Objective　To detect clustered regularly interspaced short palindromic repeats (CRISPR) in Listeria monocytogenes, and analyze the structure and homology of CRISPR loci. Methods Totally 34 strains of Listeria monocytogenes isolated in our laboratory were identified, PCR amplified and sequenced. The repeat sequence structure and spacer sequence homology in CRISPR loci were analyzed by bioinformatics software. Results A total of 7 CRISPR loci were detected in 34 strains. The mutation rate of the first 2 and last 2 bases of the Repeat sequence of CRISPR loci was higher, while the mutation rate of the middle part was lower. Seven CRISPR sites form eight CRISPR structural types, among which the Repeat sequences of CRISPR1 and CRISPR2 are relatively conserved, while the Repeat sequences of CRISPR1 and CRISPR5 can form dumbbell shaped secondary structures. The number of Spacer sequences contained in each CRISPR site ranges from 2 to 15, with an average of 2.43. The 136 Spacer sequences detected were not only homologous to Listeria plasmids and bacteriophages, but also homologous to uncultured virus sequences, staphylococcal bacteriophages, and Listeria innocua. The same CRISPR genotype did not show large-scale clustering, but some strains in the same year were in the same evolutionary cluster with close genetic relationships. Conclusion The CRISPR structure of Listeria monocytogenes in this study exhibits high specificity, and its homology with bacteriophages provides a theoretical basis for the application of bacteriophages in the control and prevention of Listeria monocytogenes.

Cholangiocarcinoma (CCA) is a fatal malignancy with steadily increasing incidence and poor prognosis. Since most CCA cases are diagnosed at an advanced stage, systemic therapies, including chemotherapy, radiotherapy, targeted therapy, and immunotherapy, play a crucial role in the management of unresectable CCA. The recent advances in targeted therapies and immunotherapies brought more options in the clinical management of unresectable CCA. This review depicts the advances of targeted therapies and immunotherapies for unresectable CCA, summarizes crucial clinical trials, and describes the efficacy and safety of different drugs, which may help further develop precision and individualization in the clinical treatment of unresectable CCA.
Humans ; Cholangiocarcinoma/drug therapy* ; Immunotherapy/methods* ; Bile Duct Neoplasms/drug therapy* ; Molecular Targeted Therapy/methods*

Objective:To analyze the clinical features of patients with severe dengue (SD) in Guangdong Province, and to improve the understanding of the diagnosis and treatment of SD in China.Methods:The clinical data, laboratory examination and etiological test results of 257 SD cases from 29 dengue fever designated hospitals in Guangdong Province from January 1, 2013 to December 31, 2019 were respectively collected. The relevant indicators of the criteria for severe organ involvement were quantified. Logistic regression analysis was performed to analyze the risk factors for the development of multiple organ failure in SD patients.Results:Among the 257 SD patients, age was (64.1±20.1) years old, with 65.4%(168/257) of them ≥60 years old, 142 were male and 115 were female. One hundred and fifty-two (59.1%) patients had underlying conditions, including 115(44.7%) patients with hypertension. The clinical manifestations were mainly fever (98.4%(253/257)), fatigue (70.0%(180/257)), cough or expectoration (44.4%(114/257)), lethargy or irritability (39.3%(101/257)), vomiting (30.4%(78/257)), abdominal pain or tenderness (20.6%(53/257)), hepatomegaly (2.3%(6/257)), bleeding tendency (59.5%(153/257)), and pleural effusion or ascites (43.6%(112/257)). Platelet count levels were decreased in 90.9%(231/254) of the cases, and 97.1%(234/241) of patients had normal or decreased hematocrit. The most common of severe manifestations were severe organ involvement (61.1%(157/257)), followed by severe bleeding (37.0%(95/257)) and severe plasma leakage (30.0%(77/257)). Severe organ involvements were more common in the kidney (27.6%(71/257)) and heart (26.8%(69/257)). Multivariate logistic regression analysis showed that age (odds ratio ( OR)=1.051, 95% confidence interval ( CI) 1.004 to 1.100, P=0.035), hypertension ( OR=5.224, 95% CI 1.272 to 21.462, P=0.022), elevated aspartate aminotransferase (AST) level ( OR=1.002, 95% CI 1.001 to 1.003, P=0.001), blood urea nitrogen (BUN) ( OR=1.050, 95% CI 1.005 to 1.098, P=0.030), and international normalized ratio (INR) ( OR=4.604, 95% CI 1.601 to 13.238, P=0.005) were risk factors for the development of multiple organ failure in SD patients. The detection results of serum samples form 113 SD patients in acute phase showed that dengue virus (DENV)-1 accounted for 89.4%(101/113), DENV-2 accounted for 9.7%(11/113), and DENV-3 accounted for 0.9% (1/113). Conclusions:Elderly and those with co-existing conditions such as hypertension in SD patients in Guangdong Province are more common. Severe organ involvement such as kidney and heart is the main cause of SD. DENV-1 infection is predominant. Significant elevated levels of AST, BUN and INR may be related to a poor prognosis.

