Objective:To establish a rapid pipeline for whole genome sequencing of Chikungunya virus (CHIKV) by combining imbricated multiplex-PCR amplification and Illumina high-throughput sequencing platform.Methods:The primary reference sequences of CHIKV were downloaded from the National Center for Biotechnology Information (NCBI) database, covering all genotypes of CHIKV. After multiple alignments using the Mafft software and phylogenetic analysis, the 20 CHIKV references were selected for primer design. The Primal Scheme tool and Geneious Prime software were used to design, evaluate and optimize the primer panel. Finally, seven CHIKV-positive samples were involved in the validation of the primer panel.Results:All the amplicons of the designed panel were generated successfully. The consensuses generated from the mapping results could cover 100.00% of the coding region of the CHIKV genome when the Ct-value of the sample was less than 33, as the percentage would decrease to 99.38% when the Ct-value reached 35. The mapping percentage could be increased by 5.70%-25.43% when using the stepwise correction mapping strategy.Conclusion:The multiplex-PCR amplification method for CHIKV whole genome sequencing is relatively simple and convenient, which only requires two tubes of PCR amplification and performs well on CHIKV-positive clinical samples with different concentration levels of virus.

Objective:To establish a rapid pipeline for whole genome sequencing of Chikungunya virus (CHIKV) by combining imbricated multiplex-PCR amplification and Illumina high-throughput sequencing platform.Methods:The primary reference sequences of CHIKV were downloaded from the National Center for Biotechnology Information (NCBI) database, covering all genotypes of CHIKV. After multiple alignments using the Mafft software and phylogenetic analysis, the 20 CHIKV references were selected for primer design. The Primal Scheme tool and Geneious Prime software were used to design, evaluate and optimize the primer panel. Finally, seven CHIKV-positive samples were involved in the validation of the primer panel.Results:All the amplicons of the designed panel were generated successfully. The consensuses generated from the mapping results could cover 100.00% of the coding region of the CHIKV genome when the Ct-value of the sample was less than 33, as the percentage would decrease to 99.38% when the Ct-value reached 35. The mapping percentage could be increased by 5.70%-25.43% when using the stepwise correction mapping strategy.Conclusion:The multiplex-PCR amplification method for CHIKV whole genome sequencing is relatively simple and convenient, which only requires two tubes of PCR amplification and performs well on CHIKV-positive clinical samples with different concentration levels of virus.

Objective To investigate the changes in blood microbiota in patients with high altitude polycythemia(HAPC)and the correlation with the risk of HAPC.Methods A cross-sectional trial was carried out among 41 HAPC patients(HAPC group)and 41 healthy plateau individuals(control group)who took physical examination in the Health Management Department of No.953 Hospital of PLA Army in 2021.High-throughput sequencing technology was used to detect the V3-V4 variable region of the 16S ribosomal RNA(16S rRNA)gene in the blood smaples,and the composition and difference of the blood microbiota were compared and analyzed between the 2 groups.Results All the participants were male and Han people,and there were no significant differences in baseline data such as age,body mass index and plateau migration time between the 2 groups(P＞0.05).The α-diversity of blood microbiota in the HAPC group,including the Simpson index(0.931±0.005 vs 0.907±0.008,P＜0.05),Goods Coverage index(0.998±0.001 vs 0.997±0.001,P＜0.001)and Pielou index(0.597±0.011 vs 0.567±0.009,P＜0.05)were significantly higher than those in the control group.Meanwhile,obvious difference was observed in the β diversity between the 2 groups(P＜0.01).The relative abundance analysis of bacteria showed that Pelomonas(0.046±0.004 vs 0.033±0.003,P＜0.05),Azospirillum(0.046±0.006 vs 0.021±0.003,P＜0.01),Acidovorax(0.032± 0.003 vs 0.019±0.002,Azospirillum(0.046±0.006 vs 0.021±0.003,P＜0.01)and Acidovorax(0.032± 0.003 vs 0.019±0.002,P＜0.05)were statistically higher in the HAPC group than the control group.LEfSe analysis showed that the characteristic blood microbiota of the HAPC group were α-Proteobacteria,and those of control group were Trichospiridae.Conclusion Significant changes are found in diversity,relative abundance and characteristic bacteria of the blood microbiota between the HAPC patients and healthy people at the high altitude,which might be closely associated with the occurrence and development of HAPC.

