The application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) can be limited due to a lack of compatible protospacer adjacent motif (PAM) sequences in the DNA regions of interest. Recently, SpRY, a variant of Streptococcus pyogenes Cas9 (SpCas9), was reported, which nearly completely fulfils the PAM requirement. Meanwhile, PAMs for SpRY have not been well addressed. In our previous study, we developed the PAM Definition by Observable Sequence Excision (PAM-DOSE) and green fluorescent protein (GFP)‍-reporter systems to study PAMs in human cells. Herein, we endeavored to identify the PAMs of SpRY with these two methods. The results indicated that 5'-NRN-3', 5'-NTA-3', and 5'-NCK-3' could be considered as canonical PAMs. 5'-NCA-3' and 5'-NTK-3' may serve as non-priority PAMs. At the same time, PAM of 5'-NYC-3' is not recommended for human cells. These findings provide further insights into the application of SpRY for human genome editing.
CRISPR-Associated Protein 9/metabolism* ; CRISPR-Cas Systems ; DNA ; Gene Editing/methods* ; Humans ; Streptococcus pyogenes/metabolism*

OBJECTIVE: To compare the effects of robotic and laparoscopic-assisted radical total gastrectomy on lymph node dissection and short-term outcomes in patients with Siewert type II adenocarcinoma of esophagogastric junction (AEG). METHODS: Inclusion criteria: the tumor center was located between 2 cm above and below the esophagogastric junction and was confirmed as adenocarcinoma by endoscopic biopsy. EXCLUSION CRITERIA: tumor with local invasion of the liver,spleen, pancreas or other organs; intraoperative finding of tumor dissemination or distant metastasis; patients undergoing palliative surgical treatment or preoperative neoadjuvant chemotherapy; patients with serious heart diseases, lung diseases, liver diseases, kidney diseases and other comorbidities; patients with multiple primary cancers;patients receiving emergency surgery. According to the above criteria, 82 patients with Siewert type II AEG who underwent gastrointestinal surgery at the Affiliated Hospital of Qingdao University from October 2014 to October 2018 were enrolled in the study. They were randomly divided into robotic surgery groups (41 cases) and laparoscopic group (41 cases) according to a computer-generated randomized allocation table. Both groups underwent radical total gastrectomy plus D2 lymph node dissection through the transabdominal esophageal hiatus approach. The intraoperative conditions and postoperative short-term outcomes were compared between two groups, including surgery time, intraoperative blood loss, length of esophagectomy, postoperative complications, postoperative gastrointestinal recovery time, length of hospital stay, postoperative unplanned reoperation rate and rehospitalization rate. Mean±SD is used for the measurement data that conforms to the normal distribution, and two independent sample t-tests are used to compare the two groups; the comparison of the count data is performed by the χ² test. RESULTS: There were 35 males (85.4%) with age of (62.3±10.0) years and body mass index of (24.4±3.2) kg/m² in the robotic surgery group. There were 37 males (90.2%) with age of (62.5±10.0) years and body mass index of (23.8±2.6) kg/m² in the laparoscopic group. No significant differences in the baseline data between two groups were found (all P>0.05). All the patients of both groups completed R0 resection successfully without conversion to laparotomy or perioperative death. Compared with the laparoscopic group, the robotic group had less intraoperative blood loss [(70.7±39.9) ml vs. (110.2±70.6) ml, t=3.118, P=0.003], longer resected esophagus [(3.0±0.7) cm vs. (1.9±0.5) cm, t=8.759, P<0.001], but longer setup time [(56.5±7.4) minutes vs. (36.0±6.6) minutes, t=4.241, P<0.001], and higher hospitalization costs [(122 317.31±57 789.33) yuan vs. (99 401.56±39 349.53) yuan, t=2.099, P=0.039], whose differences were statistically significant (all P<0.05). The total number of harvested lymph node in the robotic surgery group was 39.2±15.3,which was significantly higher than that in the laparoscopic group (33.0±12.1) (t=0.733, P=0.047). In the robotic group and the laparoscopic group, the mediastinal lymph node No.110 and No.111 were 3.6±1.2 vs. 1.5±1.0 and 3.7±2.0 vs. 1.8±1.1, respectively, with significant difference (t=10.138, P<0.001, t=8.227, P<0.001); axillary lymph node No.19 and No.20 were 2.3±1.2 vs. 1.1±0.9 and 2.0±1.0 vs. 1.0±0.1, respectively, with significant difference (t=7.082, P<0.001,t=8.672,P<0.001). There were no significant differences in the total number of abdominal lymph node and the number of lymph node in abdominal stations between two group (all P>0.05). The highest lymph node metastasis rate was approximately 20% and observed in No.1, No.2, No.3, and No.7, followed by No.8a, No.9, No.11p, and No.110 with around 5%. The lymph node metastasis rate in other stations (No.4sa, No.4sb, No.4d, No.5, No.6, No.11d, No.12a, No.19, No.20 and No.111) was less than 5%.There were no significant differences in postoperative complication rate, postoperative fever time, postoperative exhaust and defecation time, fluid diet time, and postoperative hospital stay (all P>0.05). There were 2 patients(4.9%) with unplanned reoperation and 1 patient (2.4%) with unplanned re-admission in the laparoscopic group,while 3 patients (7.3%)with unplanned reoperation and 2 patients (4.9%)with unplanned re-admission in the robotic surgery group, whose differences were also not statistically significant (χ²=0.240,P=0.675;χ²=0.346,P=1.000). CONCLUSION Robot-assisted radical total gastrectomy for Siewert II AEG is safe and feasible, which is characterized by more sophisticated operation, less blood loss and higher quality of lymph node dissection, especially for subphrenic and inferior mediastinal lymph nodes.
Adenocarcinoma ; classification ; pathology ; surgery ; Aged ; Esophageal Neoplasms ; classification ; pathology ; surgery ; Esophagectomy ; Esophagogastric Junction ; pathology ; surgery ; Female ; Gastrectomy ; Humans ; Laparoscopy ; Lymph Node Excision ; methods ; Male ; Middle Aged ; Retrospective Studies ; Robotic Surgical Procedures ; Stomach Neoplasms ; classification ; pathology ; surgery ; Treatment Outcome

