Directed evolution was used to improve the performance of beta-1,3-1,4-glucanase (designated as PtLicl6A) from Paecilomyces thermophila J18 under acidic condition. A mutant library was constructed by error-prone PCR and DNA shuffling, and positive clones were screened by Congo red staining. More than 1 500 mutants were selected. One mutant (PtLic16AM1) exhibited an optimal activity at pH 5.5, while the optimal pH of the wild-type enzyme was 7.0. The mutant PtLic16AM1 kept the high specific activity and thermotolerence of the wild-type enzyme. Sequence analysis revealed that the mutant enzyme has four sense substitutions which caused four amino acid substitutions - namely T58S, Y110N, G195E and D221G.. Homology modeling showed that among the four amino acid substitutions, Y110N was near the active site of the enzyme, while the other three was distant. T58S and G195E may play key roles in the change of optimal pH. This study provided a new perspective of obtaining applicable 3-1,3-1,4-glucanase for industrial use.
Catalysis ; Directed Molecular Evolution ; methods ; Endo-1,3(4)-beta-Glucanase ; biosynthesis ; genetics ; metabolism ; Enzyme Stability ; Hot Temperature ; Hydrogen-Ion Concentration ; Mutant Proteins ; metabolism ; Mutation ; Paecilomyces ; classification ; enzymology ; genetics ; Protein Engineering ; methods

Objective To explore the features of peripheral blood immune cells in long-term survival recipients after liver transplantation.Methods The expression of T subsets (Th1,Th2,Th17,Th22,Tregs),NK cells,NKt cells,Bregs,MDSC in long-term survival recipients (postoperative follow-up time ≥5 years,30 cases),short-term survival recipients(postoperative follow-up time ≤1 year,15 cases) and healthy control (15 cases) were determined by flowcytometry.Results Th17 cells were significantly higher in the long-term group compared with short-term group and healthy control group(P ＜0.01).Tregs in long-term group compared with short-term group were significantly higher (P ＜ 0.01),but the difference was not statistically significant compared with healthy control group (P ＞ 0.05).NK cells were significantly higher in long-term group compared with short-term group and healthy control group (P ＜ 0.01).MDSC were significantly higher in long-term group compared with short-term group and healthy control group (P ＜0.01).Conclusions Th17,Tregs,NK cells and MDSC were significantly higher in long-term survival of liver recipients,which may be related to immune tolerance.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease characterized by excessive fat accumulation in the liver. More and more evidence suggests that NAFLD is a multisystem disease that affects multiple extra-hepatic organs. Recent studies have shown that NAFLD may be associated with the risk, severity, and outcome of ischemic stroke. The article provides a summary of these aspects.

Objective To evaluate the clinical efficacy of application of hepatitis B surface antigen (HBsAg)-positive donor liver in adult liver transplantation. Methods Clinical efficacy of 28 recipients with liver diseases induced by virus B hepatitis (hepatitis B) undergoing liver transplantation using HBsAg-positive donor liver from July 2012 to October 2015 was retrospectively analyzed. Clinical prognosis and postoperative complications of the recipients were summarized. The changing features of serum levels of HBsAg and hepatitis B virus (HBV) DNA was investigated. Results After liver transplantation, 28 recipients were orally administered with entecavir to prevent the recurrence of hepatitis B. During perioperative period, 2 recipients died from sepsis and acute heart failure. During postoperative follow-up, 2 cases died from the recurrence of hepatocellular carcinoma (liver cancer). The remaining 24 patients were followed up for 12-26 months. Throughout the follow-up, 24 recipients were positive for serum HBsAg. After treatment, the titre of HBV DNA was significantly declined to <1×102 copies/mL at postoperative 12 months. No graft dysfunction induced by hepatitis B recurrence occurred in 24 recipients alive. Conclusions As a marginal donor liver, HBsAg-positive liver graft is safe for liver transplantation in the recipients with hepatitis B-related liver diseases. Postoperatively, anti-HBV treatment should be strengthened and intimate follow-up should be delivered.

