Characterization of the novel HLA-DQB1*03:516 allele by next-generation sequencing.

HLA-DQB1*03:516 differs from DQB1*03:03:02:03 by one nucleotide substitution at position 197G>A in exon 2.

Determination of the 3'UTR region of HLA-B*53:01:03 sequence by next generation sequencing.

The 3'UTR of the HLA-B*53:01:03 allele has been determined by next generation sequencing.

Research progress on stable gene transfection methods of Plasmodium falciparum / 中国热带医学

@#Abstract: Transfection of Plasmodium falciparum is helpful to study the function of its genes, such as drug resistance. However, transgenic manipulation has been very challenging, mainly due to the high A/T base sequence structure (A+T content of about 82%) and low transfection efficiency of the Plasmodium genome. Electroporation-based transfection of Plasmodium falciparum has been successfully applied in the study of certain genes, and electroporation by preloading is currently the preferred method for introducing foreign DNA into Plasmodium falciparum. The site-directed editing of Plasmodium genes mostly adopts the method of two-plasmid transfection. It is generally believed that successful transfection of Plasmodium requires a large amount of high-purity plasmid DNA and an accurate transfection system. In addition to the evaluation of the current commonly used electrotransfection methods, this paper also introduces a new transfection method, namely lyse-reseal erythrocytes for transfection (LyRET). This paper also review the role of factors such as plasmid DNA concentration, the use of transfection reagents, the setting of transfection parameters, the addition of fresh red blood cells, and the markers of successful transfection in improving the success rate and efficiency of Plasmodium transfection, in the hope of providing a reference for study in this field.

Research progress on artemisinin antimalarial resistance of Plasmodium falciparum / 中国热带医学

@#Abstract: Malaria, an infectious disease caused by Plasmodium infection, is one of the most important public health problems worldwide. Artemisinin-based combination therapies (ACTs) are recommended by WHO as the first-line treatment for uncomplicated P. falciparum malaria in malaria-endemic areas. The application of artemisinin and its derivatives has played an integral role in reducing the global incidence of malaria. However, in recent years, the emergence and spread of artemisinin resistance has brought great challenges to global malaria control and elimination. At present, the mutation of K13 gene on chromosome 13 of Plasmodium falciparum is most closely related to artemisinin resistance, but in recent years, studies have shown that K13 cannot explain all artemisinin resistance. This article reviews the recent research progress in the field of artemisinin resistance in Plasmodium falciparum, including definition of artemisinin resistance, detection methods and molecular markers related to resistance. In addition, some of the issues discussed in this review remain controversial and require further study.

A 5-year molecular epidemiology survey of human enterovirus 71 before vaccine application in Yunnan Province, China.

Enterovirus A71 (EV-A71) infection is known to cause hand, foot, and mouth disease (HFMD). Last year, an inactivated EV-A71 whole virus vaccine was used to prevent this disease in Yunnan, China. To obtain a viral genetic background for evaluating vaccine protection and monitor the adaptive evolution of the virus after the vaccination, a 5-year molecular epidemiology survey was performed before the vaccination. Twenty-six EV-A71 strains were separated from 561 stool specimens of patients with serious HFMD. The whole-genomic sequences of these strains were sequenced. Phylogenetic trees were constructed, and the mutation spectra were analyzed based on these viral sequences. There was no obvious mutation for the circular EV-A71 strains of the same year. Pathogenic EV-A71 strains may arise from a "subgroup" randomly each year. Whole-genomic analyses showed that a hotspot nonsynonymous substitution potentially affecting the immunogenicity of vaccines was found in the 2A gene, but not in genes of the viral capsid proteins, and the genetic diversity of whole viral genomes associated with the incidence of HFMD. Therefore, it will be valuable to monitor the genome-wide changes of EV-A71 to detect the adaptive mutations affecting immunogenicity or perform investigations using genetic diversity as a parameter.

[Phenotype Analysis of 78 Cases of Abnormal Hemoglobin E Homozygotes].