This study was aimed to systematically evaluate the clinical efficacy and safety of brucea javanica oil emulsion injection combined with gemcitabine plus cisplatin (GP) regimen in the treatment of non-small cell lung cancer.Literatures were retrieved from databases of CNKI,Wanfang database,VIP,CBM,Pubmed from the date of database establishment to March 2017.Journals on related fields were also manually searched.The randomized controlled trials (RCTs) of brucea javanica oil emulsion injection combined with GP regimen in the treatment of non-small cell lung cancer were collected.The results showed that there were 12 included RCTs with 1 118 patients.The meta-analysis results showed that compared with single using of GP regimen,there were statistical significance in the partial remission rate [OR =1.59,95％ CI (1.23,2.05),P =0.000 4],the total remission rate [OR =1.72,95％ CI (1.33,2.22),P ＜ 0.000 1],the KPS (Karnofsky) score improvement [OR =2.59,95％ CI (1.96,3.43),P ＜ 0.000 01],the deterioration of KPS score [OR =0.33,95％ CI (0.25,0.46),P ＜ 0.000 01],the incidence of bone marrow suppression [OR =0.48,95％ CI (0.35,0.65),P ＜ 0.000 01],and the incidence of gastrointestinal adverse reactions [OR =0.46,95％ CI (0.23,0.91),P =0.03] by brucea javanica oil emulsion injection combined with GP regimen in the treatment of non-small cell lung cancer.It was concluded that brucea javanica oil emulsion injection combined with GP regimen can increase clinical efficacy,decrease myelosuppression rate and gastrointestinal adverse reactions.

BACKGROUND AND PURPOSE: Encephalitis caused by Listeria monocytogenes (L. monocytogenes) is rare but sometimes fatal. Early diagnosis is difficult using routine cerebrospinal fluid (CSF) tests, while next-generation sequencing (NGS) is increasingly being used for the detection and characterization of pathogens. METHODS: This study set up and applied unbiased NGS to detect L. monocytogenes in CSF collected from three cases of clinically suspected listeria meningoencephalitis. RESULTS: Three cases of patients with acute/subacute meningoencephalitis are reported. Magnetic resonance imaging and blood cultures led to a suspected diagnosis of L. monocytogenes, while the CSF cultures were negative. Unbiased NGS of CSF identified and sequenced reads corresponding to L. monocytogenes in all three cases. CONCLUSIONS: This is the first report highlighting the feasibility of applying NGS of CSF as a diagnostic method for central nervous system (CNS) L. monocytogenes infection. Routine application of this technology in clinical microbiology will significantly improve diagnostic methods for CNS infectious diseases.
Central Nervous System ; Cerebrospinal Fluid ; Communicable Diseases ; Diagnosis ; Early Diagnosis ; Encephalitis ; Humans ; Listeria monocytogenes* ; Listeria* ; Magnetic Resonance Imaging ; Meningitis, Listeria ; Meningoencephalitis* ; Methods

