9α-hydroxy-4-androstene-3,17-dione (9-OH-AD) is an important intermediate in the steroidal drugs production. 3-ketosteroid-9α-hydroxylase (KSH), a two protein system of KshA and KshB, is a key-enzyme in the microbial steroid ring B-opening pathway. KSH catalyzes the transformation of 4-androstene-3,17-dione (AD) into 9-OH-AD specifically. In the present study, the putative KshA and KshB genes were cloned from Mycobacterium smegmatis mc(2)155 and Gordonia neofelifaecis NRRL B-59395 respectively, and were inserted into the expression vector pNIT, the co-expression plasmids of kshA-kshB were obtained and electroporated into Mycobacterium sp. NRRL B-3805 cells. The recombinants were used to transform steroids, the main product was characterized as 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), showing that kshA and kshB were expressed successfully. Different from the original strain Mycobacterium sp. NRRL B-3805 that accumulates 4-androstene-3,17-dione, the recombinants accumulates 9α-hydroxy-4-androstene-3,17-dione as the main product. This results indicates that the putative genes kshA, kshB encode active KshA and KshB, respectively. The process of biotransformation was investigated and the results show that phytosterol is the most suitable substrate for biotransformation, kshA and kshB from M. smegmatis mc(2)155 seemed to exhibit high activity, because the resultant recombinant of them catalyzed the biotransformation of phytosterol to 9-OH-AD in a percent conversion of 90%, which was much higher than that of G. neofelifaecis NRRL B-59395. This study on the manipulation of the ksh genes in Mycobacterium sp. NRRL B-3805 provides a new pathway for producing steroid medicines.
Androstenedione ; analogs & derivatives ; biosynthesis ; Bacterial Proteins ; genetics ; metabolism ; Biotransformation ; Ketosteroids ; Mixed Function Oxygenases ; genetics ; metabolism ; Mycobacterium ; metabolism ; Mycobacterium smegmatis ; enzymology ; Plasmids

Objective To diagnose 6 LQTS families by genetic analysis. Methods A total aof 6 LQTS pedigrees with 43 family members were brought together for genetic diagnosis by using short-sequence tandem-repeat (SIR) markers or sequencing. Genomic DNA was extracted from blood samples by standard procedure. STR markers or KCNQ1, KCNH2 and SCN5A were amplified. The haplotype analysis for LQTS was performed. If the family got the negative haplotype analysis, the sequencing was performed. Results LQTS patients were always linkaged with the SCNSA gene in family 1. KCNH2 was linkaged with the disease in family 2 to 5.21 gene carriers were identified from these 5 families. A mutation (A561V-KCNH2) was only found in the proband of family 6 and an SNP (G1691A) was found in all the members of the family. Conclusion Genetic diagnosis can not only improve presymptomatic diagnosis,bnt also provide the basis for personal therapy and research on disease-causing mutations.

AIM: To observe the effect of hypoxia on cardiaomyocytes apoptosis and the role of ALDH2 in the process. METHODS: Cultured cardiomyocytes of neonatal rats were used. Hypoxia was imposed to the cardiomyocytes with or without daidzin pretreatment. ALDH2 activity was measured by the method of acetaldehyde metabolism. Apoptosis was measured by Hoechest 33324 staining, fluorescence activated cell sorting (FACS) and the DeadEnd~ TM fluorometric TUNEL system. RESULTS: ALHD2 enzyme activity in myocytes was inhibited by daidzin (24 h, 60 ?mol/L) without induction of apoptosis. When exposed to hypoxia, however, the apoptisis was significantly increased in the cells pretreated with daidzin compared to those without the pretreatment. CONCLUSION: The reduction of ALDH2 activity might increase the susceptivity of myocytes to apoptosis following hypoxia, suggesting a protective role of ALDH2 in hypoxia-induced myocardial injury.

