In order to analyze the correlation between critical residues in the catalytic centre of BSH and the enzyme substrate specificity, seven mutants of Lactobacillus salivarius bile salt hydrolase (BSH1) were constructed by using the Escherichia coli pET-20b(+) gene expression system, rational design and site-directed mutagenesis. These BSH1 mutants exhibited different hydrolytic activities against various conjugated bile salts through substrate specificities comparison. Among the residues being tested, Cys2 and Thr264 were deduced as key sites for BSH1 to catalyze taurocholic acid and glycocholic acid, respectively. Moreover, Cys2 and Thr264 were important for keeping the catalytic activity of BSH1. The high conservative Cys2 was not the only active site, other mutant amino acid sites were possibly involved in substrate binding. These mutant residues might influence the space and shape of the substrate-binding pockets or the channel size for substrate passing through and entering active site of BSH1, thus, the hydrolytic activity of BSH1 was changed to different conjugated bile salt.
Amidohydrolases ; genetics ; metabolism ; Bile Acids and Salts ; metabolism ; Escherichia coli ; metabolism ; Gene Expression ; Lactobacillus ; enzymology ; genetics ; Substrate Specificity

ObjectiveTo investigate the effect of focal ischemic preconditioning (IPC) on the expression of protein kinase-like endoplasmic reticulum kinase ( PERK ) and glucose-regulated protein 78 (GRP78) mRNA and protein after focal cerebral ischemia/reperfusion (I/R) in rats.MethodsAll 120 male SD rats were randomly divided into three groups: sham-operation group,middle cerebral artery occlusion (MCAO) group and brain ischemia preconditioning (BIP) group.Each group was further divided into 4 subgroups according to 12 h,1,2 and 3 d after I/R.The IPC models were made in order to measure the expression of PERK,GRP78 mRNA and protein by in situ hybridization and Western blot,and the apoptosis rate of neuron by flow cytometry. Results ①The expression of PERK mRNA increased and reached the peak at 12 h,then decreased continuously after 1 d.BIP could decrease its expression.The expression of PERK protein increased at 12 h and reached the peak at 24 h,then decreased continuously after 2 d.BIP could decrease its expression.②The expression both of GRP78 mRNA and its protein all increased and reached the peak at 12 h,then decreased continuously.BIP could increase their expression (mRNA:12 h: 136.70±9.53,F=32.265; 24 h:147.54 ±9.97,F=54.920; 2 d:158.16 ±9.44,F=45.374; 3d: 165.85±10.26,F=16.493,P＜0.05; protein:12 h: 1.319±0.116,F=5.619,P＜0.05; 24 h: 1.226±0.108,F=33.742,P＜0.01; 2 d:1.183 ±0.112,F =46.556,P ＜0.01; 3 d:1.115± 0.098,F =11.730,P＜0.05).③The rate of apoptosis neuron of rats in MCAO increased markedly at 12 h after reperfusion,and reached the peak at 1 d,then decreased continuously.BIP could decrease the rate of apoptosis neuron. Conclusion BIP can protect neurons through inhibiting the expression of PERK and inducing the expression of GRP78 after endoplasmic reticulum stress in rats.

Objective To investigate the changes of the axial length,corneal curvature and refractive power in infants with monocular congenital cataract.Methods A retrospective cohort study was carried out.The clinical data of 105 eyes of 105 cases with monocular congenital cataract who were received cataract extraction during January 2015 to December 2017 were collected in the First Affiliated Hospital of Zhengzhou University.According to the initial operation age,the infants were assigned to 3-6 month old group,7-12 month old group and 13-18 month old group.The patients were followed-up for 12 months.Axial length,corneal curvature and refractive power were recorded in 3,6 and 12 months after surgery.This study followed the Declaration of Helsinki,and written informed consent was obtained from each guardian prior to any medical examination.Results The increasing value of axial length in 3-6 month old group was larger than that in 7-12 month old group,and increasing value in 7-12 month old group was larger than that in 13-18 month old group (all at P＜0.001).The increasing value of axial length of operated eyes was larger than that of fellow eyes in both 3-6 month old group and 7-12 month old group during following-up,and so was in the 13-18 month old group at 6 months and 12 months after surgery (all at P＜0.001).The ocular axial length of operated eyes was significantly shorter than that of the fellow eyes in three groups before operation (t =-10.420,P＜0.001;t=-32.288,P＜0.001;t =-2.370,P =0.024),and at 12 months after operation,the axial length of operated eyes was longer than that of the fellow eyes in the 13-18 month old group (t =18.513,P＜0.001).No significant difference was found in the axial length between operated eyes and fellow eyes in both 3-6 month old group and 7-12month old group (P =0.051,0.062).The pre-operative corneal curvature was significantly larger than that of fellow eyes in both 3-6 month old group and 7-12 month old group (both at P＜0.01),and was not significantly different in 13-18 month old group (P =0.433).There were not significant differences in corneal curvature in the three groups at 12 months after surgery (all at P＞0.05).The change value of refractive power was gradually increased as aging at 12 months after surgery (all at P＜0.001).The surgical eye amplitude of change in refractive diopter was higher than healthy eye,with significant difference between them (P＜0.001).Conclusions The developing process of axial length and corneal curvature are interacted and coordinated in bilateral eyes in infants who received extraction surgery of monocular congenital cataract.It is better to remove the cataract as early as possible in infants.Refraction of bilateral eyes still has a trend to myopia,and the shifting amplitude of myopic power decreases with aging.