The three-in-one immunoaffinity column ( IAC ) for the determination of aflatoxin B1 ( AFB1 )-zearalenone (ZEN)-deoxynivalenol (DON) was prepared with rProtein A-sepharose 4B as the column matrix. The comprehensive performance ( such as nonspecific adsorption, column blank, column capacity, column efficiency and sample standard addition recovery rate) was evaluated and investigated. The results showed that, the column capacities of AFB1 , ZEN, DON were 295 ng per 0. 25 mL gel, 905 ng per 0. 25 mL gel, 2342 ng per 0. 25 mL gel, respectively, and the column blank was 0. The average recoveries of AFB1 , ZEN and DON were 97. 4%, 98. 0% and 98. 4%, respectively. By optimizing conditions, the samples were extracted using the mixture of methanol and water (80:20, V/V), and diluted with phosphate buffered saline (contain 0. 1% Tween-20, PBST). The detection results of FAPAS (Food Analysis Performance Assessment Scheme) by different batch three-in-one columns were close to the target value. The prepared three-in-one immunoaffinity column which could take the place of conventional single immunoaffinity column was able to meet the requirement for treatment of food and feed samples, and lay a foundation for one step enrichment, purification and detection of multi-mycotoxins.

Objective To evaluate the clinical efficacy and safety of capecitabine continuous maintenance in treatment of re-currence of triple-negative breast cancer,which had responded to combined chemoradiotherapy.Methods The triple-negative breast cancer was defined as negative of ER,PR and HER2.A total of 46 patients of triple-negative breast cancer were divided into the treatment group (23 patients)and the control group (23 patients).The treatment group was given capecitabine continuous mainte-nance after 6 cycles of chemotherapy.The control group was given 6 cycles of combined chemotherapy only.The clinical efficacy and safety was evaluated between the two groups.Results The PR,PD,RR,and DCR in the treatment group were significantly higher than those in the control group(P <0.05).The acute toxic effects (except for hand-foot syndrome)were similar in two group(P >0.05).Conclusion Capecitabine continuous maintenance after combined chemoradiotherapy in treatment of recurrence of triple-neg-ative breast cancer is more effective,lower toxicity and tolerable.

PurposeTo investigate the clinical value of inverted X-ray plain film and MRI examination in the diagnosis of congenital anorectal malformation (CARM). Materials and Methods Thirty-eight cases with operatively proved anorectal malformation were reviewed; inverted X-ray plain film and MRI examination were performed in all patients before surgery. The relationship between the rectum blind side and pubococcygeal line (PC line), and the type of anal atresia was determined, to compare the diagnostic accuracy of inverted plain film with MRI for CARM typing. Results Of all the 38 cases, 19 cases were with low imperforate anus, 8 cases with median imperforate anus, and 11 cases with high imperforate anus. The accuracy rate of inverted X-ray plain film and MRI examination for the diagnosis of CARM typing was 92.1% (35/38) and 97.4% (37/38) respectively, and the difference between them was not statistically significant (χ2=1.37, P>0.05). 7 cases of fistula, 5 cases of spinal cord malformations and 1 case of right kidney agenesis can be clearly demonstrated on MRI. Conclusion Both inverted X-ray plain film and MRI can diagnose the typing of CARM accurately, but MRI is also able to diagnose the fistula, visceral, spinal cord lesion and other abnormalities accompanied with CARM, while reducing the dose of X-ray radiation and damage in children, thus has higher clinical application value compared with inverted X-ray plain film.