AlM: The Chinese medicinal herb Hanfangji is dried roots of Stephania tetrandra S. Moore (Family, Menispermaceae). Tetrandrine and fangchinoline are two major constituents of Hanfangji and these bisbenzyltetrahydroisoquinoline alkaloids possess anti - cancer and other pharmacological activities. To facilitate further pharmacodynamic investigation of these compounds, a pharmacokinetic investigation was performed in rats and in vitro. METHODS: Pharmacokinetics of tetrandrine and fangchinoline were characterized in rats p.o. or i.v. dosing an aqueous extract of Hanfangji or the individual compound. Unbound levels of systemic exposure to these two alkaloids were assessed using in vitro studies of plasma protein binding, blood-plasma partition, and lysosomal trapping. All the study samples were analyzed by liquid chromatography/mass spectrometry.RESULTS: We found two pharmacokinetic features of tetrandrine and fangchinoline. First, the two compounds had blood levels of systemic exposure substantially higher than the respective plasma levels of systemic exposure. Second, the two compounds exhibited significantly higher systemic exposure levels after p.o. dosing an aqueous extract of Hanfangji than the respective exposure levels after p.o. dosing the individual compound, at the same compound dose levels and under the same conditions for analytical measurement and the same conditions for animal study. Unbound fractions of tetrandrine and fangchinoline in rat plasma were 2%-5% and the concentrations of the alkaloids in rat erythrocytes were 5-times higher than those in rat plasma. Lysosomal inhibitor could block their trapping in lysosomes and significantly reduce their concentrations in HEK-293 cells. CONCLUSlON: The following pharmacokinetic aspects should be noted in pharmacodynamic investigation of tetrandrine and fangchinoline: extensive binding with plasma proteins, extensive binding with erythrocytes, and trapping by lysosomes of tissue cells substantially reduce the levels of unbound tetrandrine and fangchinoline in the systemic circulation.

Objective:To evaluate the effect of electroacupuncture postconditioning on the expression of microRNA-1 (miRNA-1) in rats with arrhythmias induced by myocardial ischemia-reperfusion (I/R).Methods:Twenty-four clean-grade healthy adult male Sprague-Dawley rats, aged 2-3 months, weighing 280-320 g, were divided into 3 groups ( n=8 each) using a random number table method: sham operation group (S group), I/R group and electroacupuncture postconditioning group (EP group). The model of myocardial reperfusion arrhythmia was established by ligating the left anterior descending branch of coronary artery for 30 min followed by 30-min reperfusion in anesthetized rats.The artery was only isolated without ligation after thoracotomy in group S. Bilateral Neiguan acupoints were stimulated for 30 min starting from the time point immediately after reperfusion in EP group.The occurrence of arrhythmia during reperfusion was recorded.The rats were sacrificed at 30 min of reperfusion, and hearts were removed for examination of the pathological changes of the myocardium and for determination of miRNA-1 expression (by quantitative real-time polymerase chain reaction) and expression of Kir2.1 and connexin 43 (Cx43) (by Western blot). Results:Compared with S group, the incidence of arrhythmia was significantly increased, the expression of miRNA-1 was up-regulated, and the expression of Kir2.1 and Cx43 was down-regulated in I/R and EP groups ( P<0.05). Compared with I/R group, the incidence of arrhythmia was significantly decreased, the expression of miRNA-1 was down-regulated, and the expression of Kir2.1 and Cx43 was up-regulated in EP group ( P<0.05). The pathological changes of myocardium were accentuated in I/R and EP groups when compared with S group, and the pathological changes were significantly attenuated in EP group when compared with I/R group. Conclusion:The mechanism by which electroacupuncture postconditioning reduces the occurrence of reperfusion arrhythmia is related to up-regulation of Kir2.1 and Cx43 expression after down-regulation of miRNA-1 expression in rats.

