Objective:To observe any effect of electroacupuncture (EA) on the expression of phosphorylated extracellular signal-regulated protein kinase (p-ERK1/2) and phosphorylated cyclic adenosine monophosphate response element binding protein (p-CREB) in the spinal dorsal horns of diabetics experiencing neuropathic pain.Methods:Eight rats were randomly selected from 30 healthy male Sprague-Dawley rats as the normal group (N), and the remaining twenty-two rats were treated with a single high-dose intraperitoneal injection of streptozotocin (STZ) to establish a neuropathic pain model. The rats modeled successfully were randomly divided into a model group (M, n=8) and an EA group ( n=8). In the EA group, electroacupuncture was applied at the bilateral Hou san li and Kunlun acupoints starting on the 15th day after the STZ injection. The daily sessions lasted 30 minutes for 1 week. Body weight (BW), fasting blood glucose (FBG) and paw withdrawal latency (PWL) were observed before the STZ injection and on the 7th, 14th, and 21st days afterward. The expression of p-ERK1/2 and p-CREB in the dorsal horns of the rats′ spinal cords was detected using western blotting. The count of p-CREB-positive cells in the dorsal horns and their co-localization with neurons was detected using immunofluorescence. Results:In comparison with the N group, the average BW of the M group on the 7th, 14th and 21st days after the STZ injection was significantly lower, while the average FBG was significantly higher. There was no significant difference between the M and N groups in the average PWL on the 7th day after the STZ injection, but it had decreased significantly in the M group on the 14th and 21st days. Compared with the M group, the average PWL of the EA group was significantly longer on the 21st day after the injection. The expression of p-ERK1/2 and p-CREB protein in the spines of the M group was significantly higher than in the N group. p-CREB positive cells were more numerous in the M group compared with the N group, while in the EA group they were fewer. P-CREB was co-located with neurons in the spinal dorsal horn.Conclusion:EA can alleviate neuropathic pain effectively, perhaps by inhibiting the expression of p-ERK1/2 and p-CREB in the dorsal horns of the spinal cord.

Objective:To study the risk factors of massive bleeding in patients with acute Stanford type A aortic dissection undergoing moderate hypothermic circulatory arrest repair.Methods:From January 2016 to October 2017, 486 consecutive patients with acute type A aortic dissection were included in the study. All operations were performed with moderate hypothermic circulatory arrest. The basic clinical data of patients were collected retrospectively. Massive bleeding was defined according to definition of Universal Definition of Perioperative Bleeding(UDPB) 4 class and the Blood Conservation Using Antifibrinolytics in a Randomized Trial(BART). Significant variables in univariate analysis were included in multivariate logistic regression analysis. Results:Thirty-four patients(7.00%) died in hospital. A total of one hundred and eighty-seven patients(38.48%) fulfilled criteria of the definition of BART massive bleeding. Forty-five patients(9.26%), 8 patients(1.65%), 114 patients(23.46%), 147 patients(30.25%) and 172 patients(35.39%) were in grade 0, grade 1, grade 2 and grade 4, respectively. With BART as the end point, the result of multivariate logistic regression showed that female gender( OR=3.32, P<0.001), anemia( OR=2.24, P=0.04), clearance creatine≤85 ml/min( OR=1.93, P=0.01), D-dimer level(every 500 ng/ml increase, OR=1.02, P=0.003), cardiopulmonary bypass(CPB) time( OR=1.01, P<0.001), total arch replacement(TAR, OR=2.40, P=0.02) were independent risk factors for massive bleeding, and the time from onset to operation( OR=0.86, P=0.01) was protective factor. With UDPB 4 class as the end point, multivariate logistic regression showed that creatinine clearance≤85 ml/min( OR=2.05, P=0.001), CPB time( OR=1.01, P=0.04) were independent risk factors for massive bleeding. The time from anset to operation( OR=0.85, P=0.002) and Bentall procedure( OR=0.65, P=0.04) were the protective factors. Conclusion:Massive bleeding was more common in acute Stanford type A aortic dissection. Female gender, poor preoperative renal function, high D-dimer level, early time accepting surgical operation and long CPB were independent risk factors. For high-risk patients, simple and effective surgical methods should be taken to reduce the risk of bleeding.