Objective: To study the aortic root imaging and clinical application in 15 patients with transcatheter aortic valve implantation (TAVI).
Methods: A total of 15 patients with severe aortic valve stenosis received TAVI in our hospital from 2011-03 to 2013-11 were studied. The CT scan and transthoracic echocardiography were conducted to measure the aortic root anatomy and the differences of annulus size between CT and echocardiography were calculated. The prosthetic valves were selected based on CT measurement. The pre-operative accuracy of measurement was evaluated by the follow-up study at 6 months after operation.
Results: The CT measured pre-operative aortic annulus short diameter was (21.5 ± 2.4) mm, long diameter was (27.3 ± 2.7) mm, the average inner diameter was (24.4 ± 2.4) mm, left ventricular out lfow (LVOF) tract long diameter was (28.3 ± 4.5) mm, the average inner diameter of LVOF was (24 ± 3.5), ascending aorta diameter was (35.3 ± 4.4) mm. The Venus Medtech A-Valve implanted in 8 patients with #26 and in 7 patients with #29. The average inner diameter of aortic annulus measured by CT was larger than transthoracic echocardiography, P<0.001. During 6 months follow-up period, no patients had aortic root rupture, coronary obstruction, moderate and severer aortic and peri-aortic regurgitation. There were 4 patients with atrio-ventricular block and received permanent pacemaker implantation.
Conclusion: There is a difference for aortic annulus size by CT and transthoracic echocardiography measurements. CT may presisely assess the aortic root morphology and provide strong support for TAVI.

ObjectiveTo investigate the status and explore methods to improve the quality of diabetic education for young and middle-aged patients.MethodsOutpatients took part in diabetic education program and young and middle-aged diabetic patients that visited the eastern district of affiliated hospital of Qingdao university medical college from May 1st,2010 to Oct.30th,2010 were investigated with questionnaire for their diabetic education status and demands.ResultsA total of 381 patients were enrolled in this study.59.1％ clinical diabetic education program participants were aged 60 or older and only 40.9％ were aged 20 to 59 years,of whom 66.7％ had been diagnosed with diabetes more than 5 years and only 33.3％ had been diagnosed with diabetes less than 5 years.According to the survey of 101 young and middle-aged diabetic patients,only 16.8％ patients younger than 60 took part in different kinds of diabetic education programs but about 95％ young and middle aged patients wished to participate in diabetic education programs.ConclusionYoung and middle-aged diabetic patients have received much less diabetic education than elderly patients.Diabetic education for young and middle-aged patients needs to be strengthened urgently.

Objective To test the antimicrobial susceptibility of Staphylococcus aureus from children with impetigo,and to assess the differences in randomly amplified polymorphic DNA profiles between sensitive and resistant Staphylococcus aureus strains.Methods Secretion specimens were obtained from the impetiginous lesions of 178 children,and subjected to bacterial culture.The susceptibility of 162 Staphylococcus aureus isolates against 21 antibiotics was tested.Randomly amplified polymorphic DNA PCR(RAPD-PCR)was performed to characterize the genotype of Staphylococcus aureus.Results Totally,180 bacterial strains were isolated from 178 children with impetigo in Chengdu,including 162(90.00％)Staphylococcus aureus strains.Of the 162 Staphylococcus aureus strains,148 were methicillin sensitive Staphylococcus aureus(MSSA),14 methicillin resistant Staphylococcus aureus(MRSA).The most active antibiotic was minocycline,followed by teicoplanin,quinupristin,vancomycin and nitrofurantoin,while the resistance rate to penicillin was highest,followed by that to erythromycin,clindamycin,compound sulfamethoxazole and tetracycline.All the Staphylococcus aureus isolates were sensitive to fusidic acid,nitrofurantoin,vancomycin,minocycline and teicoplanin.According to RAPD-PCR,the 162 Staphylococcus aureus strains were divided into 8 genotypes,with the three most prevalent genotypes being Ⅲ(31.48％),Ⅱ(26.54％)and Ⅵ(25.93％),which accounted for 65.43％(106/162)in all the strains.The 148 MSSA strains fell into 8 genotypes,with genotype Ⅲ(50 strains,33.78％),Ⅵ(39 strains,26.35％)and Ⅱ(33 strains,22.30％)being the most prevalent genotypes;the 14 MRSA strains fell into 3 genotypes,i.e.,genotype Ⅱ(10 strains,71.43％),Ⅵ(3 strains,21.43％),and Ⅲ(1 strain,7.14％).Conclusions Staphylococcus aureus is the most prevalent pathogenic bacteria in children with impetigo in Chengdu area,which is highly sensitive to minocycline,teicoplanin and quinupristin,and falls into 8 genotypes according to RAPD-PCR with genotype Ⅲ being the most common genotype.