We constructed a recombinant strain BL21 (DE3) (pETST3LTBalphabeta) including ST1-LT(B)-alpha-beta fusion gene via molecular technology. The SDS-PAGE and Western blotting indicated that the ST1-LT(B)-alpha-beta fusion protein was highly expressed in Escherichia coli and the molecular weight of the fusion protein was about 110 kD. The recombinant strain was induced in different concentrations of lactose and different aeration rate. The optimal culture conditions in 20 L fermentor were 1% inoculation (V/V), initial aeration 5 L/min, 0.03 mol/L lactose addition 3 hours after inoculation, and increased the aeration to 12.5 L/min for the following 6 hours. The fusion protein was about 38.53% of total cellular protein. It was nontoxic, immunogenic and protective against enterotoxigenic E. coli and Clostridium perfringens infection. The constructed recombinant strain BL21 (DE3) (pETST3LTBalphabeta) could serve as a candidate vaccine strain against diarrhea of piglet.
Animals ; Bacterial Toxins ; biosynthesis ; genetics ; Calcium-Binding Proteins ; biosynthesis ; genetics ; Clostridium perfringens ; genetics ; Dysentery ; prevention & control ; veterinary ; Enterotoxins ; biosynthesis ; genetics ; Escherichia coli ; genetics ; immunology ; Escherichia coli Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Transformation, Bacterial ; genetics ; Type C Phospholipases ; biosynthesis ; genetics ; Vaccines, Synthetic ; immunology

BACKGROUND:During ordinary plate fixation, the soft tissues around the fracture of the proximal humerus are nearly stripped to impact blood supply, and moreover, an ordinary steel plate cannot meet with the fixed requirements for severe osteoporosis, large bone defects and comminuted fractures. OBJECTIVE: To observe the functional recovery and complications in middle-aged patients with proximal humeral fractures undergoing stainless steel locking plate implantation. METHODS:From March 2011 to March 2014, 48 patients with proximal humeral fractures were treated in the 306th RESULTS AND CONCLUSION: The 48 patients were folowed up for 6-17 years, and the mean healing time was (15.3±1.2) weeks. At the last folow-up, the Neer scores were excelent in 12 cases, good in 22 cases, fair in 11 cases and poor in 3 cases, with an excelent-good rate of 71%. After internal fixation, there was one case of Hospital of PLA, including 20 males and 28 females, with an average age of 58 years. Of the 48 cases, there were 9 cases of Neer 2 fractures, 26 cases of Neer 3 fractures, and 13 cases of Neer 4 cases, al of which belonged to closed injuries. Al the patients were subject to locking plate implantation for repair of fractures of the proximal humerus, and evaluated based on Neer scores. soft tissue infection, two cases of traumatic arthritis, and no bone ununion and osteomyelitis. These findings suggest that the stainless steel locking plate implantation for repair of fractures of the proximal humerus can achieve a good anatomic reduction under minimaly invasive conditions and produce a stable rehabilitation environment for the soft tissue around the shoulder joint by internal fixation. Hence, the factures can recover faster with less complications. The stainless steel locking plate implantation can obtain good achievements in the repair of proximal humeral fractures of Neer 2, 3 as wel as Neer 4 in young patients with good bone quality.

