient 1 was found to harbor a 190 kb deletion at 9q34.3, which encompassed most of EHMT1 (OMIM 607001), the key gene for Kleefstra syndrome (OMIM 610253). Patients 2 and 3 were siblings. CMA showed that they have shared four chromosomal copy number variations (CNVs) including a deletion at 9q34.3 which spanned 154 kb and 149 kb, respectively, and encompassed the EHMT1 and CACNA1B (OMIM 601012) genes. The remaining 3 CNVs were predicted to be with no clinical significance. CONCLUSION Microdeletions at 9q33.4 probably underlay the pathogenesis of DD/ID in the three children, for which EHMT1 may be the key gene.
Child ; Chromosome Deletion ; Chromosomes, Human, Pair 9 ; Craniofacial Abnormalities/genetics* ; DNA Copy Number Variations ; Developmental Disabilities/genetics* ; Heart Defects, Congenital ; Humans ; Intellectual Disability/genetics*

OBJECTIVE: To verify a HLA-DQB1*03:90N allele and method to improve the accuracy of HLA typing. METHODS: A total of 2265 hematopoietic stem cell donors from Shenzhen Branch of China Marrow Donor Program in 2018 were initially detected by a PCR sequence-specific oligonucleotide probe (SSOP) method. Among these, a rare HLA-DQB1 allele was identified by sequence-based tying (SBT) and Ion Torrent S5 next generation sequencing (NGS). RESULTS: The SSOP typing result suggested the HLA-DQB1 to be a rare allele, while an insertion and a deletion was suspected in its exon 2 by SBT, which were confirmed by NGS as DQB1*03:90N and DQB1*06:01, respectively. CONCLUSION Rare alleles suspected by the SSOP method should be verified by other methods to ensure the accuracy of HLA genotyping. Rare alleles formed by deletions can be detected by NGS with accuracy.

OBJECTIVE: To assess the association of KIR/HLA alleles with hepatocellular carcinoma (HCC) and hepatitis B virus (HBV) infection among ethnic Han Chinese patients from southern China. METHODS: For 95 patients with HCC and 171 healthy controls, the genotype of HLA-C alleles was determined with a PCR sequence-specific oligonucleotides typing method on an Illumina GenDx NGSgo platform. Genotypes comprised of HLA-C and KIR gene alleles were also subjected to statistical analysis. RESULTS: In total 16 KIR genes (2DL2, 2DS2, 2DS3, 2DS5, 3DS1, 2DS1, 2DL5, 2DS4, 3DL1, 3DP1, 2DL3, 2DP1, 3DL3, 2DL1, 3DL2 and 2DL4) were discovered in the two groups. The frequencies of KIR2DL3 alleles and combinational genotypes of KIR2DL3/HLA-C1C2 were significantly lower in the patient group compared with the controls (0.9368 vs. 0.9883, χ²>3.84; P<0.05, OR = 0.1; 0.0112 vs. 0.2663, χ²>3.84; P<0.05, RR = 0.03). The frequency of HLA-C2C2 genotype of the patient group was significantly lower than that of the controls (0.0316 vs. 0.2690, P<0.05, RR = 0.09), while the frequency of HLA-C1C2 genotype was significantly higher than that of the controls (0.2316 vs. 0.0058, P<0.05, RR = 51.23). CONCLUSION Above results suggested that the KIR2DL3 allele is associated with lower risk for HCC. There may be individual difference in patients with HCC and HBV infection but various combinations of KIR/HLA alleles.
Alleles ; Carcinoma, Hepatocellular ; genetics ; China ; Gene Frequency ; Genotype ; Humans ; Liver Neoplasms ; genetics ; Polymorphism, Genetic ; Receptors, KIR

OBJECTIVETo study the distribution of MICA alleles among ethnic Han Chinese blood donors from Shenzhen and their linkage disequilibrium with HLA-B gene.