Objective:To study the effect of MYD88 L265P mutation on the expression of PD-L1 in tumor cells and tumor microenvironment in diffuse large B-cell lymphoma (DLBCL), and to provide theoretical basis for immunotherapy for patients.Methods:Multiplex ligation-dependent probe amplification (MLPA) was used to detect the frequency of MYD88 L265P mutation in 72 cases of DLBCL diagnosed by pathologists in Cancer Hospital of Chinese Academy of Medical Sciences from August 2008 to May 2010. Expression of PD-L1 in tumor cells and tumor microenvironment in all samples was evaluated using PD-L1 (22C3) and PD-L1 (SP142) with Ventana automatic immunohistochemical (IHC) platform. The relationship between MYD88 L265P mutation and the expression of PD-L1 in DLBCL tumor cells and tumor microenvironment was assessed.Results:Of the 72 cases of DLBCL, MYD88 L265P mutation was detected in 15 (20.8%) cases. Nine cases with JAK2 amplification were excluded, and the remaining 63 cases of DLBCL were divided into MYD88 L265P mutant group ( n=14) and MYD88 L265P wild-type group ( n=49). IHC results showed that among the 14 cases of MYD88 L265P mutant groups, PD-L1 (22C3) was positive in 7 cases (7/14) of tumor cells and PD-L1 (SP142) was positive in 4 cases (4/14) of tumor microenvironment. Among the 49 cases of MYD88 L265P wild-type group, 9 cases (18.4%) were positive for PD-L1 (22C3) in tumor cells, and 38 cases (77.6%) were positive for PD-L1(SP142) in tumor microenvironment. In addition, among the 16 cases with PD-L1(22C3) expression in tumor cells, only 2 of the 7 cases with MYD88 L265P mutation were positive for PD-L1 (SP142) in tumor microenvironment. All 9 cases with wild-type MYD88 L265P were positive for PD-L1 (SP142) in tumor microenvironment. Statistical analysis showed that the expression level of PD-L1 (22C3) in tumor cells in the MYD88 L265P mutant group was significantly higher than that in the MYD88 L265P wild-type group ( P=0.017). The expression level of PD-L1 (SP142) in tumor microenvironment in the MYD88 L265P mutant group was significantly lower than that in the MYD88 L265P wild-type group ( P=0.001). Conclusions:MYD88 L265P mutation may play an important role in the regulation of PD-L1 expression in DLBCL tumor cells and tumor microenvironment. Further studies will provide a theoretical basis for immunotherapy of DLBCL patients with MYD88 L265P mutation.

Mutants of proteins are the basis for studying their structure and function, this work aimed to establish an efficient and rapid method for constructing multi-site mutants. When four or more adjacent amino acid residues need to be mutated, firstly, two long and two short primers (long primers Ⅰ/Ⅰ, short primersⅡ/Ⅱ) were designed: the long primers contain mutated sites, and the number of mutant bases is ≤20 bp, the short primers do not contain mutated sites; GC contents of the long and short primers are ≤80%, and the difference of annealing temperature is ≤40 °C. Then two sets of reverse PCR amplifications were performed using primer pairs (Ⅰ/Ⅱand Ⅰ/Ⅱ) and templates, respectively. After amplification, each system can obtain non-methylated linear plasmids which contain mutated sites, and the breakpoints of the two sets of linear plasmids amplified by primers Ⅰ/Ⅱ and Ⅲ/Ⅳ were distributed on both sides of the mutated sites. Followed by digested by DpnⅠ to remove the methylated templates, the recovered PCR products, which were mixed in an equimolar ratio, were performed another round of denaturation and annealing: the two sets of linear plasmids were denatured at 95 °C and then annealed with each other's single-stranded DNA as templates to form open-loop plasmids, and then the transformants containing the mutations will be obtained after transformed the open-loop plasmids into Escherichia coli competent cells. Results showed that, this method can mutate 4 to 11 consecutive amino acid residues (8-20 bp) simultaneously, which will greatly simplify the construction of multi-site mutants, Thereby improve the efficiency of protein structure and function research further.
DNA Primers ; genetics ; Escherichia coli ; Mutagenesis, Site-Directed ; methods ; Plasmids ; genetics ; Polymerase Chain Reaction

