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

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.

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.

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

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

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.

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 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.

Objective:To explore the macroporous resin adsorption and the membrane separation technologies for the purification of saponins water extract of Anemarrhena asphodeloides. Methods:Ten-fold amount of water was used to extract twice for 120 min each time to extract saponins from Anemarrhena asphodeloides. The macroporous resin adsorption(HP-20,HPD-600,D101,AB-8) and the membrane separation technologies (ceramic microfiltration membranes 0.8 μm and 0.05 μm, and hollow fiber ultrafiltration mem-branes 50,10 and 6 kDa) were adopted to purify the saponins water extract liquid. The physicochemical parameters including electri-cal conductivity,viscosity and turbidity were measured,as well as the contents of total saponins,proteins and polysaccharides were de-termined. Results:The viscosity and turbidity decreased,the value of pH increased and the electrical conductivity of the saponins puri-fication liquid changed irregularly after the membrane filtration. The microfiltration membrane was more advantageous than the ultrafil-tration membrane in removing macromolecular substances. The smaller the pore diameter of microfiltration membrane, the smaller the intercepted molecular weigh,the higher the removal ratio of proteins and the higher the penetration rate of the total saponins,while the polysaccharides content was stable, which was consistent with the results of physicochemical parameters. The ceramic microfiltration membrane could obtain clearer extract,while the ultrafiltration membrane was more suitable for the enrichment of saponins when the in-tercepted molecular weight was 6 kDa. The macroporous resin HPD-600 was the best for the purification of timosaponin water extraction liquid.Conclusion:The selection of membrane for the separation and purification of different substances is particularly important. The change of physicochemical parameters and the content decrease of macromolecular substances have obvious corresponding relationship. Ultrafiltration membrane is better than microfiltration membrane for the purification of timosaponin water extract liquid.