A droplet digital polymerase chain reaction ( ddPCR) method for quantifying E. coli O157:H7 by targeting rfbE gene was developed. The probe concentration in ddPCR was optimized and the linearity range, precision, limit of detection ( LOD) and limit of quantification ( LOQ) were also evaluated. The optimized probe concentration was 300 nmol/L. The ddPCR response was linear over the E. coli O157:H7 genome DNA concentration range from 4 to 1. 25×105 copies in 20 μL ddPCR system and the linear correlation coefficient (R2) was 0. 999. The ddPCR precision (RSD) was less than 5% over the DNA concentration range from 760 to 88400 copies/20 μL. The LOD and LOQ was 3 copies in 20 μL and 4 copies in 20 μL, respectively. Specificity test showed that the ddPCR was specific for detecting E. coli O157:H7. Both ddPCR and standard real time quantitative PCR showed the same results for 16 real samples of chicken meat, pork and beef, which indicated that ddPCR method was suitable for detection of E. coli O157:H7 in food.

Objective To discuss the therapeutic strategy and the clinical efficacy of percutaneous cholecystostomy in treating high-risk patients with acute cholecystitis. Methods During the period of Jan. 2006-June 2008,percutaneous cholecystostomy was performed in 27 high-risk patients with acute cholecystitis,consisting of lithic cholecystitis (n = 21) and non-lithic cholecystitis (n = 6). Of 27 patients,percutaneous cholecystostomy via transhepatic approach was performed in 22 and via transperitoneal approach in 5. The 7 F drainage catheter was used. Cholecystography was conducted before the drainage catheter was extracted. Results Percutaneous cholecystostomy was successfully accomplished in all 27 cases,with a technical success rate of 100%. Postoperative patency of gallbladder drainage was obtained in 25 patients,with the relieving or subsiding of abdominal pain and the restoring of temperature and leukocyte account to normal range within 72 hours. In one patient,as the abdominal pain relief was not obvious 72 hours after the procedure,cholecystography was employed and it revealed the obstruction of the drainage catheter. After reopening of the drainage catheter,the abdominal pain was relieved. In another case,cholecystography was carried out because the abdominal pain became worse after the procedure,and minor bile leak was demonstrated. After powerful anti-infective and symptomatic medication,the abdominal pain was alleviated. The drainage catheter was extracted in 25 patients 6-7 weeks after the treatment. Of these 25 patients,12 accepted selective cholecystectomy,7 received percutaneous cholecystolithotomy and 6 with non-lithic cholecystitis did not get any additional surgery. The remaining two patients were living with long-term retention of the indwelling drainage-catheter. Conclusion Percutaneous cholecystostomy is a simple,safe and effective treatment for acute cholecystitis in high-risk patients. This technique is of great value in clinical practice.

Objective To research the effect of cavity under Bolus to anisotropic analytical algorithm (AAA) on calculation precision of dose in shallow tissue based on Monte Carlo method;Methods A 30 cm × 30 cm × 30 cm water phantom with the upper surface was constructed which was located at the source-axis distance (SAD) of the medical linear accelerator and the center as well as coincided with the central axis of the radiation field in Eclipse treatment panning system. Above the water phantom, a water film of 1 cm thick with or without different cavities was constructed or. AAA was used to calculate the dose distribution on the central axis and the x-axis of different depth of the water model with different cavities respectively. The accelerator model, the same water phantom and the water film were constructed and the dose distributions of the same positions were calculated with Geant4. Based on the Geant4 calculation result, the calculation precision of AAA with different cavity were compared;Results For cavities with area of 2 cm × 2 cm, if the thickness is smaller than 0.5 cm, the AAA calculation error is about 2%. with the cavity thickness increase, the AAA would overestimate the dose in the shallow area under the cavity. With the cavity area increase, the area where AAA overestimate the shallow dose gradually moved out until near the edge of the radiation field, and the calculation error on the central area gradually reduced until there is basically no error. Conclusions The shallow dose would be increased according to the cavity size when planning with AAA; If there are cavities with large volume, it is better to reposition.

More than 150 glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are expressed in mammalian cells and involved in various physiological processes such as immune recognition, cell communication and signal transduction. GPI is transferred to proteins in the endoplasmic reticulum (ER). When GPI-anchoring is impaired, precursor proteins are thought to be degraded through ER-associated degradation (ERAD). However, the mechanism of their degradation in ERAD remains unclear. To investigate the impact of ERAD pathways on degradation of GPI precursor proteins, we used series of knockout (KO) human embryonic kidney 293 (HEK293) cells defective in PIGS gene, which encodes a GPI transamidase complex subunit, combined with KO in HRD1 (PIGS-HRD1-KO) or GP78 (PIGSGP78-KO), which encodes the E3 ubiquitin ligases for the ERAD pathways. We compared the stability of 16 GPI precursor proteins in the ERAD-deficient cells with the parental PIGS-KO cells. Western blotting data showed that the GPI precursor proteins were stabilized in either PIGS-HRD1-KO (I

Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.
Bacillus subtilis/metabolism* ; Vitamin K 2/metabolism* ; Bioreactors/microbiology* ; Membrane Microdomains/metabolism*