Objective:To investigate the cytotoxicity of a recombinant Vibrio vulnificus cytolysin membrane pore-forming peptide (rMpf) to J774A.1 cells as well as its influences on reactive oxygen species (ROS) and lysosomes, and to analyze the cellular localization of the rMpf. Methods:The rMpf was expressed using an Escherichia coli expression system and then used to treat murine J774A.1 cells in vitro. The viability of rMpf- and PBS-treated J774A.1 cells and the levels of ROS and lysosomes in these cells were analyzed by flow cytometry. Confocal microscopy was used to observe the cellular localization of rMpf in the J774A.1 cells. Results:No significant differences in cell viability, ROS production or lysosome formation in J774A.1 cells were found between low-dose (4 μg/ml) rMpf-treated and untreated groups. However, the cell viability, ROS production and lysosome formation were significantly decreased after treating J774A.1 cells with higher doses of rMpf (20 and 60 μg/ml). Moreover, the rMpf was found to localize in both the cytoplasm and nuclei of J774A.1 cells in a dose-dependent manner.Conclusions:The rMpf could localize in both the cytoplasm and nuclei of J774A.1 cells. It would inhibit the cellular ROS production and lysosome formation to damage macrophage function. This study provided a novel sight for understanding the cytotoxic domain and pathogenesis of Vibrio vulnificus cytolysin and other membrane pore-forming cytolysins.