In order to explore new ways in restraining the ascending blood pressure, this paper reports a research on the effects of multi-mode audio frequency pulse modulating laser irradiation with electrical stimulation on the hemorrheology and blood pressure of the spontaneously hypertensive rats (SHR). Forty male SHR were randomly divided into four groups: Group A (control), Group B (treated with electrical stimulation on stomach 36 point (ST-36)), Group C (treated with low level pulse laser irradiation on Erjian acupuncture point), Group D (low level laser irradiation together with electrical stimulation on Erjian acupuncture point and ST-36). Laser irradiation lasted for 45 min/ day, while electrical stimulation lasted for 30 min/day. After 10 days' treatment, we compared the hemorrheology and blood pressure of the rats in the four groups. The results were that values of hemorrheology in group A and group C were obviously improved compared with those in the other two groups. Meanwhile, the blood pressures in the three treated groups (B, C, and D) were lower than in the control group after the treatment, and the value of the rats in group A was the lowest. In conclusion, the treatment the laser irradiation combined with electrodes stimulation on spontaneously hypertensive rats brought better results of hemorrheology and blood pressure in the tested rats.
Acupuncture Points ; Animals ; Blood Pressure ; physiology ; Blood Viscosity ; Electric Stimulation ; Electroacupuncture ; Hemorheology ; physiology ; Hypertension ; blood ; physiopathology ; therapy ; Laser Therapy ; Male ; Rats ; Rats, Inbred SHR

C-arm based surgical navigation system is a kind of computer assisted surgery (CAS) system. In this paper, key techniques concerning this kind of system are presented. The key techniques mainly include XRII image distortion correction and C-arm imaging system calibration. On this basis, we designed relevant tools and made them fabricated, and developed a software system. Experimental results of human fresh spine sample showed that with the surgical navigation system, screw insertion accuracy was much higher than that without the surgical navigation system.
Algorithms ; Calibration ; Humans ; Radiographic Image Enhancement ; methods ; Radiographic Image Interpretation, Computer-Assisted ; methods ; Spine ; surgery ; Surgery, Computer-Assisted ; methods

OBJECTIVETo analyze the hematological and genetic characteristics of unstable hemoglobin Rush (Hb Rush) and compound heterozygote of Hb Rush and thalassemia.

METHODSPeripheral blood samples and genomic DNA from three patients (including two ethnic Dai and one Han Chinese) with anemia of undetermined origin were collected. Hematological phenotypes of these patients were determined through red blood cell analysis and hemoglobin electrophoresis. Genotypes of alpha- and beta-globin genes, -158 XmnⅠ polymorphic site ofγ promoter region, and haplotypes of 7 polymorphic restriction sites in the beta-globin gene cluster were determined using PCR-based methods and DNA sequencing.

RESULTSAll patients have presented hypochromic microcytic anemia and hemoglobin fraction with significant increased measurement (30.5%-59.2%) in the region of fetal hemoglobin during alkaline medium electrophoresis. DNA analysis suggested that all patients have carried mutations leading to the unstable hemoglobin Rush (HBB codon 101, GAG>CAG, Glu>Gln). Two of them were compound heterozygotes of Hb Rush and thalassemia mutations of -α,CD17 and Hb E, respectively. Hb Rush mutation was associated with various haplotypes of the β-globin gene cluster. No significant association was found between increased abnormal hemoglobin fraction in the region of Hb F and the polymorphism ofγ promoter or large deletion of the beta-globin gene cluster.

CONCLUSIONThis study has confirmed the distribution of Hb Rush among various Chinese populations and is the third report of its kind. Hb Rush can result in increased measurement of hemoglobin fraction in the region of fetal hemoglobin (Hb F) during routine hemoglobin electrophoresis under alkaline condition. Hb Rush heterozygote alone can lead to hypochromic microcytic anemia and thalassemia-like phenotype. Prenatal diagnosis of Hb Rush is necessary for carriers.