OBJECTIVE: To analyze the hematological characteristics of HbE homozygotes. METHODS: Complete blood cells count and hemoglobin electrophoresis were used for phenotypic analysis of 78 cases with HbE homozygotes from Yunnan province, China. The PCR-fluorescence hybridization was used to detect the common gene mutation of thalassemia. The hematological indexes, including MCV, MCH, Hb, HbA2, HbF and HbE were statistically analyzed between groups with different sex, ages and compound α thalassemia status. RESULTS: In HbE homozygotes (HbEE), 89.5% (17/19) children presented mild to moderate microcytic hypochromic anemia, and 10.5% of them presented moderate anemia. 39.6% (19/48) of women with HbEE developed mild anemia ,while 11 cases of male with HbE homozygotes were asymptomatic. The levels of MCV and MCH in HbE homozygotes increased by co-inheritance of α thalassemia mutation. CONCLUSION: The clinical phenotype of HbE homozygote shows highly heterogeneous, which is relates with age, sex and co-inheriting α-globin genotypes. In Hb EE women and children are more likely to develop mild to moderate anemia. The microcytic hypochromic anemia degree is relieved when HbEE combined with α- thalassemia.

Characterization of the novel KIR3DL1*0150213 and KIR3DL1*112 alleles using sequence-based typing.

KIR3DL1*0150213 differs from KIR3DL1*0150211 at 15 nucleotide positions. KIR3DL1*112 differs from KIR3DL1*03101 at 19 nucleotide substitutions.

Identification of two novel KIR3DL1 subtypes, KIR3DL1*0010104 and KIR3DL1*0010105.

KIR3DL1*0010104 and KIR3DL1*0010105 share a common 4 bp deletion in their intron 2.

Synthesis and antibacterial evaluation of novel cationic chalcone derivatives possessing broad spectrum antibacterial activity.

There is an urgent need to identify new antibiotics with novel mechanisms that combat antibiotic resistant bacteria. Herein, a series of chalcone derivatives that mimic the essential properties of cationic antimicrobial peptides were designed and synthesized. Antibacterial activities against drug-sensitive bacteria, including Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Salmonella enterica, as well as clinical multiple drug resistant isolates of methicillin-resistant S. aureus (MRSA), KPC-2-producing and NDM-1-producing Carbapenem-resistant Enterobacteriaceae were evaluated. Representative compounds 5a (MIC: 1 µg/mL against S. aureus, 0.5 µg/mL against MRSA) and 5g (MIC: 0.5 µg/mL against S. aureus, 0.25 µg/mL against MRSA) showed good bactericidal activity against both Gram-positive and Gram-negative bacteria, including the drug-resistant species MRSA, KPC and NDM. These membrane-active antibacterial compounds were demonstrated to reduce the viable cell counts in bacterial biofilms effectively and do not induce the development of resistance in bacteria. Additionally, these representative molecules exhibited negligible toxicity toward mammalian cells at a suitable concentration. The combined results indicate that this series of cationic chalcone derivatives have potential therapeutic effects against bacterial infections.

Overview of the improvement of the ring-stage survival assay-a novel phenotypic assay for the detection of artemisinin-resistant Plasmodium falciparum.

Artemisinin resistance in Plasmodium falciparum threatens the remarkable efficacy of artemisinin-based combination therapies worldwide. Thus, greater insight into the resistance mechanism using monitoring tools is essential. The ring-stage survival assay is used for phenotyping artemisinin-resistance or decreased artemisinin sensitivity. Here, we review the progress of this measurement assay and explore its limitations and potential applications.

Sarcocystis menglaensis n. sp. (Apicomplexa: Sarcocystidae) from Williamson's mouse deer Tuagulus williamsoni (Kloss) (Artiodactyla: Tragulidae).

Williamson's mouse deer, Tuagulus williamsoni (Kloss), is one of the smallest ungulates among tragulid species found in northern Thailand, and Yunnan Province, China. Here we describe Sarcocystis menglaensis n. sp., infecting two of 14 (14.3%) Williamson's mouse deer from south-western China. By light microscopy, sarcocysts of S. menglaensis are microscopic, up to 2,170 µm in length, and have a striated sarcocyst wall with 1.5-3.6 µm long palisade-like protrusions. Transmission electron microscopy observations revealed that sarcocyst wall is of "type 10f", and has numerous villar protrusions folded over the cyst wall. The villar protrusions contained microtubules dispersed throughout the protrusions. Phylogenetic analysis based on 18S rDNA and mitochondrial cox1 gene sequences indicated that S. menglaensis shared a close affinity with species of Sarcocystis Lankester, 1982 from ruminants, which utilise felids as definitive hosts.