Objective:To explore the effect of somatosensory interactive games on lower-limb function after stroke. Methods:Randomized controlled trials (RCT) about the effectiveness of somatosensory interactive games on lower-limb function after stroke were retrieved from domestic and foreign databases, from inception to September, 2018. After literature quality evaluation, a meta-analysis was performed using RevMan5.3. Results:A total of 414 patients were included in eleven articles. Compared with routine rehabilitation measures, somatosensory interactive games improved Berg Balance Scale scores (WMD = 1.75, 95%CI 0.95~2.54, P < 0.001), increased stride frequency (SMD = 1.21, 95%CI 0.03~2.38, P = 0.04), decreased the time of Timed Up and Go Test (WMD = -4.21, 95%CI -7.36~-0.89, P = 0.01), and improved the motor function of lower limbs (SMD = 0.66, 95%CI 0.15~1.17, P = 0.01), but worked less in pace (SMD = 0.05, 95%CI -0.98~1.09, P = 0.92) and 10-metre Maximum Walking Speed (WMD = 3.54, 95%CI -1.12~8.20, P = 0.14). Conclusion:Compared with the routine rehabilitation, somatosensory interactive games can improve balance, walking and motor function. However, it is needed to further research on the pace and speed.

Aim To investigate the induction of ferroptosis of Triapine in non-small cell lung cancer cells A549, and its mechanism. Methods The effects of Triapine on the proliferation of A549 cells were assessed by MTT assay and colony formation assay; the effect of intracellular ROS levels of Triapine treated A549 cells was studied by DCFH-DA probe; the intracellular glutathione (GSH) and lipid peroxides (LPO) levels of A549 cells were detected by the kits after treating with Triapine; the effects of Triapine on the expression of glutathione peroxidase 4 (GPX4) in A549 cells were analyzed by Western blot; the changes of GPX4 level and cell viability were evaluated for the cells intervened with ROS inhibitor. Results Triapine could inhibit the proliferation of A549 cells, and the IC

Objective To investigate the clinical application of nasal-insertion type ileus-tube in the treatment of adhesive small intestinal obstruction. Methods A total of 221 patients with simple adhesive small intestinal obstruction, who were admitted to authors’ hospital during the period from January 2010 to Aug. 2014, were enrolled in this study. The patients were randomly divided into nasal-insertion type ileus-tube group (n=111) and nasogastric tube group (n=110). After the procedure, the patients were kept under close observation, focusing on the abdominal distention, gastrointestinal decompression amount, the recovery time of anal exhaustion and defecation, the vanishing time of intestinal air-liquid plane on erect abdominal X-ray film, etc. The cure rate, effective rate and transit-operation rate were calculated. The results were compared between the two groups. Results The tube placement operation was successfully performed in all patients. Compared with the nasogastric tube group, in the nasal-insertion type ileus-tube group the recovery time of abdominal distention, anal exhaustion and defecation and the vanishing time of intestinal air-liquid plane on erect abdominal X-ray film were obviously shorter, and the gastrointestinal decompression amount was larger. In the nasal-insertion type ileus-tube group the cure rate and effective rate were significantly increased, while the transit-operation rate was decreased; the differences between the two groups were statistically significant (P<0.05). Conclusion For the treatment of adhesive small intestinal obstruction, the placement of nasal-insertion type ileus-tube is effective and reliable. This technique can strikingly improve the clinical symptoms, therefore, it is worthy of promotion and application in clinical practice.

Nicotinamide adenine nucleotide (NADH), the key cofactor in the metabolic network, plays an essential role in biochemical reaction and physiological function of industrial strains. Manipulation of NADH availability and form is an efficient and easy way to redirect the carbon flux to the target metabolites in industrial strains. We reviewed the physiological function of NADH. Detailed strategies to manipulate NADH availability are addressed. NADH manipulation to enhance metabolic function of industrial strains was discussed and potential solutions were suggested.
Bacteria ; metabolism ; Energy Metabolism ; genetics ; physiology ; Fermentation ; Industrial Microbiology ; Lactococcus lactis ; metabolism ; NAD ; metabolism ; physiology ; Saccharomyces cerevisiae ; metabolism ; Streptococcus mutans ; metabolism

To investigate the mechanism of high product specificity of gamma-clodextrin glucanotransferase (CGTase) from alkalophilic Bacillus clarkii 7364, we aligned protein sequence and structure model, found out that loss of 6 amino acids at subsite -7 probably affected its product specificity. Using overlapping PCR method, we inserted 6 amino acids into subsite -7 of CGTase. The mutant CGTase gene was ligated with pET-20b (+) and expressed in Escherichia coli BL21 (DE3). The extracellular recombinant enzyme was used to transform soluble starch into cyclodextrins (CDs). HPLC analysis results show that, compared to wild CGTase, the gamma-CDs produced by mutant enzyme decreased from 76.0% to 12.5%, whereas the ratio of alpha- and beta-CDs increased from 8.7% and 15.2% to 37.5% and 50%. The possible mechanism was that, compared to alpha-, beta-CGTase, wild gamma-CGTase lacks 6 amino acids in its subsite -7. This conformation provided more space for glucose combination and was thus advantageous for forming gamma-CD. When the 6 amino acids were inserted into the subsite -7 of wild gamma-CGTase, the space to bind with glucose reduced and consequently resulted in less gamma-CD production.
Bacillus ; enzymology ; genetics ; Escherichia coli ; genetics ; metabolism ; Glucosyltransferases ; biosynthesis ; genetics ; Mutant Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics