Clinical feature and genetic analysis of a patient with Idiopathic hypogonadotropic hypogonadism due to a novel variant of CHD7 gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the clinical feature and genetic etiology of a patient with normosmic idiopathic hypogonadotropic hypogonadism (nIHH) due to variant of CHD7 gene.@*METHODS@#A patient who had presented at Anhui Provincial Children's Hospital in October 2022 was selected as the study subject. Clinical data of the patient was collected. The patient and his parents were subjected to trio-whole exome sequencing. Candidate variant was verified by Sanger sequencing and bioinformatic analysis.@*RESULTS@#The patient had featured delayed development of secondary sexual characteristics but normal olfactory function. Genetic testing revealed that he has harbored a c.3052C>T (p.Pro1018Ser) missense variant of the CHD7 gene, for which both of his parents were of the wild type. The variant has not been recorded in the PubMed and HGMD databases. Analysis of amino acid sequences suggested that the variant site is highly conserved, and the variant may affect the stability of protein structure. Based on the guidelines from the American College of Medical Genetics and Genomics, the c.3032C>T variant was classified as a likely pathogenic (PS2+PM2_Supporting+PP2+PP3+PP4).@*CONCLUSION@#The delayed development of secondary sexual characteristics of the patient may be attributed to the c.3052C>T (p.Pro1018Ser) variant of the CHD7 gene. Above finding has expanded the variation spectrum of the CHD7 gene.

Expressions of miRNA-146a in peripheral mononuclear cells of children with systemic juvenile idiopathic arthritis and its clinical significance / 中华实用儿科临床杂志

Objective:To investigate the role of miR-146a in the pathogenesis of systemic juvenile idiopathic arthritis (sJIA) and its clinical significance.Methods:This article is a prospective clinical cohort study.Twenty-six patients with sJIA (14 cases of initial active group and 12 cases of stable group), 15 patients with multijoint juvenile idiopathic arthritis (JIA) and 15 patients with oligojoint JIA diagnosed in the Department of Rheumatology and Immunology of Anhui Provincial Children′s Hospital from June 2018 to December 2020 were enrolled.Twenty healthy controls from the out-patient clinic were also recruited.The expression level of miR-146a in peripheral blood mononuclear cells (PBMCs) of research objects was detected by real-time fluorescence quantitative polymerase reaction (qPCR), and the serum levels of interleukin (IL) - 6, tumor necrosis factor (TNF) - α and IL-1β in sJIA patients and healthy controls were detected by enzyme-linked immunosorbent assay.The expression levels of miR-146a in PBMCs and cytokines among different groups were compared by analysis of variance. Pearson correlation analysis was used to analyze the correlation of the relative expression level of miR-146a in PBMCs with clinical inflammatory indexes and serum cytokines in sJIA patients. Results:(1) The expression level of miR-146a in PBMCs of early sJIA patients was significantly higher than that in the multijoint JIA group and oligojoint JIA group (8.77±3.15 vs.4.40±1.59, 2.55±1.15, t=6.27, 14.23; all P<0.05). The expression level of miR-146a in PBMCs of sJIA active patients was significantly higher than that in sJIA stable patients (8.77±3.15 vs.3.63±1.37, t=10.27, P<0.05). There was no significant difference in the expression level of miR-146a between the sJIA stable group and healthy control group ( P>0.05). (2) The expression levels of IL-1β, IL-6 and TNF-α were significantly higher in sJIA active patients group than those in sJIA stable group[(58.56±17.47) ng/L vs.(26.32±10.54) ng/L, (73.72±11.16) ng/L vs.(23.20±9.12) ng/L, (70.93±19.97) ng/L vs.(24.25±9.49) ng/L, all P<0.05]. There was no significant difference in the expression levels of IL-1β, IL-6 and TNF-α between the sJIA stable group and healthy control group(all P>0.05). (3)The expression of miR-146a in PBMCs of sJIA patients was positively correlated with serum ferritin levels, platelets, erythrocyte sedimentation rates, C-reactive proteins, IL-1β, IL-6 and TNF-α( r=0.542, 0.433, 0.329, 0.306, 0.333, 0.342, 0.319, all P<0.05). Conclusions:miR-146a may be involved in the inflammatory process of sJIA disease.miR-146a can well distinguish sJIA from multijoint JIA and oligojoint JIA.TNF-α, IL-1β and IL-6 are involved in sJIA inflammatory responses.