METHODSFor 143 randomly selected blood donors, the MICA and HLA-B alleles were determined with a PCR-sequence based typing (SBT) method. Allelic frequency, haplotypic diversity and linkage disequilibrium were analyzed with a Pypop software.

RESULTSThirteen MICA and 35 HLA-B alleles were identified among the 143 blood donors, among which MICA*008:01 had the highest frequency (76/286), whilst MICA*008:01-HLA-B*40:01 and MICA*010-HLA-B*46:01 were the most common haplotypes. No novel allele was identified.

CONCLUSIONThe allele frequencies, haplotype diversities and linkage disequilibrium parameters under a high resolution can facilitate further studies and applications of the MICA and HLA-B genes.

OBJECTIVETo list the key points for quality control during HLA-A, B, C, DRB1 and DQB1 allele typing by taking consideration of hardware, software and experimental procedures.

METHODSA total of 10 167 samples from randomly selected healthy blood donors and donor-recipient pairs from Shenzhen were typed for exons 2-4 of HLA-A, B, C, exon 2 of HLA-DRB1, and exons 2 and 3 of HLA-DQB1 by PCR- sequence-based typing. For 56 cases whose forward and reverse sequences were inconsistent, the samples were re-checked by a PCR-sequence specific oligonucleotide probe method. Novel alleles not included in the IMGT/HLA database were cloned and sequenced using in-house primers.

RESULTSEight novel HLA alleles were identified. A table for key positions of single nucleotide polymorphisms (SNPs) were generated, which summarized the key points for quality control during HLA-A, B, C, DRB1 and DQB1 allele typing. Among the listed SNPs, 3 were located at the HLA-A locus, 8 were at the HLA-B locus, 6 were at the C locus, 6 were at the DQB1 locus, and 4 were at the DRB1 locus. To ensure the quality control, an unique sample number for DNA transferring tubes in the process of experiment should be considered.

CONCLUSIONA protocol for quality control should be enforced by checking all of the key points. The SNPs and critical control points of the alleles should be examined to ensure the accuracy of HLA typing results.

BACKGROUND: Due to the polymorphism of HLA, a large number of ambiguities have been generated by conventional HLA typing techniques, and confirmed stereotypes of ambiguous results based on group-specific haploid full-length typing are rarely reported.OBJECTIVE: To analyze the accuracy of HLA-typing ambigulity based on group-specific haploid full-length sequencing. METHODS: The low-resolution results were used as the starting point for two ambiguous samples. Sanger sequencing (PCR-SBT) based on haploid full-length was performed after group-specific amplification. RESULTS AND CONCLUSION: One case showed a new A*02:03:01 allele, which was found a mutation in NT817 from C to T in comparison with A*11:01:01:01. The other case indicated another new C*07:02:01:01, which was found a mutation in NT879 from A to G in comparison with C*08:01:01. In conclusion, these results indicate that the group-specific haploid full-length sequencing method can be used to accurately classify HLA alleles and to discover new alleles.