OBJECTIVETo investigate the diagnostic value of imageology of giant cell tumour of tendon sheath (GCTS) including X-ray, CT and MRI.

METHODSThirty-five patients with GCTTS confirmed by operation and pathology were retrospectively analyzed. There were 16 males and 19 females. The average age was 39.4 years, ranged from 7 to 66 years. All the patients underwent X-ray examination, 8 patients underwent CT examination, and 16 patients underwent MRI examination.

RESULTSThere were 2 patients in knee joint, 6 patients in ankle joint, 1 patient in capitulum radius, 2 patients in wrist joint, 14 patients in hand and 10 patients in foot. Ten cases were the diffuse form, and 25 cases were the focal form. The X-ray results: the slightly high density soft tissue mass surrounding the bone were shown in 32 cases, 3 cases were normal. The bone erosion were shown in 9 cases, the obvious destruction of bone were shown in 5 cases. CT results: The soft tissue mass and the destruction of bone were shown clearly. MRI results: On T1WI, the signal intensity of GCTTS almost was similar to those of skeletal muscle in 9 cases and was slightly lower than those of skeletal muscle in 7 cases. On T2WI, the signal intensity presented mainly hypointensity with patchy isointensity or hyperintensity signal. A little of fluid was shown in 6 cases.

CONCLUSIONX-ray can demonstrate the lesion and erosion of bone, destruction of bone can clearly be shown on CT. The low intensity signal on MRI T1WI and T2WI is the characteristic appearance of GCTTS. And it can clearly show the lesion range and type of GCTTS.

Adolescent ; Adult ; Aged ; Child ; Diagnosis, Differential ; Female ; Giant Cell Tumors ; diagnosis ; Humans ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Soft Tissue Neoplasms ; diagnosis ; Tendons ; pathology ; Tomography, X-Ray Computed

OBJECTIVE: To analyze the phenotype and genotype of a patient affected with inherited antithrombin deficiency. METHODS: All exons and exon-intron boundaries of the AT genes were subjected to PCR amplification and Sanger sequencing. The influence of variants on the disease was predicted using bioinformatic software (MutationTaster). RESULTS: The results of all coagulation tests were normal, though the antithrombin activity and antigen content of the proband and his father have decreased significantly (34%, 48% and 12.97 mg/dL, 15.60 mg/dL, respectively). His mother was normal. Genetic analysis revealed that the proband and his father both carried a heterozygous g.2736dupT variant of the AT gene. Bioinformatic analysis suggested that the variant may be pathogenic. CONCLUSION The proband and his father both had type I hereditary antithrombin deficiency caused by a g.2736dupT variant of the AT gene. The variant was unreported previously.
Antithrombin III/genetics* ; Antithrombin III Deficiency/genetics* ; DNA Mutational Analysis ; Genetic Testing ; Heterozygote ; Humans ; Male ; Mutation ; Pedigree

Objective: Schizophrenia (SCZ) is a severe psychiatric disorder with unknown etiology and lacking specific biomarkers. Herein, we aimed to explore plasma biomarkers relevant to SCZ using targeted metabolomics. Methods: Sixty drug-naïve SCZ patients and 36 healthy controls were recruited. Psychotic symptoms were assessed using the Positive and Negative Syndrome Scale. We analyzed the levels of 271 metabolites in plasma samples from all subjects using targeted metabolomics, and identified metabolites that differed significantly between the two groups. Then we evaluated the diagnostic power of the metabolites based on receiver operating characteristic curves, and explored metabolites associated with the psychotic symptoms in SCZ patients. Results: Twenty-six metabolites showed significant differences between SCZ patients and healthy controls. Among them, 12 metabolites were phosphatidylcholines and cortisol, ceramide (d18:1/22:0), acetylcarnitine, and γ-aminobutyric acid, which could significantly distinguish SCZ from healthy controls with the area under the curve (AUC) above 0.7. Further, a panel consisting of the above 4 metabolites had an excellent performance with an AUC of 0.867. In SCZ patients, phosphatidylcholines were positively related with positive symptoms, and cholic acid was positively associated with negative symptoms. Conclusion Our study provides insights into the metabolite alterations associated with SCZ and potential biomarkers for its diagnosis and symptom severity assessment.

