Polyhydroxyalkanoate depolymerase (PHAD) can be used for the degradation and recovery of polyhydroxyalkanoate (PHA). In order to develop a PHAD with good stability under high temperature, PHAD from Thermomonospora umbrina (TumPHAD) was heterelogously expressed in Escherichia coli BL21(DE3). At the same time, a mutant A190C/V240C with enhanced stability was obtained via rational design of disulfide bonds. Characterization of enzymatic properties showed that the mutant A190C/V240C had an optimum temperature of 60 ℃, which was 20 ℃ higher than that of the wild type. The half-life at 50 ℃ was 7 hours, at 50 ℃ which was 21 times longer than that of the wild type. The mutant A190C/V240C was used for the degradation of polyhydroxybutyrate (PHB), one of the typical PHA. At 50 ℃, the degradation rate of PHB being treated for 2 hours and 12 hours was 2.1 times and 3.8 times higher than that of the wild type, respectively. The TumPHAD mutant A190C/V240C obtained in this study shows tolerance to high temperature resistance, good thermal stability and strong PHB degradation ability, which may facilitate the degradation and recovery of PHB.
Thermomonospora ; Actinomycetales ; Escherichia coli/genetics* ; Polyhydroxyalkanoates

Objective:To analyze the expression of SMARCE1 in clear cell meningioma (CCM), and evaluate the role of SMARCE1 in the differential diagnosis in morphologically similar diseases.Methods:Thirteen samples/11 cases of CCMs were collected from the First Affiliated Hospital of Fujian Medical University, Shandong Provincial Hospital, Xuanwu Hospital of Capital Medical University and Thaihe Hospital of Hubei Province from January 2000 to December 2018, as well as 17 cases of meningiomas with clear-cell-like morphology, 782 cases of other types of meningiomas and other intracranial tumors with clear-like morphology. A tissue microarray was made using these cases, on which immunohistochemical/histochemical staining of SMARCE1, SSTR2, EMA, Ki-67, p53, PAS and D-PAS were performed.Result:The tumor cells of CCM had sheet-like architecture, without typical whorl formation.The CCM had round to polygonal cells, with clear, glycogen-rich cytoplasm and prominent blocky perivascular and interstitial collagen. The immunohistochemistry staining showed that none of the CCMs expressed SMARCE1(0/13).However, all of the other types of lesions, including meningioma(782/782), meningiomas with clear-like morphology(17/17), intracranial metastatic clear cell renal cell carcinoma(10/10), haemangioblastoma(10/10), central neurocytoma(10/10), oligodendroglioma(10/10), ependymoma(13/13), lioblastoma(42/42), and solitary fibrous tumor/hemangiopericytoma(35/35) showed positive nuclear staining of SMARCE1. Ki-67 index were 1%-5%, and p53 positive-rate were 0-40% in CCMs. PAS stain showed cytoplasmic granular positive and D-PAS were negative in all CCMs and meningiomas with clear-like morphology.Conclusion:SMARCE1 is a useful marker for the diagnosis of CCM and its mimickers.

Objective To investigate whether Wnt/β-catenin signaling pathway mediating the neuroprotection of isoflurane post-conditioning in hippocampal neurons damage induced by ischemia/reperfusion injury in rats.Methods According to the randomized principle, 60 male Sprague-Dawley rats were randomly divided into five groups (12 rats in each group):sham group (group S), model group (group M), ISO+model group (group MI), ISO+model+DKK-1 group (group MDI) and model+DKK-1 group (group MD).A rat model of middle cerebral artery occlusion (MCAO) was established with 90 min ischemia followed by 24 hreperfusion.Group S was only exposed to one side of the internal carotid artery without fishing line.Isoflurane post-conditioning groups (group MI, MDI) were immediately treated with 1.5％isoflurane for 60 min at the onset of reperfusion.DKK-1 (5μg/kg) was injected intracerebroventricularly 30 min before the model established in group MDI and group MD.After reperfusion for 24 h, Longa score method was used for neurological deficit score.HE staining and Tunel fluorescence was employed to observe the morphological changes of neurons.Immunohistochemistry and Western Blot were applied to detect the expression of target protein in CA1 region.Results Compared with group S, the neurobehavioral score, the number of apoptosis and the expression of Bax and GSK-3βprotein in group M all increased (P<0.05), while the expression ofβ-catenin and Bcl-2/Bax ratio decreased (P<0.05) ;Compared with group M, the neurobehavioral score, the number of apoptosis and the expression of Bax protein were significantly decreased (P<0.05), while the expression of Bcl-2, β-catenin protein and the Bcl-2/Bax ratio were significantly increased (P<0.05) in group MI.Compared with group MI, the neurobehavioral score, the number of apoptosis, Bax and GSK-3βprotein in group MDI were significantly increased (P<0.05), while the Bcl-2, β-catenin protein expression, and Bcl-2/Bax ratio were significantly decreased (P<0.05).Conclusion Isoflurane post-conditioning may protect the hippocampus neurons against cerebral ischemic reperfusion-induced damage via the way that the Wnt/β-catenin signaling pathway regulates the expression levels of Bcl-2 and Bax proteins in rats.