{L-End}Objective To analyze the prevalence of multi-site work-related musculoskeletal disorders (WMSDs) and their influencing factors among workers in the chemical pharmaceutical industry. {L-End}Methods A total of 563 workers from three chemical pharmaceutical factories in Guangzhou City were selected as the research subjects using the judgment sampling method. The prevalence of multi-site WMSDs in the past 12 months was investigated using the electronic questionnaire of Chinese Version of Musculoskeletal Disorders Questionnaire. {L-End}Results The prevalence of multi-site WMSDs in the research subjects was 30.0% (169/563). The results of multivariable logistic regression analysis showed that workers who smoked regularly/frequently and those who had their hands above shoulder level for long periods of time had a lower risk of multi-site WMSDs than those who never/occasionally smoked (all P≤0.05). Workers with a high school, secondary specialty, college degree, or above had a lower risk of multi-site WMSDs than those with a middle school education or below (all P<0.05). The longer the daily working hours, the higher the risk of multiple WMSDs (all P<0.05). Workers with regular/frequently work shifts had a lower risk of multi-site WMSDs than those with none/occasional work shifts (P<0.05). {L-End}Conclusion The prevalence of multi-site WMSDs among workers in the chemical pharmaceutical industry is relatively high. The influencing factors are individual factors, unreasonable work organization and poor ergonomics factors.

Objective:To evaluate the accuracy of pRIFLE criterion and KDIGO criterion for the diagnosis of acute kidney injury (AKI) after radical operations for tetralogy of Fallot in children from the perspective of postoperative outcomes.Methods:A total of 375 children, aged<8 yr, undergoing radical operations for tetralogy of Fallot, were selected continuously and retrospectively. According to the pRIFLE and KDIGO diagnostic criteria, postoperative AKI was diagnosed, and the children were classified into different AKI grades. The prognostic parameters (postoperative mechanical ventilation time, duration of intensive care unit (ICU) stay, postoperative length of hospital stay, total costs of hospitalization, and in-hospital mortality, etc.) were collected, and the differences among different AKI grades were compared. Logistic regression method was used to analyze the risk factors for prolonged postoperative length of hospital stay (≥14 days) when two different criteria were used to diagnose AKI. The children diagnosed as non-AKI by KDIGO criterion were further confirmed using pRIFLE criterion, and the prognostic parameters in the children who were diagnosed as AKI and non-AKI were compared.Results:When two different criteria were used to diagnose AKI after radical resection for tetralogy of Fallot, the incidence was 56.8% (pRIFLE criterion) and 40.0% (KDIGO criterion). AKI diagnosed according to the two criteria was the independent risk factor for prolonged postoperative length of hospital stay, and the levels of all the prognostic parameters (postoperative mechanical ventilation time, duration of ICU stay, postoperative length of hospital stay, total costs of hospitalization, and in-hospital mortality) were significantly higher in AKI children than in non-AKI (AKI grade 0) children ( P<0.01). Among the 225 children diagnosed as non-AKI according to the KDIGO criterion, 63 cases were diagnosed as AKI and 162 cases as non-AKI according to the pRIFLE criterion, however, there was no significant difference in each prognostic parameter between children with AKI and non-AKI ( P>0.05). Conclusion:The pRIFLE criterion has a higher sensitivity, while the KDIGO criterion produces better accuracy when used to evaluate the diagnosis of AKI after radical operation for tetralogy of Fallot in children from the perspective of postoperative outcomes.

Recent studies have shown that stress can substantially facilitate breast cancer metastasis,which can be reduced by nonselective β1/β2-adrenergic receptor(β1/β2-AR)blocker.However,several side effects were identified.Thus,it is extremely warranted to explore more effective and better-tolerated β2-AR blocker.Currently,we demonstrated that baicalin(BA),a major bioactive component of Scutellaria bai-calensis Georgi,could significantly attenuate stress hormones especially epinephrine(Epi)-induced breast cancer cell migration and invasion in vitro.Mechanistically,we identified that β2-AR was a direct target of BA via the drug affinity responsive target stability(DARTS)combined with mass spectrum assay,and BA photoaffinity probe with pull-down assay,which was further confirmed by a couple of bio-physical and biochemical assays.Furthermore,we demonstrated that BA could directly bind to the Phe-193 and Phe-289 of β2-AR,subsequently inhibit cyclic adenosine monophosphate-protein kinase A-focal adhesion kinase(cAMP-PKA-FAK)pathway,and thus impede epithelial-mesenchymal transition(EMT),thereby hindering the metastatic progression of the chronic stress coupled with syngeneic and xenograft in vivo orthotopic and tail vein mouse model.These findings firstly identify BA as a potential β2-AR inhibitor in the treatment of stress-induced breast cancer metastasis.