Strong association of SLC1A1 and DPF3 gene variants with idiopathic male infertility in Han Chinese.

Male infertility is a multifactorial syndrome encompassing a wide variety of disorders. In recent years, several genome-wide single-nucleotide polymorphism (SNP) association studies (GWAS) have been performed on azoospermia and/or oligozoospermia in different populations including two GWAS on nonobstructive azoospermia in China; however, the association of SNPs with idiopathic male infertility, especially asthenozoospermia and oligozoospermia, and their correlation with semen parameters are still not clear. To investigate genetic variants associated with idiopathic male infertility (asthenozoospermia, oligozoospermia, and oligoasthenozoospermia) in Chinese Han people, 20 candidate SNPs were selected from GWAS results and genotyped using the Sequenom MassARRAY assay. A total of 136 subfertile men and 456 healthy fertile men were recruited. rs6476866 in SLC1A1 (P = 1.919E-4, OR = 0.5905, 95% CI: 0.447-0.78) and rs10129954 in DPF3 (P = 0.0023, OR = 2.199, 95% CI: 1.311-3.689) were strongly associated with idiopathic male infertility. In addition, positive associations were observed between asthenozoospermia and rs215702 in LSM5 (P = 0.0016, OR = 1.479, 95% CI: 1.075-2.033) and between oligoasthenozoospermia and rs2477686 in PEX10 (P = 0.0011, OR = 2.935, 95% CI: 1.492-5.775). In addition, six SNPs (rs215702 in LSM5, rs6476866 in SLC1A1, rs10129954 in DPF3, rs1801133 in MTHFR, rs2477686 in PEX10, and rs10841496 in PED3A) were significantly correlated with semen quality alterations. Our results suggest that idiopathic male infertility in different ethnic groups may share the same mechanism or pathway. Cohort expansion and further mechanistic studies on the role of genetic factors that influence spermatogenesis and sperm progressive motility are suggested.

[Association study of telomere length with idiopathic male infertility].

Telomeres are evolutionary conserved, multifunctional DNA-protein complexes located at the ends of eukaryotic chromosomes. Telomeres maintain chromosome stability and genome integrity and also play an important role in meiosis which aid in synapsis, homologous recombination, and segregation. Sperm telomere has been reported to play an important role in fertilization and embryo development. Nowadays, the association between telomere and reproduction is one of the major areas of interest, however whether sperm telomere associated with male infertility is not clear. In this study, in order to find out the association between Chinese idiopathic infertility and sperm telomere length, we analyzed the difference of sperm telomere length between idiopathic infertile men and normal fertile men, as well as the correlations between sperm telomere length and human semen characteristics. We analyzed 126 Chinese idiopathic infertile men and 138 normal fertile men for sperm telomere length by using quantitative PCR. We found that the relative sperm mean telomere length of infertile men was significantly shorter than that of fertile men (2.894 ± 0.115 vs. 4.016 ± 0.603, P=5.097 x 10⁻5). Both sperm count and semen progressive motility are related with telomere length. Our results suggest that sperm telomere length is associated with idiopathic male infertility of China and we proposed the possibility that shorter telomeres in sperm chromosome will reduce spermatogenesis and sperm functions, which finally affected the fertility of male.

[Association between six single nucleotide polymorphisms of EGLN1 gene and adaptation to high-altitude hypoxia].