Objective To establish a fast and quantitative light initiated chemiluminescent assay (LICA) method for high mobility group box1 (HMGB1) determination.Methods Two strains of paired HMGB 1 monoclonal antibodies were used.One was used to coat receptor microspheres.The other was labeled with biotin first and then composed with chain mildew element of affinity donor microsphere to form LICA method for HMGB1.After optimizing the reaction system,the technical specifications of the method was evaluated.Serum HMGB1 levels of common pneumonia patients (CPP) and severe pneumonia patients (SPP) were measured and compared with that of health controls.Two-sample t test was used.Results The sensitivity of LICA was 0.1 μg/L,with linear measurement ranging from 0.1 to 1 000 μg/L.The precisions of intra-and inter-analysis were 1.74％-2.92％ and 1.93％-3.73％ respectively,both were lower than 5％.The recovery rate was 99.74％ (range:94.53％-106.37％).The correlation coefficient of LICA and enzyme-linked immunosorbent assay (ELISA) was 0.888 2.The LICA method had good specificity and no obvious cross reaction with HMGB2 and HMGB3.The serum HMGB1 level in CPP (n=35) and SPP (n=25) was significantly higher than that in health controls (n=35):(6.76±3.13),(19.69±+9.04) vs (1.49±+0.74) μg/L;t values:-5.447 and-5.186,both P＜0.01.The HMGB1 levels between CPP and SPP were also significantly different (t=-3.500,P＜0.01).Conclusions The established LICA method of HMGB1 has high sensitivity and specificity with reliable results.This method is also homogeneous,fast and cleaning-free,thus has a good prospect in clinical application.

Objective To determine the effects of different doses of cisatracurium on motor e-voked potential of neurosurgery operation.Methods Sixty patients,36 males and 24 females,aged 18 to 65 years,ASA physical status Ⅰ or Ⅱ,scheduled for spinal surgery with motor evoked potential monitoring,were included and randomly assigned to three groups.A single dose of cisatra-curium besilate for injection was given by intravenous injection in 5 s after the induction of general an-esthesia,respectively 0.1 mg/kg (group A),0.1 5 mg/kg (group B)and 0.2 mg/kg (group C).Cas-cade Elite 32 channel monitor was used to monitor MEPs,the electrode was stimulated for once two minutes after given the muscle relaxant,and the leading time of the wave of MEPs was recorded. Cooper’s score was used to evaluate the intubation conditions.Results The appearance time of the wave of motor evoked potentials was significantly longer in group C [(39.60±1.79)min]than that in groups A [(20.10 ± 1.89 )min]and B [(20.50 ± 1.93 )min](P < 0.05 ).The intubation conditions was significantly better in group B (100%)and C (100%)than that in group A (65%)(P<0.05).Conclusion The shortest time to elicit waveform of MEPs using the dose of cisatracurium is 0.1 5 mg/kg at induction of general anesthesia,which is better for tracheal intubation.The dose 0.1 5 mg/kg of cisatracurim is recommended as the initial dose on neurosurgery operation with motor e-voked potential monitoring.

Objective To evaluate the effect of ropivacaine combined with low-dose naloxone or sufentanilropivacaine mixture on brachial plexus block carried under the guidance of ultrasound.Methods A total of 100 patients of our hospital undergoing upper limb surgery was randomly divided into four groups with 25 patients in each group.Four groups are patients receiving 20 mL of 0.375％ mesylate ropivacaine (Group D),20 mL of 0.375％ mesylate ropivacaine + 10 μg sufentanil (Group S),20 mL of 0.375％ mesylate ropivacaine + 100 ng naloxone (Group N) and 20 mL of 0.375％ mesylate ropivacaine + 10 μg sufentanil +100 ng naloxone (Group N+S).All patients underwent interscalene brachial plexus block under ultrasound guidance.The sensory block,motor block and other adverse reactions were observed and recorded at 5min,6,12,18,24 h.Results The sensory and motor block time of group D was (435.5 ± 77.9) min and (350.2 ± 69.8) min,group S (831.7 ± 52.0)min and (675.8 ± 48.1)min,group N (933.0 ± 117.1) min and (499.0 ± 40.5) min,group N+S (919.3 ± 59.0) min and (534.8 ± 56.6)min.The sensory block time of group N and group N + S were significantly longer than that of group D and S (P ＜0.05).The sensory and motor block time of group D were obviously shorter than that of other groups (P ＜ 0.05).There were no significant difference in the onset time of sensory and motor block in all groups.Conclusion Low dose of naloxone combined with ropivacaine or sufentanil-ropivacaine mixture can increase the duration of sensory block on brachial plexus.