Adult ; Base Sequence ; Blood Protein Electrophoresis ; methods ; Female ; Fetal Hemoglobin ; genetics ; metabolism ; Genotype ; Haplotypes ; Hemoglobins, Abnormal ; genetics ; metabolism ; Heterozygote ; Humans ; Infant ; Mutation ; Phenotype ; Polymorphism, Genetic ; Sequence Analysis, DNA ; methods ; Thalassemia ; blood ; diagnosis ; genetics ; Young Adult ; alpha-Globins ; genetics ; metabolism ; beta-Globins ; genetics ; metabolism

AIM:We investigated how AT 1-R stimulated by mechanical stresses induces cardiac fibrosis .METHODS:We produced in vivo cardiac pressure overload model in angiotensinogen knockout ( ATG-/-) mice and in vitro mechanically-stretched cell model in cultured neonatal cardiac cells of ATG-/-mice both lack the participation of Ang II .RESULTS: Pressure overload for 4 weeks in ATG-/-mice induced myocardial hypertrophy accompanied by the significant interstitial fibrosis , however , the TGF-β, a key regulatory factor of fibrosis, was not significantly increased in these ATG-/-mice.Meanwhile, the inhibitor for AT1-R significantly inhibited mechani-cal stress-induced cardiac fibrosis in these ATG-/-models whereas inhibition of TGF-βdid not.CONCLUSION:The results showed that mechanical stress-induced fibrotic responses through AT 1-R required the phosphorylation of Smad 2 but not the involvement of TGF-β.

Objective To explore the value of axis shift between the baseline normal sinus rhythm (NSR) and WCT in diagnosis of wide QRS-complex tachycardia (WCT). Methods 390 surface ECGs of 186 patients with WCT were obtained from April 2012 to April 2018 at Ningbo Medical Center Lihuili Hospital at which the arrhythmia diagnosis was proven by intracardiac electrophysiological study. The axis shift between the baseline NSR and WCT was calculated by table lookup method. Then we analyzed the role of axis shift in diagnosis of WCT. Results Among the 186 patients with WCT, 147 (79.03％) were ventricular tachycardia (VT) , and 39 (20.97％) were supraventricual tachycardia (SVT) with conduction abnormalities. In the 95％ confidence interval, the axis shift showed an outstanding discrimination performance. The area under the ROC curve is 0.708 (0.579-0.817, P =0.007). Compared with left axis deviation, right axis deviation, the right axis deviation of LBBB morphology, the axis shift> 68 degree is more sensitive (53.06％) , and the specificity (91.43％) is also more desirable. Moreover, if the axis shift set> 130 degree, the specificity can reach 100％, and the sensitivity (12.24％) is equivalent to northwestern axis. Conclusion A significant axis shift between the baseline NRS and WCT can distinguish WCT accurately. Given the ease of grasping, it can probably be feasible to popularize as a routine diagnosis method for WCT in primary hospitals.

Objective To investigate the influence of type 2 diabetes mellitus(T2DM)on cognitive function and the effective connectivity with in the default mode network(DMN)in the brain.Methods A total of 93 hospitalized patients diagnosed with T2DM were enrolled in this study as T2DM group from The First Affiliated Hospital of Guangzhou University of Chinese Medicine during September 2021 to December 2022.Simultaneously,108 healthy individuals were recruited from the community as normal control(NC)group.The cognitive functions were evaluated in the two groups.A random dynamic causal modeling approach was employed to analyze the effective connectivity within DMN in both groups.Additionally,Pearson correlation analysis was performed to examine the association between differential connectivity,clinical indicators,and cognitive scores in both groups.Results In comparison to the NC group,T2DM individuals exhibited statistically significant reductions in scores in the auditory verbal learning test(AVLT)for immediate recall and the digit symbol substitution test(DSST)(P＜0.05).Additionally,they displayed a notable decrease in effective connectivity from the left lateral parietal cortex(LLPC)to the posterior cingulate cortex(PCC),as well as from the LLPC to the right lateral parietal cortex(RLPC)within the DMN(P＜0.05).Pearson correlation analysis unveiled a negative association between HbA1c levels and the strength of effective connectivity from LLPC to PCC.Conversely,a positive correlation was observed between AVLT(immediate)scores and the strength of effective connectivity from LLPC to PCC and LLPC to RLPC.Additionally,DSST scores displayed a positive correlation with the strength of effective connectivity from LLPC to PCC(P＜0.05).Conclusion Patients with T2DM display compromised effective connectivity from LLPC to PCC and LLPC to RLPC within the DMN network,and this alteration may associated with cognitive impairment.