Objective:To explore the potent effects of denatonium benzoate(DB)on Mas-related G protein-coupled receptor-B2(MrgprB2)-mediated rat mast cell degranulation.Methods:RBL-2H3 cells were treated with DB overnight,before challenged with MrgprB2 ligands substance P(SP).The release of β-hex from MrgprB2-activated RBL-2H3 was detected by substrate method.Detec-tion of LTC4,IL-6,TNF-α and cPLA2 activity were performed by ELISA.The Ca2+influx and the expression of RBL-2H3 MrgprB2 re-ceptors were measured by fluorescence assay.Results:The results showed 10 μmol/L,50 μmol/L,80 μmol/L,100 μmol/L DB treat-ments promoted β-hex and LTC4 releases from activated RBL-2H3,accompanied by increased Ca2+mobilization and cPLA2 activa-tion.DB treatments did not affect IL-6 and TNF-α LTC4 releases in MrgprB2-activated RBL-2H3,as well as the levels of MrgprB2 ex-pression in mast cells.Conclusion:Taken together,DB enhanced the MrgprB2-mediated RBL-2H3 degranulation in vitro,probably by up-regulating Ca2+mobilization in activated cells.

BACKGROUND: The presence of fibrosis is a criterion for subtype classification in the newly updated hypersensitivity pneumonitis (HP) guidelines. The present study aimed to summarize differences in clinical characteristics and prognosis of non-fibrotic hypersensitivity pneumonitis (NFHP) and fibrotic hypersensitivity pneumonitis (FHP) and explore factors associated with the presence of fibrosis. METHODS: In this prospective cohort study, patients diagnosed with HP through a multidisciplinary discussion were enrolled. Collected data included demographic and clinical characteristics, laboratory findings, and radiologic and histopathological features. Logistic regression analyses were performed to explore factors related to the presence of fibrosis. RESULTS: A total of 202 patients with HP were enrolled, including 87 (43.1%) NFHP patients and 115 (56.9%) FHP patients. Patients with FHP were older and more frequently presented with dyspnea, crackles, and digital clubbing than patients with NFHP. Serum levels of carcinoembryonic antigen, carbohydrate antigen 125, carbohydrate antigen 153, gastrin-releasing peptide precursor, squamous cell carcinoma antigen, and antigen cytokeratin 21-1, and count of bronchoalveolar lavage (BAL) eosinophils were higher in the FHP group than in the NFHP group. BAL lymphocytosis was present in both groups, but less pronounced in the FHP group. Multivariable regression analyses revealed that older age, <20% of lymphocyte in BAL, and ≥1.75% of eosinophil in BAL were risk factors for the development of FHP. Twelve patients developed adverse outcomes, with a median survival time of 12.5 months, all of whom had FHP. CONCLUSIONS Older age, <20% of lymphocyte in BAL, and ≥1.75% of eosinophil in BAL were risk factors associated with the development of FHP. Prognosis of patients with NFHP was better than that of patients with FHP. These results may provide insights into the mechanisms of fibrosis in HP.
Humans ; Bronchoalveolar Lavage Fluid ; Prospective Studies ; Alveolitis, Extrinsic Allergic/diagnosis* ; Fibrosis ; Carbohydrates

Humans ; Asthma/drug therapy* ; Biological Therapy

High Altitude Military Hygiene is the professional core course of high-altitude medicine, which is significant to the cultivation of military medical talents urgently needed by plateau troops. Under the background of "golden course" construction and army curriculum reform, aiming at the problems such as outdated content of course materials, single teaching mode and insufficient capacity of practical courses, we actively explored the effective path of "golden course" construction, including the renovation of the curriculum-construction concept, the optimization and reorganization of the teaching content, the expansion of case teaching and equipment teaching methods, and the implementation of curriculum ideological and political education and examination reforms. The reform has further improved the learning effect of students and the level of curriculum construction, and also provided beneficial reference for the construction of similar courses in military colleges and universities.

Objective:To explore the application of reflective circulation fusion link teaching in operating room nursing teaching.Methods:A total of 60 nursing students of operating room accepted by our unit from October 2019 to October 2020 were divided into the control group (30 cases, using routine nursing instruction of operating room) and the experimental group (30 cases, taking reflective circulation fusion link teaching). The assessment results, self-directed learning ability and satisfaction with the teaching before and after the teaching were compared between the two groups. SPSS 26.0 was used for t test and chi-square test. Results:The scores of theoretical knowledge and nursing skills assessment in the two groups were improved, and the scores of the experimental group after teaching were higher than those of the control group[(92.84±5.91) vs. (87.25±7.29); (91.68±7.14) vs. (85.96±8.62)] ( P<0.05); the two groups were both improved in terms of learning awareness, learning behaviors, learning strategies, and learning evaluation and interpersonal skills dimensions, and the scores of the above-mentioned dimensions of the test group after teaching were higher than those of the control group ( P<0.05); the satisfaction scores of improving learning initiative, deepening the understanding and mastery of theoretical knowledge, and enhancing operational skills were higher than those of the control group ( P<0.05). Conclusion:The application of reflective circulation fusion link teaching in nursing students of the operating room can not only enhance the students' assessment results and self-directed learning ability, but also improve their satisfaction of the teaching.

