To develop a genetic transformation method of Aureobasidium pullulans and T-DNA insertion for high-efficient screening of polymalic acid (PMA) producing strain. Agrobacterium tumefaciens-AGL1, containing the selection genes encoding hygromycin B phosphotase or phosphinothricin acetyltranferase, was used to transform Aureobasidium pullulans CCTCC M2012223 and transformants were confirmed by colony PCR method. Transferred DNA (T-DNA) insertional mutants were cultured in microwell plate, and screened for high-titer PMA producing strain according to the pH response model. DNA walking was used to detect the insertion sites in the mutant. Results show that the selection markers could stably generated in the transformants, and 80 to 120 transformants could be found per 10(7) single cells. A high-titer PMA mutant H27 was obtained, giving a good PMA production caused by the disruption of phosphoglycerate mutase, that increased by 24.5% compared with the control. Agrobacterium tumefaciens-mediated transformation and high-efficient screening method were successfully developed, which will be helpful for genetic transformation of Aureobasidium pullulans and its functional genes discovery.
Agrobacterium tumefaciens ; Ascomycota ; genetics ; metabolism ; DNA, Bacterial ; Malates ; metabolism ; Polymerase Chain Reaction ; Polymers ; metabolism ; Transformation, Genetic

Objective To investigate the efficacy of dexmedetomidine on sedation in post-cardiac surgery patients with NIV intolerance.The changes of respiratory function and hemodynamics of the patients as well as non-invasive ventilation (NIV) failure rate were also under evaluation.Methods Thirty-five post-cardiac surgery patients with NIV intolerance and hypoxemia were enrolled in this prospective study.All patients were sedated with dexmedetomidine.NIV was standardized according to the uniform protocol.The main outcome was NIV success (avoiding endotracheal intubation) or NIV failure (requiring endotracheal intubation or die).The cardiorespiratory parameters (BP,HtR and RR) and artery blood gas analysis were prospectively recorded before and after sedation.The respiratory function and hemodynamics changes in both groups (NIV success group and NIV failure group) were then evaluated.Factors independently associated with NIV failure were identified using a logistic regression model.Results Twenty out of 35 patients (57.14％) survived while 15 (42.86％) patients failed NIV.After 1 h and 4 h of NIV with dexmedetomidine sedation,respiratory rate in both groups were decreased compared with baseline,especially in NIV success group.The PaO2/FiO2 was also improved after 1h and 4h of NIV treatment compared with baseline.The improvement was more significantly in NIV success group.The heart rate was decreased compared with baseline with no differences between two groups.There were no significant changes on PaCO2 and mean arterial pressure (MAP) during the treatment.The respiratory and hemodynamics variables identified as predictors of NIV failure were included in a multivariate logistic regression.RR ＞ 23 time/min (OR =3.2,95％ CI:2.043 ～ 4.301,P ＜ 0.01) 1 h after NIV,RR ＞ 20 time/min (OR =2.1,95％ CI:1.659～3.231,P=0.025) 4 h after NIV,PaO2/FiO2 ＜178 mmHg (OR=2.4,95％CI:1.892 ～ 3.287,P ＜0.01) 1 h after NIV and PaO2/FiO2 ＜ 185 mmHg (OR =1.7,95％ CI:1.243 ～ 2.365,P =0.041) 4 h after NIV independendy predicted NIV failure.Conclusions Dexmedetomidine might be considered as an effective and safe sedative for post-cardiac surgery patients with NIV intolerance.Early identification of predictors of NIV failure may facilitate early intervention.

Objective To study the incidence,possible risk factors,and influence on patient outcomes of diaphragmatic dysfunction in patients after surgical treatment of type A aortic dissection using ultrasound.Methods Patients who received replacement of hemiarch or total arch with concomitant procedures concerning aortic pathology,and an elephant trunk procedure for the descending aorta were prospectively enrolled in this study from February to May 2017.After surgery,they were transferred to the cardiac surgical intensive care unit.They were divided into two groups based on diaphragmatic excursion:diaphragmatic dysfunction (DD) group and diaphragmatic function normal (DN) grouBilateral diaphragmatic excursions were evaluated using ultrasound during spontaneous breathing trial by T-tube.The differences in demographic characteristics,operation-related variables and outcomes were compared between the two groups.Results A total of 42 patients were enrolled in this study,and 32 of them suffered from diaphragmatic dysfunctions.Compared with DN group,the excursion of the influenced diaphragm in DD group was significantly reduced[(0.450 ± 0.331)cm vs.(1.801 ± 0.616)cm,P＜0.01],while the excursion of the non-influenced diaphragm was not reduced[(2.013±0.655)cm vs.(1.801 ±0.616) cm,P=0.254].Diaphragmatic thickness was comparable [(0.184±0.028)cm vs.(0.189±0.028)cm,P=0.559 ] between the two groups while thickening fraction was significantly reduced in DD group[(4.67％±3.63)％ vs.(23.58％±10.69)％,P＜0.01].Meanwhile,respiratory rate was significantly higher in DD group as compared to DN group [(24.13 ± 4.98)times/min vs.(20.50 ± 3.17)times/min,P=0.037].Patients in DD group showed longer cross-clamp duration[(121.78±27.75)min vs.(93.10±18.84)min,P=0.004] and longer cardiopulmonary bypass duration [(208.09±32.78)min vs.(182.70±24.38)min,P=0.03] than patients in DN grouFurthermore,binary logistic analysis indicated that longer cross-clamp duration was the potential risk factor for diaphragmatic dysfunction after type A aortic dissection surgery.Mechanical ventilation duration was longer in DD group than in DN group (88 h vs.37 h,P=0.194) but without statistical significance.The usage of noninvasive ventilation was significantly increased in DD group as compared to DN group (46.88％ vs.10％,P=0.036).Other outcomes such as post-operative complications,mortality,ICU length of stay were comparable between the two groups.Conclusions Diaphragmatic dysfunction was very common after surgical treatment of type A aortic dissection.Longer duration of cross-clamp was considered as a potential risk factor of diaphragmatic dysfunction.A sequential management of noninvasive ventilation after extubation was feasible for diaphragmatic dysfunction after surgical treatment of type A aortic dissection.