Clinical characteristics and genetic analysis of an ethnic Han Chinese child with Keppen-Lubinsky syndrome due to a de novo KCNJ6 mutation / 中华医学遗传学杂志

OBJECTIVE@#To investigate the clinical characteristics and genetic basis for a child with Keppen-Lubinsky syndrome (KPLBS).@*METHODS@#Trio-whole exome sequencing (Trio-WES) was carried out for the proband and her parents. Candidate variant was verified by Sanger sequencing and bioinformatic analysis.@*RESULTS@#The child has featured peculiar facies including large eyes, alar hypoplasia, microretrognathia, premature aging appearance in addition with growth delay and mental retardation. Trio-WES has identified that she has carried a de novo variant of the KCNJ6 gene, namely c.460G>C (p.Gly154Arg). The variant has not been recorded in the database. Prediction of protein structure indicated that the variant may affect the potassium ion selective filtration structure channel in the transmembrane region of KCNJ6 protein, which may result in up regulation of the function of the channel.@*CONCLUSION@#The de novo c.460G>C (p.Gly154Arg) variant of the KCNJ6 gene probably underlay the KPLBS in this child. Above finding has enriched the genotypic and phenotype spectrum of this syndrome.

Correlation of type 2 diabetes and impaired glucose regulation with chronic kidney disease in middle-aged and elderly individuals / 南方医科大学学报

OBJECTIVE@#To explore the correlation of different glucose metabolism statues with chronic kidney disease (CKD) in middle-aged and elderly individuals in Lanzhou.@*METHODS@#Based on the baseline data of REACTION Study in Lanzhou area, we randomly sampled 10 038 residents aged 40-75 years in 3 communities in Lanzhou, who were classified into normal glucose tolerance (NGT), impaired glucose regulation (IGR) and diabetes groups. The estimated glomerular filtration rate (eGFR) and urinary albumin-to-creatinine ratio (ACR) were used to assess the renal function and albuminuria, respectively. Binary logistic regression was performed to analyze the contribution of the risk factors to CKD. Polynominal regression was used to determine the trends of eGFR with the increment of ACR.@*RESULTS@#Among all the participants, the prevalences of albuminuria, CKD and renal insufficiency (RI) were 26.2%, 27.4% and 2.5%, respectively. The prevalence of albuminuria, CKD and RI were significantly higher in the diabetes group than in IGR and NGT groups (@*CONCLUSIONS@#Diabetes mellitus is a significant risk factor for albuminuria and RI, while IGR is not. Screening for albuminuria and eGFR is highly recommended for individuals with diabetes, hypertension, and obesity, especially in women and the elderly population.

Two-plasmid scarless genetic modification in Escherichia coli hfq and rne-710 / 生物工程学报

Gene modification is an important technique to understand gene function. We firstly constructed Δhfq::Spe and Δrne-710::Spe mutant strains of Escherichia coli MG1655. The fragment lacking of hfq and rne-710 was ligated to the auxiliary plasmid and separately replace the spectinomycin box by homologous recombinase system to obtain the Δhfq and Δrne-710 mutant strains. The combination of two-plasmid scarless genetic modification and fusion PCR led to a new way for the long DNA fragment gene deletions.

Advance in research on regulation of sRNAs in bacterial biofilm formation / 军事医学

Small RNAs(sRNAs) play a significant role in the regulation of bacterial growth.When sensing certain environmental cues such as fluctuation of nutrient concentration, temperature, pH, and osmolarity, sRNAs can influence the expression of target genes.The formation of biofilms is initiated by bacteria transitioning from the planktonic to the surface-associated mode of growth, which is a self-produced extracellular matrix composed of proteins, polysaccharides, and DNA.Recent evidences have shown that small RNA plays an important role in the regulation of bacterial biofilm formation.sRNAs have key roles in biofilm formation process by base pairing with target mRNAs or interaction with modulating proteins.This review discussed the regulation mechanism and pathway of sRNAs in bacterial biofilms formation, and summarized three classical regulatory models of sRNAs in bacterial biofilms formation, this review also gives the research status and development direction of sRNAs in bacterial biofilms formation.