OBJECTIVETo investigate the positive rate of hepatitis B surface antibody (HBsAb) in children.

METHODSBlood samples from 3022 children who received a physical examination in outpatient departments from 2009 to 2011 were subjected to serological test using ELISA to measure the positive rate of HBsAb.

RESULTSThe positive rate of HBsAb decreased with age (P<0.01). There was no significant difference in the positive rate of HBsAb between boys and girls (P>0.05), however the positive rate of HBsAb in boys aged one year and over was lower than in girls of the same age (P<0.01). The positive rate of HBsAb in boys aged between 3 and 4 years was higher than in girls of the same age (P<0.01). The positive rate of HBsAb decreased with age in boys, and was lower in those aged two years and over than in those aged one year and over (P<0.01). The positive rate of HBsAb also decreased with age in girls, with significant differences between different age groups (P<0.01).

CONCLUSIONSThe positive rate of HBsAb decreases with age in children, so younger children have a higher risk of infection with hepatitis B virus. Serological monitoring of hepatitis B needs to be enhanced.

Age Distribution ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis B Antibodies ; blood ; Hepatitis B Surface Antigens ; immunology ; Humans ; Infant ; Male

Objective To establish the method for extracting exogenous short DNA fragments of Schistosoma japonicum from urine samples, and to evaluate the efficiency of this method for extraction from urine samples treated with various methods. Methods The S. japonicum SjG28 gene fragment was selected as a target sequence, and the 81 bp short DNA fragment was amplified on the target sequence using PCR assay. Following characterization using sequencing, the short DNA fragment was added into the urine samples as an exogenous short DNA fragment. Primers and probes were designed with SjG28 as a target gene, to establish the real-time fluorescent quantitative PCR (qPCR) assay. The sensitivity of this qPCR assay was evaluated with exogenous short DNA fragments that were diluted at a 1:10 dilution ratio as the DNA template, and the specificity of the qPCR assay was evaluated with the genomic DNA of S. mansoni, S. haematobium, Babesia, Ancyiostoma duodenaie, Cionorchis sinensis, and Paragonimus westermani as DNA templates. Exogenous short DNA fragments were added into artificial and healthy volunteers’ urine samples, followed by pH adjustment, centrifugation and concentration, and the efficiency of extracting exogenous short DNA fragments from urine samples was compared with the QIAmp Viral RNA Mini Kit (Qiagen kit) and BIOG cfDNA easy kit (BIOG kit). Results An 81 bp small DNA fragment of S. japonicum was successfully prepared, and the lowest detection limit of the established qPCR assay was 100 copies/μL of the 81 bp small DNA fragment of S. japonicum. If the genomic DNA of S. japonicum, S. mansoni, S. haematobium, Babesia, A. duodenaie, C. sinensis, and P. westermani served as DNA templates, the qPCR assay only detected fluorescent signals with S. japonicum genomic DNA as the DNA template. If the pH values of artificial urine samples were adjusted to 5, 6, 7 and 8, the recovery rates were (49.12 ± 2.09)%, (84.52 ± 4.96)%, (89.38 ± 3.32)% and (87.82 ± 3.90)% for extracting the exogenous short DNA fragment of S. japonicum with the Qiagen kit, and were (2.30 ± 0.07)%, (8.11% ± 0.26)%, (13.35 ± 0.61)% and (20.82 ± 0.68)% with the BIOG kit, respectively (t = 38.702, 26.955, 39.042 and 29.571; all P values < 0.01). If the Qiagen kit was used for extracting the exogenous short DNA fragment from artificial urine samples, the lowest recovery rate was seen from urine samples with a pH value of 5 (all P values < 0.05), and there were no significant differences in the recovery rate from urine samples with pH values of 6, 7 and 8 (all P values > 0.05). Following centrifugation of artificial [(64.30 ± 1.00)% vs. (58.87 ± 0.26)%; t = 12.033, P < 0.05] and healthy volunteers’ urine samples [(31 165 ± 1 017) copies/μL vs. (28 471 ± 818) copies/μL; t = 23.164, P < 0.05]. In addition, concentration of artificial urine samples with the 10 kDa Centrifugal Filter and concentration of healthy volunteers’ urine samples with the 100 kDa Centrifugal Filter were both effective to increase the recovery of the Qiagen kit for extracting the exogenous short DNA fragment of S. japonicum (both P values < 0.01). Conclusions A method for extracting exogenous short DNA fragments of S. japonicum from urine samples has been successfully established, and the Qiagen kit has a high extraction efficiency. Adjustment of urine pH to 6 to 8 and concentration of healthy volunteers’ urine samples with the 100 kDa Centrifugal Filter are both effective to increase the efficiency of extracting exogenous short DNA fragments of S. japonicum.