Objective: To investigate the incidence of postoperative pancreatic fistula (POPF) and its risk factors after radical gastrectomy. Methods: The prospective study was conducted. The clinicopathological data of 2 089 patients who underwent radical gastrectomy in 22 medical centers between December 2017 and November 2018 were collected, including 380 in the Zhongshan Hospital of Fudan University, 351 in the Renji Hospital of Shanghai Jiaotong University School of Medicine, 130 in the Ruijin Hospital of Shanghai Jiaotong University School of Medicine, 139 in the Peking University Cancer Hospital, 128 in the Fujian Provincial Cancer Hospital, 114 in the First Hospital Affiliated to Army Medical University, 104 in the First Affiliated Hospital of Nanchang University, 104 in the Affiliated Hospital of Qinghai University, 103 in the Weifang People′s Hospital, 102 in the Fujian Medical University Union Hospital, 99 in the First Affiliated Hospital of Air Force Medical University, 97 in the Sir Run Run Shaw Hospital Affiliated to Zhejiang University School of Medicine, 60 in the Hangzhou First People′s Hospital Affiliated to Zhejiang University School of Medicine, 48 in the Fudan University Shanghai Cancer Center, 29 in the First Affiliated Hospital of Xi′an Jiaotong University, 26 in the Lishui Municipal Central Hospital, 26 in the Guangdong Provincial People′s Hospital, 23 in the Jiangsu Province Hospital, 13 in the First Affiliated Hospital of Sun Yat-Sen University, 7 in the Second Hospital of Jilin University, 4 in the First Affiliated Hospital of Xinjiang Medical University, 2 in the Beijing Chao-Yang Hospital of Capital Medical University. Observation indicators: (1) the incidence of POPF after radical gastrectomy; (2) treatment of grade B POPF after radical gastrectomy; (3) analysis of clinicopathological data; (4) analysis of surgical data; (5) risk factors for grade B POPF after radical gastrectomy. Measurement data with normal distribution were represented as Mean±SD, and comparison between groups was analyzed using ANOVA. Count data were described as absolute numbers or percentages, and comparison between groups was analyzed using the chi-square test. Univariate analysis was conducted using the t test or chi-square test based on data excluding missing data of clinico-pathological and surgical data. Multivariate analysis was conducted using the Logistic regression model based on factors with P<0.20 in univariate analysis. Results: There were 2 089 patients screened for eligibility, including 1 512 males, 576 females and 1 without sex information, aged (62±11)years. The body mass index (BMI) was (23±3)kg/m². (1) The incidence of POPF after radical gastrectomy: the total incidence rate of POPF in the 2 089 patients was 20.728%(433/2 089). The incidence rates of biochemical fistula, grade B pancreatic fistula, and grade C pancreatic fistula were 19.627%(410/2 089), 1.101%(23/2 089), 0, respectively. (2) Treatment of grade B POPF after radical gastrectomy: 2 of 23 patients with grade B POPF after radical gastrectomy had drainage tube placed for more than 21 days and received anti-infective therapy. Four of 23 patients with grade B POPF after radical gastrectomy had ascites detected by imaging examination, of which 2 received peritoneal drainage guided by ultrasound, 1 received failed puncture drainage, 1 received no puncture drainage, and they were given anti-infective therapy. Eleven of 23 patients with grade B POPF after radical gastrectomy had no ascites detected by imaging examinations, and they were given anti-infective therapy and inhibitors of pancreas secretion for clinical manifestation as fever or elevated white blood cells. Six patients with no typical clinical manifestations were given somatostatin to inhibite pancreas secretion and prolonged duration of abdominal drainage tube placement (with a median time of 7 days). All the 23 patients recovered well after treatment, without reoperation. (3) Analysis of clinicopathological data: for the 2 089 patients, BMI, cases with or without neoadjuvant therapy were (23±3)kg/m^2, 1 487, 160 of patients without pancreatic fistula, (23±3)kg/m^2, 386, 22 of patients with biochemical fistula, and (24±3)kg/m^2, 22, 1 of patents with grade B pancreatic fistula, showing significant differences between the three groups (F=5.787, χ²=8.269, P<0.05). (4) Analysis of surgical data: for the 2 089 patients, cases with open surgery, laparoscopic assisted surgery, totally laparoscopic surgery (surgical method), cases with D₁ lymph lode dissection, D₂ lymph lode dissection, and other lymph lode dissection (range of lymph lode dissection), cases with no omentectomy, partial omentectomy, and total omentectomy (range of omentectomy), cases with no usage of energy facility, usage of CUSA, LigaSure, LigaSure+ CUSA as energy facility, cases with or without biological glue, the number of lymph node dissection were 737, 624, 292, 24, 1 580, 51, 418, 834, 381, 63, 1 530, 23, 16, 1 431, 201, 33±14 of patients without pancreatic fistula, 146, 189, 74, 11, 389, 9, 110, 171, 128, 35, 359, 6, 9, 378, 31, 31±14 of patients with biochemical fistula, and 14, 5, 4, 0, 20, 3, 6, 13, 4, 2, 18, 1, 2, 22, 1, 37±16 of patients with grade B pancreatic fistula, showing significant differences between the three groups (χ²=15.578, 9.397, 15.023, 28.245, 8.359, F=4.945, P<0.05). (5) Risk factors for grade B POPF after radical gastrectomy: results of univariate analysis showed that usage of energy facility was a related factor for grade B POPF after radical gastrectomy (χ²=9.914, P<0.05). Results of multivariate analysis showed that laparoscopic assisted surgery, combined evisceration, application of LigaSure + CUSA, the number of lymph lode dissection were independent factors for for grade B POPF after radical gastrectomy (odds ratio=0.168, 3.922, 9.250, 1.030, 95% confidence interval: 0.036-0.789, 1.031-14.919, 1.036-82.602, 1.001-1.059, P<0.05). Conclusions The incidence of grade B POPF after radical gastrectomy is relatively low. Laparoscopic assisted surgery, combined evisceration, application of LigaSure + CUSA, and the number of lymph lode dissection are independent risk factors for grade B POPF. Trial Registration: This study was registrated at ClinicalTrial.gov in United States with the registration number of NCT03391687.