Analysis on Heart Rate Variability and Its Influencing Factors of Hypertensive Patients from Different TCM Syndrome / 中国中医药信息杂志

Objective To investigate the syndrome type distribution of hypertensive patients; To analyze the correlation of characteristics of HRV time domain parameters and its influence factors. Methods Totally 515 cases of hypertensive patients were included and were put under syndrome type distribution. Demographic information, laboratory test parameters, risk factors and clinical symptoms were collected for correlation analysis. HRV time domain parameters were recorded by using 24 h ambulatory electrocardiogram. The differences in SDNN, SDNN Index, HRV Index, PNN50, and RMSSD of different TCM syndrome types were compared. Results Among 515 patients: 160 cases with hyperactivity of yang due to yin deficiency syndrome, 136 cases with turbid phlegm and blood stasis syndrome, 83 cases with overabundant liver-fire syndrome, 69 cases with deficiency of kidney qi, and 67 cases with abundant phlegm-dampness syndrome. By comparing different TCM syndromes, the level of SDNN was significantly reduced in the hyperactivity of yang due to yin deficiency syndrome, overabundant liver-fire syndrome,deficiency of kidney qi syndrome compared with turbid phlegm and blood stasis syndrome, abundant phlegm-dampness syndrome (P<0.05); SDNN Index and HRV Index decreased significantly in the hyperactivity of yang due to yin deficiency and overabundant liver-fire syndrome compared with abundant phlegm-dampness syndrome (P<0.05). SDNN Index decreased significantly in the deficiency of kidney qi compared with abundant phlegm-dampness syndrome (P<0.05). The level of PNN50 was significantly reduced in the deficiency of kidney qi compared with hyperactivity of yang due to yin deficiency syndrome (P<0.05). RMSSD decreased significantly in the hyperactivity of yang due to yin deficiency syndrome, deficiency of kindney qi syndrome, overabundant liver-fire syndrome compared with turbid phlegm and blood stasis syndrome (P<0.05). Discriminant analysis showed that SBP, DBP, MBPS, SDNN, SDNN Index, HRV Index, PNN50, RMSSD were correlated with the diagnosis of five syndrome types. Logistic regression analysis showed that the factors including gender (female), insomnia, elevated systolic blood pressure, MBPS, decreased SDNN Index and PNN50 were positively correlated to hyperactivity of yang due to yin deficiency; other factors including gender (female), advanced age, elevated blood pressure, decreased SDNN, HRV Index and RMSSD were positively correlated with turbid phlegm and blood stasis syndrome. And the study also showed that advanced age, family history of hypertension, elevated blood pressure, decreased SDNN Index, HRV Index and PNN50 were positively correlated to abundant phlegm-dampness syndrome. Conclusion HRV time domain parameters can be significantly reduced in the hyperactivity of yang due to yin deficiency, overabundant liver-fire syndrome, and deficiency of kidney qi syndrome. The autonomic nerve function is damaged seriously. Hyperactivity of yang due to yin deficiency syndrome, abundant phlegm-dampness syndrome turbid phlegm and blood stasis syndrome are closely related to the influencing factors that lead to cardiovascular and cerebrovascular events.

Association between serum Klotho and oxidative stress in metabolically healthy obese individuals / 安徽医科大学学报

Objective To investigate the association between serum Klotho and oxidative stress in metabolically healthy obese individuals. Methods 180 physicalexamination subjects were investigated. They were devided into 3 groups:60 cases of normal weight and metabolic normality,60 cases of metabolically healthy but obese( MHO) ,60 cases of obesity with metabolic syndrome. Fasting blood glucose(FBG),2 h blood glucose(2 hBG),triglyceride ( TG) ,high densitylipoprotein cholesterol( HDL-C) , systolic blood pressure, diastolic blood pressure,body height, body weight,waist circumference,body mass index( BMI) were recorded. Malondialdehyde( MDA) ,superoxide dis-mutase( SOD) ,total antioxidation capacity( TAOC) ,Klotho were detected by ELISA. The difference of clinical pa-rameters,metabolic parameters,oxidative stress and Klotho among these three groups was compared by the methods of covariance analysis. Regression analysis and pearson correlation were used to evaluate the relationship of Klotho with oxidative stress. Results Compared with the control group, TAOC, SOD, Klotho were decreased significantly while MDA elevated significantly in both MHO group and obesity with metabolic syndrome group(P<0. 05). Com-pared with MHO group, TAOC, Klotho were significantly lower in obesity with metabolic syndrome ( P<0. 05 ) . Klotho protein was significantly positively associated with SOD, TAOC, negatively associated with waist circumfer-ence,BMI,FBG,2 hBG(r= -0. 182,-0. 225,-0. 221,-0. 202,-0. 188,P<0. 05). SOD,TAOC were deter-minants for Klotho. Conclusion The balancebetween oxidative and antioxidative system is disturbed in subjects with MHO. Klotho protein may maintain the normal metabolism of thebody by regulating the oxidative stress.