Objective: To analyze the clinical epidemiological characteristics including composition of pathogens , clinical characteristics, and disease prognosis acute bacterial meningitis (ABM) in Chinese children. Methods: A retrospective analysis was performed on the clinical and laboratory data of 1 610 children <15 years of age with ABM in 33 tertiary hospitals in China from January 2019 to December 2020. Patients were divided into different groups according to age,<28 days group, 28 days to <3 months group, 3 months to <1 year group, 1-<5 years of age group, 5-<15 years of age group; etiology confirmed group and clinically diagnosed group according to etiology diagnosis. Non-numeric variables were analyzed with the Chi-square test or Fisher's exact test, while non-normal distrituction numeric variables were compared with nonparametric test. Results: Among 1 610 children with ABM, 955 were male and 650 were female (5 cases were not provided with gender information), and the age of onset was 1.5 (0.5, 5.5) months. There were 588 cases age from <28 days, 462 cases age from 28 days to <3 months, 302 cases age from 3 months to <1 year of age group, 156 cases in the 1-<5 years of age and 101 cases in the 5-<15 years of age. The detection rates were 38.8% (95/245) and 31.5% (70/222) of Escherichia coli and 27.8% (68/245) and 35.1% (78/222) of Streptococcus agalactiae in infants younger than 28 days of age and 28 days to 3 months of age; the detection rates of Streptococcus pneumonia, Escherichia coli, and Streptococcus agalactiae were 34.3% (61/178), 14.0% (25/178) and 13.5% (24/178) in the 3 months of age to <1 year of age group; the dominant pathogens were Streptococcus pneumoniae and the detection rate were 67.9% (74/109) and 44.4% (16/36) in the 1-<5 years of age and 5-<15 years of age . There were 9.7% (19/195) strains of Escherichia coli producing ultra-broad-spectrum β-lactamases. The positive rates of cerebrospinal fluid (CSF) culture and blood culture were 32.2% (515/1 598) and 25.0% (400/1 598), while 38.2% (126/330)and 25.3% (21/83) in CSF metagenomics next generation sequencing and Streptococcus pneumoniae antigen detection. There were 4.3% (32/790) cases of which CSF white blood cell counts were normal in etiology confirmed group. Among 1 610 children with ABM, main intracranial imaging complications were subdural effusion and (or) empyema in 349 cases (21.7%), hydrocephalus in 233 cases (14.5%), brain abscess in 178 cases (11.1%), and other cerebrovascular diseases, including encephalomalacia, cerebral infarction, and encephalatrophy, in 174 cases (10.8%). Among the 166 cases (10.3%) with unfavorable outcome, 32 cases (2.0%) died among whom 24 cases died before 1 year of age, and 37 cases (2.3%) had recurrence among whom 25 cases had recurrence within 3 weeks. The incidences of subdural effusion and (or) empyema, brain abscess and ependymitis in the etiology confirmed group were significantly higher than those in the clinically diagnosed group (26.2% (207/790) vs. 17.3% (142/820), 13.0% (103/790) vs. 9.1% (75/820), 4.6% (36/790) vs. 2.7% (22/820), χ²=18.71, 6.20, 4.07, all P<0.05), but there was no significant difference in the unfavorable outcomes, mortility, and recurrence between these 2 groups (all P>0.05). Conclusions: The onset age of ABM in children is usually within 1 year of age, especially <3 months. The common pathogens in infants <3 months of age are Escherichia coli and Streptococcus agalactiae, and the dominant pathogen in infant ≥3 months is Streptococcus pneumoniae. Subdural effusion and (or) empyema and hydrocephalus are common complications. ABM should not be excluded even if CSF white blood cell counts is within normal range. Standardized bacteriological examination should be paid more attention to increase the pathogenic detection rate. Non-culture CSF detection methods may facilitate the pathogenic diagnosis.
Adolescent ; Brain Abscess ; Child ; Child, Preschool ; Escherichia coli ; Female ; Humans ; Hydrocephalus ; Infant ; Infant, Newborn ; Male ; Meningitis, Bacterial/epidemiology* ; Retrospective Studies ; Streptococcus agalactiae ; Streptococcus pneumoniae ; Subdural Effusion ; beta-Lactamases