Objective: To explore the effects of microRNA-34a on regulating silent information regulator 1 (SIRT1) and influence of SIRT1 on myocardial damage of rats with severe burns at early stage. Methods: (1) Twenty-four Sprague-Dawley (SD) rats were divided into sham injury (SI) group, simple burns (SB) group and SIRT1 agonist (SA) group according to the random number table (the same grouping method below), with 8 rats in each group. Rats in groups SB and SA were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burns) on the back, and rats in group SI were sham injuried on the back. Immediately after injury, rats in groups SI and SB were intraperitoneally injected with normal saline of 50 mL/kg, and rats in group SA were intraperitoneally injected with normal saline of 50 mL/kg and 1 mg/mL resveratrol of 50 mg/kg. At 6 h post injury, abdominal aortic blood was collected to make serum and myocardial tissue of rats was collected. (2) Myocardial cells of twelve neonatal SD rats were collected and divided into microRNA-34a mimic control (MMC) group, microRNA-34a mimic (MM) group, microRNA-34a inhibitor control (MIC) group, and microRNA-34a inhibitor (MI) group, which were respectively transfected with gene sequences of mimic control, mimic, inhibitor control, and inhibitor of microRNA-34a. The microRNA-34a expression level and protein expression level of SIRT1 in myocardial cells were respectively detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Another batch of myocardial cells were divided into microRNA-34a inhibitor control+ burn serum (MCB) group, microRNA-34a inhibitor+ burn serum (MB) group, and microRNA-34a inhibitor+ burn serum + EX527 (MBE) group. Myocardial cells in group MCB were transfected with gene sequence of inhibitor control, and myocardial cells in the later groups were transfected with gene sequence of inhibitor of microRNA-34a. After transfection of 48 h, myocardial cells in group MBE were cultured in Dulbecco′s modified Eagle′s medium (DMEM) solution for 6 hours, with serum in group SB of volume fraction of 10% and final amount-of-substance concentration of 1 mol/L, and myocardial cells in the other 2 groups were cultured in DMEM solution with serum from rats of group SB of volume fraction of 10%. The protein expression levels of myocardial cells of SIRT1, cleaved-caspase-3, and Bax were detected by Western blotting. (3) Myocardial tissue from (1) was collected to detect expression levels of microRNA-34a and mRNA of SIRT1 in groups SI and SB by real-time fluorescence quantitative RT-PCR. Morphology of myocardial tissue of rats in groups SI, SB, and SA was observed with biological image navigator. The mRNA expression levels of interleukin 1β (IL-1β) and tumor necrosis factor (TNF-α) of rats in groups SI, SB, and SA were detected by real-time fluorescence quantitative RT-PCR. The expression levels of cleaved-caspase-3, and Bax of myocardial tissue of rats in groups SI, SB, and SA were detected by Western blotting. Data were processed with one-way analysis of variance and least-significant difference test. Results: (1) After transfection of 48 h, the expression level of microRNA-34a of myocardial cells in group MM was 4.67±0.92, significantly higher than 1.03±0.04 in group MMC (P<0.01); the protein expression level of SIRT1 of myocardial cells in group MM was 0.35±0.06, significantly lower than 1.12±0.11 in group MMC (P<0.01). After transfection of 48 h, the expression level of microRNA-34a of myocardial cells in group MI was 0.26±0.07, significantly lower than 1.33±0.07 in group MIC (P<0.01); the protein expression level of SIRT1 of myocardial cells in group MIC was 1.12±0.16, significantly lower than 1.74±0.34 in group MI (P<0.01). At 6 h after culture, compared with those in group MCB, the SIRT1 protein expression level of myocardial cells in group MB was significantly increased (P<0.05), while cleaved-caspase-3 and Bax protein expression levels of myocardial cells in group MB were significantly decreased (P<0.05). Compared with those in group MB, the SIRT1 protein expression level of myocardial cells in group MBE was with no significantly statistical difference (P>0.05), and cleaved-caspase-3 and Bax protein expression levels were significantly increased (P<0.05). (2) At 6 h post injury, compared with that in group SI, the microRNA-34a expression level of myocardial tissue in group SB was significantly increased (P<0.01), and the mRNA expression level of SIRT1 of myocardial tissue in group SB was significantly decreased (P<0.01). At 6 h post injury, myocardial cells in group SI arranged neatly with normal nucleus and no inflammatory cells infiltration; myocardial cells in group SB arranged disorderly, with no abnormal nucleus, and obvious inflammatory cells infiltration; myocardial cells in group SA arranged neatly, with normal nucleus and little inflammatory cells infiltration. At 6 h post injury, compared with those in group SB, the mRNA expression levels of IL-1β and TNF-α, and the protein expression levels of cleaved-caspase-3 and Bax of myocardial tissue in groups SI and SA were significantly decreased (P<0.01). Conclusions The microRNA-34a expression level of myocardial tissue of rats with severe burns at early stage increases, which decreases the expression level of SIRT1, and increases the expression levels of IL-1β, TNF-α, cleaved-caspase-3 and Bax, leading to obvious myocardial damage. Activation of SIRT1 can alleviate myocardial damage of rats with severe burns at early stage through decreasing expression levels of IL-1β, TNF-α, cleaved-caspase-3, and Bax.

Clinical advances in the treatment of intracranial hemorrhage (ICH) are restricted by the incomplete understanding of the molecular mechanisms contributing to secondary brain injury. Acrolein is a highly active unsaturated aldehyde which has been implicated in many nervous system diseases. Our results indicated a significant increase in the level of acrolein after ICH in mouse brain. In primary neurons, acrolein induced an increase in mitochondrial fragmentation, loss of mitochondrial membrane potential, generation of reactive oxidative species, and release of mitochondrial cytochrome c. Mechanistically, acrolein facilitated the translocation of dynamin-related protein1 (Drp1) from the cytoplasm onto the mitochondrial membrane and led to excessive mitochondrial fission. Further studies found that treatment with hydralazine (an acrolein scavenger) significantly reversed Drp1 translocation and the morphological damage of mitochondria after ICH. In parallel, the neural apoptosis, brain edema, and neurological functional deficits induced by ICH were also remarkably alleviated. In conclusion, our results identify acrolein as an important contributor to the secondary brain injury following ICH. Meanwhile, we uncovered a novel mechanism by which Drp1-mediated mitochondrial oxidative damage is involved in acrolein-induced brain injury.

Objective: To investigate the incidence and rank of chronic obstructive pulmonary disease and pneumoconiosis to the workers in different occupational positions in Jinchang Cohort. Methods: In January 2014, a cohort of follow-up population in jinchang city was taken as the research object, 17843 individuals among follow-up populations in Jinchang Cohort Study, removed the individuals with chronic obstructive pulmonary disease and pneumoconiosis before 2013, and counted the new incidence individuals diagnosed by the A-Class hospital in Grade III in Jinchang City, Gansu Province, as the investigation objects to investigate the incidence rate & rank of chronic obstructive pulmonary disease and pneumoconiosis. The statistical significance was tested by chi-square test. Results: The 2-year incidence rate of Chronic Obstructive Pulmonary Disease and Pneumoconiosis in the population of Jinchang Cohort Study were 11.60‰, 13.51‰ for male and 8.46‰ for female. the ranks of 2-year incidence rates of chronic bronchitis, emphysema, pneumoconiosis and other phenotypes of chronic obstructive pulmonary disease were 7.06‰、3.42‰、0.84‰、0.34‰, respectively. Incidence rate of chronic bronchitis among administrators and executive staffs were 10.45‰; incidence rate of chronic bronchitis among service staffs were 10.45‰; incidence rate of pneumoconiosis among mining staffs were 3.44‰. Conclusion The first incidence rank of chronic obstructive pulmonary disease and pneumoconiosis in Jinchang cohort is chronic bronchitis, and the risk factors are smoking and occupational exposure.