Objective To investigate the safety and feasibility of a simplified protocol combining with mesenchymal stem cell (MSC) in ABO-incompatible (ABO-I) liver transplant patients. Methods Twelve ABO-I liver transplant patient who received the therapy of a simplified protocol combining with MSC in the Third Affiliated Hospital of Sun Yat-sen University between January 2014 and September 2015 were recruited in this prospective study. Ten cases were male and 2 were female, with a mean age of (39±13) years old. The informed consents of all patients or their families were obtained and the local ethical committee approval was received. A immunologic tolerance induction protocol, plasma exchange + rituximab + intravenous immunoglobin + MSC (simplified protocol combining with MSC and without splenectomy and graft local infusion), was used to prevent the antibody-mediated rejection (AMR) after liver transplantation (LT). The perioperative condition and postoperative outcome of the patients were observed. Results Three death cases were observed after LT including 2 cases died of multiple organ failure and 1 of gastrointestinal hemorrhage. The other cases survived. Two cases developed acute cellular rejection and no AMR case was observed. Biliary complication was observed in 3 cases, hepatic artery stenosis in 1 case and infection in 6 cases. Conclusion The simplified protocol combining with MSC is safe and effective in preventing the AMR after ABO-I LT.

Ideal osseointegration is intimately related to favorable osteoimmune properties around dental implants. An increasing number of in vitro and in vivo studies have indicated that the Hippo-YAP signaling pathway is involved in this biological process. In this article, the implicated roles of Hippo-YAP the signaling axis in peri-implant osteoimmunology were summarized by reviewing relevant evolving literature. The discrepancy concerning the Hippo-YAP signaling regulatory effect on osteogenesis and polarization direction were analyzed as well as propose the potential mechanism, which may be caused by the maturation of osteogenesis-related cells and heterogeneity of macrophages. More attention should be given to the requirements of promoting osteogenesis and patterns of regulating the immune microenvironment by Hippo-YAP in future studies.

OBJECTIVETo evaluate the differences in the clinical therapeutic effects on spasmodic hemiplegia after stroke among the alliance therapy of scalp acupuncture, music therapy combined with rehabilitation, the simple rehabilitation therapy and the combination of music therapy and rehabilitation.

METHODSA total of 76 patients of post-stroke spasmodic hemiplegia were randomized into a rehabilitation group (25 cases), a combination group with music therapy and rehabilitation (25 cases) and an alliance therapy group with scalp acupuncture, music therapy and rehabilitation (26 cases). In the rehabilitation group, the routine rehabilitation therapy was applied, including the removal of various incentives that cause spasm, the correction of body position and the physical therapy. In the combination group, the music therapy was added on the basis of the treatment as the rehabilitation group. The music physician used the rhythmic auditory stimulation, the patterned sensory enhancement and the therapeutic instrumental music playing to set up the task in the treatment. In the alliance therapy group, scalp acupuncture was added on the basis of the treatment as the combination group. The anterior oblique line of vertex-tempora (MS 6) and the posterior oblique line of vertex-tempora (MS 7) on the contralateral side were selected and stimulated with penetrating needling technique. The needles were retained. During the needling retaining, the needles were rotated once every 10 min, for 2 min each time. The treatment was given one session a day, totally for 5 sessions a week, continuously for 4 weeks. The Fugl-Meyer assessment (FMA), Barthel index (BI) and the modified Ashworth scale (MAS) of the affected elbow and the passive knee movement at static condition were observed in the patients before and after treatment.

RESULTSThe results of FMA, BI and MAS were not different before treatment in the patients among the three groups (all>0.05), indicating the comparability among groups. After treatment, FMA and BI scores were all increased apparently in the three groups as compared with those before treatment (all<0.05). MAS grade was reduced remarkably as compared with that before treatment (all<0.05). After treatment, FMA and BI scores in the alliance therapy group were higher than those in the combination group and the rehabilitation group (all<0.05). FMA and BI scores in the combination group were higher than those in the rehabilitation group (both<0.05). MAS grade in the alliance therapy group was lower than those in the combination group and the rehabilitation group (both<0.05). MAS grade in the combination group was lower than that in the rehabilitation group (<0.05).

CONCLUSIONThe alliance therapy with scalp acupuncture, music therapy and rehabilitation achieve the remarkable clinical therapeutic effects on post-stroke spasmodic hemiplegia as compared with the routine rehabilitation and the combination of music therapy and rehabilitation.