Expression and purification of virulence protein Pla of Yersinia pestis and its activity / 军事医学

Objective To prepare recombinant plasminogen activator(Pla) protein in E.coli BL21 cells that can be used in studying interactions between Yersinia pestis proteins and immunologic diagnosis of plague.Methods The pla gene was amplified by PCR and cloned into the pET28a expression vector.E.coli BL21 competent cells were transformed with the recombinant vectors, and isopropyl-β-D-thiogalactopyranoside ( IPTG) was added to induce expression of Pla protein. The expressed protein was detected by SDS-PAGE electrophoresis.The inclusion bodies of Pla protein were denatured in 8 mol/L urea, and then refolded using gradient urea solutions.The purified protein was identified by SDS-PAGE electrophoresis and Western blot.Results and Conclusion The constructed expression vector was demonstrated to be correct through agarose gel electrophoresis and sequencing.The recombinant Pla protein was accumulated as an inclusion body in E.coli, and the overexpression product was mainly a target protein, the yield of which was very high.SDS-PAGE purity of the bioactive Pla protein was obtained by denaturing and refolding the inclusion bodies.This study provides a simple and quick method for highly efficient preparation of biologically active Pla protein.

Screening, identification and function of pPCP1 plasmid-derived sRNA in Yersinia pestis:a preliminary study / 军事医学

Objective To identify small non-coding RNAs encoded by plasmid pPCP1 and investigate their roles in biofilm formation, stress tolerance and/or virulence in Yersinia pestis.Methods Seven plasmid pPCP1-encoded sRNAs were identified by RNA-seq results in Y.pestis in our previous studies.Northern blot was used to validate the presence of the seven sRNAs.The sRNA-deletion mutants were constructed via λ-Red homologous recombination system.The biofilm formation, high salt tolerance and virulence of the phenotypes were compared between Y.pestis WT strain and sRNA mutants.Results and Conclusion The expression of seven pPCP1-encoded sRNAs was validated and the transcript length detected by Northern blotting corresponded to the length observed by RNA-seq.On this basis, five sRNA-deletion mutants were obtained.The capacity of biofilm formation was weakened upon deletion of sR3446.The tolerance of sR3446, sR3457, sR4338 and sR4340 mutants was found weakened in vitro compared to that of wild-type strain,but the tolerance of sR6143 was found increased.Slight virulence attenuation was found in two sRNA mutants ( sR4338 and sR4340 ) .The results suggest that pPCP1-deriving sRNA might be implicated in stress response, biofilm and virulence in Y.pestis.

Loop-mediated isothermal amplification for quick detection of Yersinia pestis / 军事医学

Objective To establish a simple and quick loop-mediated isothermal amplification (LAMP)method for detection of Yersinia pestis.Methods LAMP Primers were designed based on the specific sequence 3a in Y.pestis chromosome.LAMP reaction results were detected using turbidity meter or visual method.The specificity of the constructed method was evaluated by detecting Y.pestis and its closely-related bacteria.The different dilution DNA template was detected with LAMP and PCR to evaluate the sensitivity of the method.Results Thirty strains of bacteria closely related to Y.pestis were detected by the constructed LAMP,and all the results were negative,indicating that the method had a very high specificity.The detection sensitivity of this LAMP assay was 20 pg of DNA per reaction,which was ten-fold that of the regular PCR.The detection reaction was completed in 25 min.Conclusion This LAMP method is quick,sensitive, specific and simple,which is expecked to become an effective method for rapid detection of Y.pestis on the scene.