OBJECTIVETo analyze the clinical manifestations and intervention against fulminant scrub typhus-associated hemophagocytic syndrome.

METHODThe medical records for the onset time of hemophagocytic syndrome, the clinical course, the chest radiographic findings, laboratory data, antibiotic therapy, clinical outcome and its prognosis were retrospectively reviewed.

RESULT(1) Four patients were diagnosed as scrub typhus based on clinical manifestations only, while 15 patients met the criteria of laboratory diagnosis. All 19 patients with scrub typhus had hemophagocytic syndrome. Eschar lesion was identified in 12 patients, 7 patients were described as an ulcer. A seasonal pattern (78.9% from June through September in 15 patients) was observed. Clinical misdiagnosis was common (all 19 cases). There were 9 patients with admitting diagnosis of scrub typhus, 10 patients were not diagnosed as scrub typhus after admission. In 5 cases within 3 days after admission diagnosis was corrected as scrub typhus. Until discharge from the hospital, 5 cases were not diagnosed with scrub typhus. In this study, the length of time from the illness onset (beginning of fever) to the occurrence of clinical symptoms was (9 ± 4) days. (2) All 19 patients had changed AST levels (149 ± 37) U/L, albumin levels (23 ± 4) g/L, C-reactive protein levels (103 ± 51) mg/L, and platelet count (48 ± 41) × 10⁹/L; bone marrow aspiration revealed in 16 patients marked hemophagocytosis. Weil-Felix agglutination test revealed positive results in 6 of 15 cases. Diagnostic IFA results were positive for 14 patients; 19 patients had interstitial pneumonitis and 17 patients had pleural effusion. (3) Five cases with failure to diagnose the disease had ineffective antibiotics treatment (imipenem or β-lactam-based regimens). These patients did not receive appropriate treatment with antibiotics against scrub typhus. Fourteen patients with admitting diagnosis of scrub typhus were successfully treated with appropriate antibiotics, 8 cases with chloramphenicol, 3 cases with azithromycin, and in 3 patients (2 cases of azithromycin and one case of erythromycin), therapy was then switched to chloramphenicol. Four patients were treated with methylprednisolone and 10 patients with dexamethasone. (4) During their hospitalization, the clinical course in five cases with failure to diagnose the disease rapidly developed and progressed to the life-threatening MODS, four of five cases died. However, the course in 14 patients were relieved and did not progress to MODS.

CONCLUSIONThe diagnosis of scrub typhus was frequently delayed, the early course of scrub typhus could be associated with hemophagocytic syndrome. Serious complications of MODS generally occur without antibiotic treatment. Scrub typhus-associated hemophagocytic syndrome should be taken into consideration among patients with acute systemic febrile illness, significant increases in levels of CRP, hypoalbuminemia, thrombocytopenia, splenomegaly, pneumonitis with pleural effusion, especially those with suspected exposure history. It was not easily recognized without careful observation and was present for a few days in each patient.

Anti-Bacterial Agents ; therapeutic use ; Azithromycin ; therapeutic use ; C-Reactive Protein ; analysis ; Clinical Laboratory Techniques ; Diagnosis, Differential ; Erythromycin ; therapeutic use ; Humans ; Imipenem ; therapeutic use ; Lymphohistiocytosis, Hemophagocytic ; epidemiology ; Pneumonia ; Retrospective Studies ; Scrub Typhus ; diagnosis ; drug therapy ; epidemiology