OBJECTIVE: To analyze the genotype and phenotype of a sibpair with partial deletion of SATB2 gene caused by 2q33.1 microdeletion. METHODS: Both children have featured mental retardation and development delay, and were subjected to karyotyping, single nucleotide microarray (SNP array) and real-time fluorescence quantitative PCR analysis. Karyotyping and SNP Array analysis were also carried out on their parents to verify the origin of mutation. RESULTS: Both sibs had a normal karyotype. SNP array showed that sib 1 had arr[hg19]2q33.1(200 192 328 - 200 197 269)×1 (4.9 kb), 2q35 (218 105 663 - 218 816 675)×3 (711 kb), while sib 2 had arr[hg19]2q33.1(200 192 328 - 200 197 269)×1 (4.9 kb), 2q35 (218 105 663-218 810 908)×3 (705.2 kb). The deletion has partially overlapped with that of 2q33.1 microdeletion syndrome and involved part of the SATB2 gene. The result of real-time fluorescence quantitative PCR assay was consistent with that of SNP assay. The duplication has originated from their father and has not been associated with any disease phenotypen. CONCLUSION Both sibs have carried partial deletion of SATB2 gene and had similar clinical phenotypes. Haploinsufficiency of such gene probably underlies the clinical manifestations in both patients.
Child ; Chromosome Deletion ; Chromosome Disorders ; Chromosomes, Human, Pair 2 ; Genetic Testing ; Humans ; Karyotyping ; Matrix Attachment Region Binding Proteins ; genetics ; Phenotype ; Transcription Factors ; genetics

Objective    To evaluate the involvement of renal artery in acute Stanford type A aortic dissection (TAAD) using CT angiography (CTA) and to analyze the difference of renal function among different types of renal artery involvement. Methods    From January 2016 to November 2017, 151 patients of acute TAAD with renal artery involvement were included in the study. There were 118 males and 33 females, with an average age of 47.93±10.53 years. All patients underwent aortic CTA to confirm the TAAD. According to CTA，involvement of one side of renal artery can be divided into four types: type A, large tear near renal artery orifice, difficult to distinguish true or false lumen; type B, the orifice of the renal artery originates entirely from the false lumen; type C, the orifice of the renal artery originates entirely from the true lumen; type D, renal artery dissection is observed, renal artery intima can be seen. The levels of serum creatinine (sCr) and creatinine clearance (CC) in all groups were analyzed and compared. Results    The results of one-way ANOVA analysis showed that there was no significant difference in sCr or CC among the groups (P>0.05). There was no significant difference in age, sex, proportion of hypertension history and onset time among the above groups (P>0.05). Conclusion    The three most common types of renal artery involvement were BC type, CC type, and AC type. The types of renal artery involvement do not affect renal function.

KylinRay-IMRT is the advanced radiotherapy treatment planning module of accurate radiotherapy system (KylinRay) aiming to provide accurate and efficient plan design platform. In this paper the system design, main functions and key technologies of KylinRay-IMRT were introduced. KylinRay-IMRT supports three dimensional conformal radiotherapy (3D-CRT), intensity modulated radiotherapy (IMRT) and many other types of treatment plan design with function modules including patient data management, image registration and fusion, image contouring, image three dimensional reconstruction and visualization, three dimensional conformal radiotherapy planning, intensity modulated radiotherapy planning, plan evaluation and comparison, and report print. KylinRay-IMRT has been tested by the national standard YY/T 0889-2013, the results showed that the performance of KylinRay-IMRT can fully meet the standard requirements.
Humans ; Radiotherapy Dosage ; Radiotherapy Planning, Computer-Assisted ; Radiotherapy, Conformal ; Radiotherapy, Intensity-Modulated ; Tomography, X-Ray Computed

Objective To explore the effect of transcranial direct current stimulation (tDCS) on the walking function of patients with early Parkinson's disease.Methods Thirty patients with early Parkinson's disease were randomly divided into an intervention group (n =15) and a control group (n =15).In addition to routine treatment,both groups were apparently provided with tDCS,though the current intensity for the intervention group was 2mA and that of the control group was zero.Both groups were treated for 20min every day for 5 days in succession.Before and after the treatment,all of the patients were evaluated using the timed up and go test (TUGT) and their gaits were analyzed to determine step velocity,cadence and width.Results Before and after the treatment there were no significant differences between the two groups in terms of average TUGT time,though the average TUGT time had decreased significantly compared with before the treatment.Before the treatment there were no significant differences between the two groups in average step velocity,cadence or width.After the treatment,significant improvements were observed only in the intervention group.The average step width of the intervention group was then significantly bigger than that of the control group.Conclusion Transcranial direct current stimulation can improve the walking function and stability of early Parkinson's patients.