Transfusion of necrotic cells redresses regulatory T cell and Th17 cell imbalance in septic mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of transfusion of necrotic cells on regulatory T (Treg) and Th17 cell balance in septic mice.</p><p><b>METHODS</b>Thirty-four C57BL/6 mice were randomized into PBS group (n=5), sham-operated group (n=5), sepsis group (n=12), and necrotic cell transfusion group (n=12) and subjected to intraperitoneal PBS injection, sham operation by separating the cecum only, cecal ligation and puncture (CLP), and injection of 2 × 10⁷ necrotic cells 5 days before CLP, respectively. All the mice were sacrificed 2 weeks after CLP for analyzing the proportion of CD4⁺Foxp3⁺Treg cells and CD4⁺IL17A⁺Th17 cells in the peripheral blood, spleen and thymus by flow cytometry.</p><p><b>RESULTS</b>The percentage of Th17 cells and Treg/Th17 ratio in the spleen was significantly higher in CLP group than in the sham-operated group and PBS group (P<0.01). The percentage of Treg cells in the thymus was significantly lower in CLP group than in the sham-operated group (P<0.01). Pre-infusion of necrotic cells redressed the abnormality of Treg and Th17 cell percentages and Treg/Th17 imbalance in mice following CLP (P<0.05).</p><p><b>CONCLUSION</b>Pre-infusion of necrotic cells can reverse Treg/Th17 imbalance in septic mice.</p>

Development of whole-genome DNA microarray of Yersinia pestis and its validity in comparative genomic analysis / 解放军医学杂志

Objective To develop a whole-genome DNA microarray based on the genomic sequences of Y. pestis CO92 and 91001 and its use in comparative genomic analysis. Methods A total number of 4 005 genes of Y. pestis were amplified by PCR and printed onto glass slides in duplicate. Fluorescently labeled probes were prepared by marking genomic DNAs with random hexamers and Klenow. Labeled DNAs were hybridized with the microarrays by the method of two-fluorescence comparative hybridization. Three sets of two-fluorescence hybridizations were performed to examine the absence/presence of each gene. Results The results agreed with those derived from the in silico genomic comparison. Conclusion The results demonstrate that the microarry can be a useful tool for comparative genomic analysis of Y. pestis.

DNA microarray analysis of genome dynamics in Yersinia pestis: insight into bacterial genome microevolution and niche adaptation / 解放军医学杂志

Objective To study the genome dynamics of Y. pestis and look for the relationship between its genome microevolution and niche adaption.Methods The DNA microarray combined with PCR was used to perform comparative genomic analysis of natural populations of Y.pestis.Results It was revealed that considerable genome dynamics of Y. pestis were the result of gene acquisition and loss in genome. We established a genomotyping system to group homologous isolates of Y. pestis, drew an outline of parallel microevolution of the Y. pestis genome, and established the link between the bacterial niche adaptation and genome microevolution.Conclusion The transmission, colonization and expansion of Y. pestis in natural foci are the results of its parallel, directional and gradual adaptation to the complex interactions among the environment, the hosts, and the pathogen itself.

Detection of DNA tag sequences for rapid identification of Yersinia pestis / 解放军医学杂志

Objective To identify the DNA tag sequences with the purpose of rapid and specific characterization of Y. pestis. Methods DNA microarray hybridization combined with PCR was used to perform genomic comparison between strains of Y. pestis and Y. pseudotuberculosis in order to screen and identify Y. pestis-specific genes. Results Twenty eight signature genes of Y. pestis were discovered. Three pairs of Y. pestis-specific primers were designed according to tag genes and proved to amplify the specific sequences of the target bacterium, showing no cross-reaction with the closely related Y. pseudotuberculosis and a large collection of genomic DNAs from other organisms. Conclusion DNA tag sequence is an ideal target for the rapid detection and identification of Y. pestis by PCR method.

DNA microarray-based comparative genomic analysis of live plague vaccines / 解放军医学杂志

Objective To identify and compare the genome differences among live plague vaccines prepared with different strains of the bacillus. Methods The whole-genome DNA microarray of Yersinia pestis was used as a tool to perform genomic comparison among live plague vaccines prepared with 19 different strains. Results Dozens of deletions and/or increased copies of the genomic fragments were identified in the studied vaccines of different strains. Conclusion The revealed genomic differences among the vaccines from different origins account for the variability of the immunogenic and protective potency of live plague vaccines. The whole-genome DNA microarray was also provesd to be an ideal tool for the pre-evaluation of a vaccine strain.