Developing a high-throughput screening method is of great importance for directed evolution of atrazine chlorohydrolase. A mutagenesis library of atzA from Pseudomonas sp. ADP and Arthrobacter sp. AD1 was constructed using error-prone PCR and DNA shuffling. Candidate mutants were screened through Haematococcus pluvialis expression system, using atrazine as selection pressure. Sequence analysis showed that mutations in the obtained 12 mutants with enhanced activity were all point-substitutions and scattered throughout the gene. Enzymatic activity analysis showed that the mutants all had higher activities than that of the wild type. The activities were 1.8-3.6 fold of the wild-type enzyme when cultured in BBM medium with 1 mg/L atrazine, whereas 1.8-2.6 fold with 2 mg/L atrazine. These results indicated that Haematococcus pluvialis expression system is an ideal high throughput screening system for directed evolution of atrazine chlorohydrolase.
Amidohydrolases ; genetics ; Atrazine ; metabolism ; Bacterial Proteins ; genetics ; Biodegradation, Environmental ; Chlorophyta ; genetics ; metabolism ; Herbicides ; metabolism ; High-Throughput Screening Assays ; Hydrolases ; biosynthesis ; genetics ; Mutagenesis, Insertional ; Pseudomonas ; enzymology ; genetics

Objective To explore the diagnostic value of endobronchial ultrasound?guided transbronchial needle aspiration ( EBUS?TBNA) in the diagnosis of enlarged mediastinal lymph nodes, and to analyze the reasons of false negative results of EBUS?TBNA. Methods A retrospective analysis of the clinical and imaging data of 389 patients who underwent EBUS?TBNA for sampling enlarged hilar and mediastinal lymph nodes from October 2009 to October 2015 in Shandong Tumor Hospital, to evaluate its significance in the diagnosis of enlarged mediastinal lymph nodes, and to analyze the reasons of false negative results. Results Among the 389 collected cases, positive biopsy results were obtained in 362 cases, and false negative results in 27 cases. The sensitivity, specificity, positive predictive value and negative predictive value were 92. 9%, 100%, 100% and 25. 0%, respectively. There was a positive correlation between the size of lymph node and biopsy positive rate (P=0.021). The subcarinal lymph nodes had the highest positive rate ( 97. 7%) , followed by the paratracheal lymph nodes ( 91. 2%) , with a statistically significant difference (P=0.006). The positive rates obtained by cytology (88.7%) and pathology (92.5%,) showed no significant difference ( P=0. 065 ) . The positive rate of EBUS?TBNA was not correlated with pathological types (P=0.932). Needle types (21G, 22G) had no significant effect on diagnosis accuracy (P=0.142). Conclusions EBUS?TBNA is a practical technology for diagnosis of enlarged mediastinal lymph nodes, with unique characteristics such as minimally invasiveness, local anesthesia, good safety and repeatability. Along with the accumulation of surgical experience, improvement of operative skills, more close cooperation between surgeons, cytologists and pathologists, false negative results will be reduced and positive rate of EBUS?TBNA examination will be further improved.

Objective To explore the diagnostic value of endobronchial ultrasound?guided transbronchial needle aspiration ( EBUS?TBNA) in the diagnosis of enlarged mediastinal lymph nodes, and to analyze the reasons of false negative results of EBUS?TBNA. Methods A retrospective analysis of the clinical and imaging data of 389 patients who underwent EBUS?TBNA for sampling enlarged hilar and mediastinal lymph nodes from October 2009 to October 2015 in Shandong Tumor Hospital, to evaluate its significance in the diagnosis of enlarged mediastinal lymph nodes, and to analyze the reasons of false negative results. Results Among the 389 collected cases, positive biopsy results were obtained in 362 cases, and false negative results in 27 cases. The sensitivity, specificity, positive predictive value and negative predictive value were 92. 9%, 100%, 100% and 25. 0%, respectively. There was a positive correlation between the size of lymph node and biopsy positive rate (P=0.021). The subcarinal lymph nodes had the highest positive rate ( 97. 7%) , followed by the paratracheal lymph nodes ( 91. 2%) , with a statistically significant difference (P=0.006). The positive rates obtained by cytology (88.7%) and pathology (92.5%,) showed no significant difference ( P=0. 065 ) . The positive rate of EBUS?TBNA was not correlated with pathological types (P=0.932). Needle types (21G, 22G) had no significant effect on diagnosis accuracy (P=0.142). Conclusions EBUS?TBNA is a practical technology for diagnosis of enlarged mediastinal lymph nodes, with unique characteristics such as minimally invasiveness, local anesthesia, good safety and repeatability. Along with the accumulation of surgical experience, improvement of operative skills, more close cooperation between surgeons, cytologists and pathologists, false negative results will be reduced and positive rate of EBUS?TBNA examination will be further improved.

Objective To observe the effect of antibacterial dressing tamponade combined with seal negative pressure drainage in the treatment of necrotizing fasciitis .Methods 52 patients with necrotizing fasciitis were selected . After full scavenging of fasciitis and necrotic tissue,then filled the whole wound with a kind of dressings with antibacterial properties-Nano silver antiseptic dressing,and a negative pressure drainage tube was placed at the bottom of the wound.Then the wound was closed with a transparent paste ,and the air was cut off,in order to make the antibacterial dressing could not only anti bacteria ,but also play a role in the growth of the wound ,and at the same time,the tissue leachate in the wound was drained through the negative pressure tube in time ,so as to accelerate the wound healing and reduce the use of antibiotics .Results Among the 52 cases,the rest were cured except for 1 case of individual cause of abandonment of treatment died of toxic shock .,the average hospitalization time was (29.0 ±15.3)days,days of using antimicrobial was (3.8 ±1.6)days,90% cases stopped using antibiotics within 1 week.Conclusion Antibacterial dressing tamponade combined with seal negative pressure drainage in the treatment of necrotizing fasciitis can reduce the switching frequency,reduced antibiotic using intensity,shorten the healing time,it is a kind of very good method.