Objective: To investigate routine blood test results and secular changes among Tibetan children and adolescents aged from 3 to 19 in the plateau, and to provide the basis for reference range of routine blood test for this population. Methods: A total of 1 568 Tibetan children and adolescents aged from 3 to 19 living in Shigatse, Tibet were selected by cluster random sampling method. Routine blood test results and its secular trends were compared by age and gender. Results: Significantly differences were found in red blood cell(RBC), hemoglobin(HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin(MCH), white blood cell(WBC), neutrophil(NEU), neutrophil percentage (NEU%), lymphocyte(LYM), lymphocyte percentage(LYM%)，monocyte percentage(MON%)，eosinophil percentage(EOS%),basophil percentage(BAS%) and platelet(PLT) among the four age groups of 3-5, 6-12, 13-15, and 16-19 years ( F/H =60.22, 179.41, 249.45, 115.03, 74.90, 14.33 , 33.46, 78.90, 49.20, 97.29, 24.45,24.28,42.65,20.10, P <0.05). Among red blood cell indexes, RBC, HGB, HCT,MCH increased with age in boys( F =148.77, 493.04, 623.14, 249.92, P <0.05), but there was no similar trend in girls( F =1.37, 0.15, 2.94, 0.11, P >0.05). HCT showed significant sex differences among the four age groups of 3-5 years, 6-12 years, 13-15 years, and 16-19 years [(41.33±2.31)% vs (41.98±2.40)%; (43.28±2.60)% vs ( 43.75 ±2.36)%; (46.20±3.11)% vs (44.83±2.67)%; (51.10±4.15)% vs (43.61±4.70)%, t =-2.10, -2.88, 3.50, 10.82, P <0.05]. WBC, NEU, NEU%, LYM, LYM%, monocyte(MON), and MON% increased significantly with age in both boys and girls ( P <0.05). From the age of 12 to 13, RBC, HGB and HCT in Tibetan male and female adolescents showed an opposite trend and widened gradually. Conclusion Red blood cell index shows significantly different trends among Tibetan adolescents and children of different ages and genders. Regional nationality, age, gender, and other factors should be considered when developing the reference value range of blood routine index.

Objective:To investigate the pathogenic spectrum of hand, foot and mouth disease(HFMD)from 2017 to 2019 in Guangzhou, and analyze molecular epidemiological characteristics of coxsackievirus A6 (CV-A6).Methods:Enterovirus A71 (EV-A71), CV-A16, CV-A6 and enterovirus were tested by reverse transcription-quantitative polymerase chain reaction(RT-qPCR). The CV-A6 representative samples were isolated and the VP1 region of isolates were amplified and analized by Mega5.0 and SeqMen.Results:A total of 7 578 enterovirus-positive specimens were detected from 2017 to 2019, 320 specimens were positive for EV-A71, 1481 specimens were positive for CV-A16, 3171specimens were positive for CV-A6, and 2606 specimens were positive for other enterovirus. Children under the age of 5 years were the most vulnerable population, and the male/female incidence ratio was 1.56∶ 1.The incidence occurred in all seasons, one peak between May and July, the other between September and November. The virus was isolated from 80 CV-A6 positive specimens and the full length of VP1 gene region was sequenced and nucleotide sequence similarity analysis was performed. The nucleotide homology in the VP1 region was 93%-100%, and the amino acid homology was 98%-100%. The nucleotide homology with the CV-A6 prototype strain (Gdula) VP1 region was 79%-81%, and the amino acid homology was 95%-97%. The nucleotide homology with the representative strain of D3 subtype was 92%-98%, and the amino acid homology was 98%-100%. Phylogenetic tree shows that all CV-A6 belonged to the sub-genotype D3 and distributed in multiple branches.Conclusions:CV-A6 is emerging as one of the major pathogen causing HFMD in Guangzhou, and all insolates belonged to D3 subtype. Closely monitoring the molecular characteristics of CV-A6 and changes in the pathogen spectrum can provide scientific basis for HFMD prevention and control.