Objective To observe the expression of PAR2 and TMEM16A in the model of chronic constriction injury (CCI) in rat dorsal root ganglion (DRG) neurons,and to explore the role of it in the neuropathic pain.Methods Rats were divided into Sham operation group (Sham) and CCI group.Both groups were observed respectively to determine thermal withdrawal latency (TWL).The expression of PAR2 and TMEM16A in the dorsal root ganglion of the rat was analyzed using Western blot and immunofluorescence.Results The difference in preoperative TWL between CCI group and Sham group rats was not statistically significant (P ＜ 0.01).TWL was signifi cantly lower at all other time points after operation (P ＜ 0.01).Immunofluorescence results showed that PAR2 and TMEM16A coexisted in rat DRG neurons.Western blot results showed that,compared with Sham group,CCI group PAR2 and TMEM16A protein expression significantly increased after 7 d and 14 d (P ＜ 0.01),and the PAR2 and TMEM16A protein expression on 14 d is higher than that of 7 d (P ＜ 0.05).Conclusions Expression level of PAR2 and TMEM16A in CCI group was significantly higher than those in Sham group.The expression level of these proteins may be the cause of rat model of neuropathic pain.

Epithelial-mesenchymal transition is the phenomenon of epithelial cells transforming to mesenchymal cells under special microenvironment,and thus tumor cells obtain characters such as difficult to adhere,motility enhancement,resistant to apoptosis.In the process of embryonic growth and tumor development,epithelial cells were found to be transformed into mesenchymal cells.Many researches showed that epithelial-mesenchymal transition plays an important role during the invasion and metastasis of colorectal cancer,and its mechanism involves many kinds of signaling pathways and related molecules.

Objective To investigate the effects of different BIS values on postoperative cogni-tive dysfunction (POCD)and S100βprotein(S100β)in the early stage of postoperation.Methods Fifty patients who were scheduled for selective abdominal surgery under general anesthesia (male 34 cases, female 1 6 cases,aged 65 to 75 years,ASA Ⅰ or Ⅱ)were randomly divided into two groups:light anesthesia group (group L,n =25,BIS value was maintained at 50 to 59)and deep anesthesia group (group D,n =25,BIS value was maintained at 30 to 39).BP,HR,SpO 2 ,ECG,PET CO 2 ,inhaled anes-thetic concentration and BIS values were recorded on time points of 5 minutes after the patients ente-ring the operating room (T0 ),before endotracheal (T1 ),intubation (T2 ),incision (T3 ),two hours after incision (T4 ),three hours after incision (T5 )and at the end of surgery (T6 ).The procedure du-ration,anesthesia time,dosages of propofol,fentanyl,midazolam and VAS scores on 1 d after sur-gery were also recorded.Blood samples were collected on time points of 10 min before anesthesia,im-mediately after surgery and 24,48 h after operation.S100β concentration were detected.Mini-mental State Examination (MMSE)score and Trail Making Test (TMT)completion time were recorded on 1 d before surgery and 1,3,7 d after surgery.Results BIS value of group D were lower than group L on T2 ,T3 and T4 .The propofol dosage of group D was significantly greater than that in group L (P <0.05 ).The concentration of serum S100βincreased significantly immediate and 48 h after operation in both groups compared with 10 min before anesthesia(P < 0.05).It was still higher 24 hours after op-eration than before anesthesia.But there was no statistic difference.Compared with the end of surger-y,the concentration of serum S100βin two groups on 24 h after surgery were significantly decreased (P < 0.05 ).The concentration of serum S100β in group L on the end of surgery and 24 h after surgery were higher than that in group D significantly (P <0.05).Compared with 1 d before surgery, postoperative 1 d MMSE scores in two groups and postoperative 3 d MMSE score in group L de-creased significantly (P <0.01).Compared with postoperative 3 d,postoperative 7 d MMSE score in group L increased significantly (P <0.01).Postoperative 1,3 d MMSE score in group D were signifi-cantly higher than group L (P <0.05).Compared with 1 d before surgery,TMT completion time in two groups on 1 d after surgery were significantly prolonged (P <0.01 ).Compared with 1 d after surgery,TMT completion time in two groups on 3 d after surgery were significantly shortened (P <0.01).Compared with 3 d after surgery,TMT completion time in group L on 7 d after surgery was significantly shortened (P <0.01 ).TMT completion time in group D on 1,3 d postoperative were significantly shorter than group L (P <0.05).POCD incidence of group D on 1 d after surgery was lower than that in group L (P < 0.05).Conclusion Different depth of anesthesia can ensure hemo-dynamic balance in old patients during surgery and after surgery.When BIS value was maintained at 30 to 39,it had lower S100βprotein levels,lower incidence of early POCD and a lesser degree of post-operative cognitive dysfunction.

Gastrointestinal neuroendocrine tumor(GI-NET)originates from peptide neurons and neu-roendocrine cells in gastrointestinal tract,and secrets peptide hormones,leading to carcinoid syndrome which rarely happens in clinical practice. Because of the improvement of diagnostic method and understanding of this rare disease,the morbidity is rising in recent years. The main treatments of GI-NET are surgery and compre-hensive therapy,consisting of chemotherapy and targeted therapy.