Objective To compare the trichloroethylene (TCE)-induced alteration in cell proliferation,cell apoptosis,histone deacetylase activity and expression levels in human hepatic L-02 cells (L-02 cells) and SET deficient cells,and reveal the TCE-induced effect in histone modification and the role of SET on epigenetic pathway.Methods The L-02 cells and preestablished SET deficient cells were treated with different TCE concentrations.For the changes of cell proliferation level and apoptosis rate,The L-02 cells and SET deficiency cells without TCE treatment were served as the control group,the TCE treatment was in the concentration of 2.0 and 8.0 mmol/L for 24 h.For histone deacetylase activity and expression levels,the TCE treatment was in the concentration of 0.25,0.50,1.0,2.0,4.0,and 8.0 mmol/L for 24 h.Results After treatment with TCE for 24 h,the cell proliferation level was significantly decreased and the apoptotic rate was significantly increased in both cell lines.When concentration of TCE were reached to 8.0 mmol/L,the difference of cell proliferation level and apoptotic rate between two groups was statistically significant (t=-4.362 for proliferation level and t =23.950 for apoptotic rate,both P＜0.05).After treatment with TCE for 24 h in various concentration (0,0.25,0.50,1.00,2.00,4.00 and 8.00 mmol/L),the activity of histone deacetylases was significantly increased in both cell lines.When the TCE concentration were high than 0.50 mmol/L,compared with control group of L-02 cells,the enzymes activity were significantly increased (F=403.26,P＜ 0.001).When TCE concentration was reached 1.00 mmol/L,the enzyme activity is highest.Compared with control group of SET deficiency cells,the enzyme activity was significantly increased when TCE concentration was reached 1.00 mmol/L (F=44.01,P＜0.001).When concentration of TCE reached 0.50 mmol/L,the difference of enzyme activity between two groups was statistically significant.For the protein expression,compared with control group of L-02 cells,TCE exposure can induced a significant increased expression level of HDAC2 in TCE-treated L-02 cells (Fvalues were 79.99,P＜0.001).But the alteration in SET deficiency cells was not significant.Conclusion TCE exposure can induce a significant alteration on cell proliferation,apoptotic rate and,the activity and expression on histone deacetylases.SET deficiency can attenuate the TCE-induced alteration in histone modification in L-02 cells.Our results indicated that SET is involved in the mechanism of TCE-induced cytotoxicity and epigenetic regulation in L-02 cells.

Objective To compare the trichloroethylene (TCE)-induced alteration in cell proliferation,cell apoptosis,histone deacetylase activity and expression levels in human hepatic L-02 cells (L-02 cells) and SET deficient cells,and reveal the TCE-induced effect in histone modification and the role of SET on epigenetic pathway.Methods The L-02 cells and preestablished SET deficient cells were treated with different TCE concentrations.For the changes of cell proliferation level and apoptosis rate,The L-02 cells and SET deficiency cells without TCE treatment were served as the control group,the TCE treatment was in the concentration of 2.0 and 8.0 mmol/L for 24 h.For histone deacetylase activity and expression levels,the TCE treatment was in the concentration of 0.25,0.50,1.0,2.0,4.0,and 8.0 mmol/L for 24 h.Results After treatment with TCE for 24 h,the cell proliferation level was significantly decreased and the apoptotic rate was significantly increased in both cell lines.When concentration of TCE were reached to 8.0 mmol/L,the difference of cell proliferation level and apoptotic rate between two groups was statistically significant (t=-4.362 for proliferation level and t =23.950 for apoptotic rate,both P＜0.05).After treatment with TCE for 24 h in various concentration (0,0.25,0.50,1.00,2.00,4.00 and 8.00 mmol/L),the activity of histone deacetylases was significantly increased in both cell lines.When the TCE concentration were high than 0.50 mmol/L,compared with control group of L-02 cells,the enzymes activity were significantly increased (F=403.26,P＜ 0.001).When TCE concentration was reached 1.00 mmol/L,the enzyme activity is highest.Compared with control group of SET deficiency cells,the enzyme activity was significantly increased when TCE concentration was reached 1.00 mmol/L (F=44.01,P＜0.001).When concentration of TCE reached 0.50 mmol/L,the difference of enzyme activity between two groups was statistically significant.For the protein expression,compared with control group of L-02 cells,TCE exposure can induced a significant increased expression level of HDAC2 in TCE-treated L-02 cells (Fvalues were 79.99,P＜0.001).But the alteration in SET deficiency cells was not significant.Conclusion TCE exposure can induce a significant alteration on cell proliferation,apoptotic rate and,the activity and expression on histone deacetylases.SET deficiency can attenuate the TCE-induced alteration in histone modification in L-02 cells.Our results indicated that SET is involved in the mechanism of TCE-induced cytotoxicity and epigenetic regulation in L-02 cells.