To investigate the association between SNPs located in 5'UTR and intron of prolyl hydroxylase 2 (EGLN1 or PHD2) and adaptation to high-altitude hypoxia, the SNPs (rs2066140, rs2808584, rs2491405, rs2486741, rs2486734 and rs21533646) of EGLN1 gene were genotyped using Sequenom MassArray genotyping system in 152 unrelated healthy Tibetan individuals (3 650 m altitude) and 192 Han (5 00 m altitude), and the haplotypes of these SNPs were constructed and analyzed. Our results showed all the homozygous genotypes of six SNPs loci were significantly different between the two groups (P<0.05). The frequencies of haplotypes G-G (rs2066140 and rs2808584) and G-C (rs2486741 and rs2486734) of high-altitude group were significantly different from low-altitude group (P<0.05). In addition, the frequencies of haplotypes C-A (rs2066140 and rs2808584) and C-T (rs2486741 and rs2486734) of high-altitude group were significantly lower than those in low-altitude group (P<0.05). Our results indicate that the polymorphism of homozygous genotype in six SNPs and their haplotypes were associated with adaptation to high-altitude hypoxia.

[Association between polymorphisms of PSMB8, PSMB9 and TAP2 genes with rheumatoid arthritis in ethnic Han Chinese from Yunnan].

OBJECTIVE: To assess the association between single nucleotide polymorphisms (SNPs) of PSMB8, PSMB9 and TAP2 genes and rheumatoid arthritis (RA) in ethnic Han Chinese from Yunnan. METHODS: A case-control study was carried out using 177 RA patients and 288 healthy controls. Genotypes of rs2071543, rs55745125 and rs138635403 loci of PSMB8 gene, and rs17587 locus of PSMB9 gene were determined with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). And a polymerase chain reaction amplification refractory mutation system (ARMS-PCR) was used for typing rs2228396 locus of TAP2 gene. Genotypic and allelic frequencies were calculated. An Epi Info 7 software was used to calculate the Odds Ratio (OR) of above SNPs between the two groups. RESULTS: Allelic and genotypic frequencies of rs138635403 and rs17587 loci have differed significantly between the two groups (P<0.05). The frequency of GG genotype for rs17587 locus was also higher in the RA group (0.672) compared with control group (0.524) (OR=1.862, 95%CI: 1.261-2.749). CONCLUSION: Genetic polymorphisms of rs17587 appeared to be associated with RA in ethnic Han Chinese from Yunnan.

[Association of polymorphisms of PTPN22 and PADI4 genes with rheumatoid arthritis in Yunnan].

OBJECTIVE: To assess the association between genetic polymorphisms of 7 SNPs in PTPN22 and PADI4 genes and susceptibility to rheumatoid arthritis in Yunnan. METHODS: A case-control study was carried out on 192 patients of rheumatoid arthritis and 288 healthy controls. Genotypes of rs33996649 and 1858 loci within PTPN22 gene, and rs11203366 and rs874881 loci within PADI4 gene were determined with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Genotypes of rs1635579, rs2428736 and rs2240340 in PADI4 gene were determined with pyrosequencing. RESULTS: The frequencies of alleles and genotypes of rs2240340 locus in PADI4 gene showed a significant difference between rheumatoid arthritis and controls in Yunnan population (P U+003C 0.05). CONCLUSION: Our results suggested that rs2240340 in PADI4 gene is associated with susceptibility to rheumatoid arthritis in Yunnan.

[The research progress of human genetic diversity in China].

Human genetic diversity refers to genomic variation among races, ethnic groups, isolated populations and individuals worldwide, and is one major resource and tool on discovering human evolution and migration, interaction between genetic background and environment, and factors associated with human diseases and health. China has abundant and valuable resource of human genetic diversity due to 56 ethnic groups and a large population accounting for one fifth of the total population in the world. After decades of efforts, a large number of research data on human genetic diversity have been accumulated in China, and some of outcomes reach advanced international level. This review mainly focuses on the recent progress and outcomes achieved in applying genetic markers including morphological markers, biochemical and immunological markers and DNA markers in research of genetic diversity, and the application of mitochondrial DNA, Y chromosomal DNA, HLA and others in research of the origin and relationship of Chinese ethic groups, and the origin and mi-of modern East Asian populations. This review also summarizes the advances in the research fields of preservation and utilization of Chinese genetic resource, identification of genes associated with disease selective and adaptive for natural pressure, application of whole genome association study and next generation sequencing, and Chinese human genome as well.