Objective:To evaluate the effect of electroacupuncture (EA) on electrophysiological characteristics of ventricular myocardium during myocardial ischemia-reperfusion (I/R) in rats.Methods:Twenty-four clean-grade healthy adult male Sprague-Dawley rats, weighing 280-320 g, were divided into 3 groups ( n=8 each) using a random number table method: sham operation group (group SH), I/R group and group EA.The model of myocardial I/R injury was established by ligating the left anterior descending branch of coronary artery for 30 min followed by 30-min reperfusion in anesthetized rats.Bilateral Neiguan acupoints in forelimbs were stimulated for 30 min during the period of reperfusion in group EA.Heart rate (HR), monophasic action potential amplitude (MAPA), maximum depolarization rate (V max), and monophasic action potential duration at 90% repolarization (MAPD 90) were recorded.The development of arrhythmias and arrhythmias score were recorded during reperfusion. Results:Compared with group SH, HR was significantly decreased, MAPA and V max were decreased, MAPD 90 was prolonged, and the incidence of ventricular premature beat, ventricular tachycardia and ventricular fibrillation and arrhythmias score were increased at T 1, 2 in I/R and EA groups ( P<0.05). Compared with group I/R, HR was significantly increased, MAPA and V max were increased, MAPD 90 was shortened, and the incidence of ventricular tachycardia and ventricular fibrillation and arrhythmias score were decreased at T 2 in EA group ( P<0.05). Conclusion:EA can accelerate myocardial depolarization and shorten repolarization, thus decreasing the occurrence of reperfusion arrhythmia in rats.

Objective To assess the capability of serum and urine neutrophil gelatinase-associated lipocalin (NGAL) in predicting acute kidney injury (AKI) in septic patients.Methods From July 1, 2014 through December 31, 2014, a prospective observational study of septic patients without AKI was carried out in Renmin Hospital, Tianjin Hospital, and Nankai Hospital, Tianjin.The patients with AKI which was developed after admission to intensive care unit (ICU) were assigned to AKI group and the patients without AKI were assigned to non-AKI group.Clinical data and serum and urine sample were collected at the admission, and at 12, 24, 36, 48, 60, 72 ,84 and 96 hours after admission for detecting creatinine and NGAL.The measurement data accorded with normal distribution were used for t test or variance analysis of repeated measures;comparison of measurement data in non-normal distribution was carried out using the Mann-Whitney U test or Fridman test;comparison of count data was performed using Fisher exact probability method.ROC curve of serum or urine NGAL was plotted and the diagnostic values of serum or urine NGAL in predicting AKI were assessed by calculation of the area under the receiver operating characteristic curve (AuROC).Results Fifty septic patients were included.Thirty-five patients were in AKI group and fifteen in non-AKI group.The median age of AKI group was 73 and the median age of non-AKI group was 60 (IQR, 47-82).The urine NGAL (uNGAL) concentration in AKI group was higher than that in non-AKI group at the every interval and serum NGAL (sNGAL) was higher in AKI group than that in non-AKI group at only first twointervals.The uNGAL showed the capability of prediction for AKI progression at the 48 hour (AuROC=0.83,95% CI:0.70-0.97), 36 hours (AuROC=0.75,95%CI:0.59-0.91), 24 hours (AuROC=0.83,95%CI:0.70-0.95), 12 hours (AuROC=0.73,95%CI:0.60-0.88) prior to AKI occurred.The sNGAL showed capability of prediction at the 48 hours (AuROC=0.69,95%CI:0.51-0.88), 36 hours (AuROC=0.69,95%CI:0.52-0.87) prior to AKI occurred.Conclusions The sNGAL and uNGAL both were useful biomarker that predicted development of AKI in early stage.But the performance of sNGAL was slightly inferior to that of uNGAL for predicting development of AKI.

OBJECTIVETo establish a systematic method for isolation and identification of aerobic and facultative anaerobic bacteria in the oral cavity.

METHODSSamples of the saliva, dental plaque and periapical granulation tissue were collected from 20 subjects with healthy oral condition and from 8 patients with different oral diseases. The bacteria in the samples were identified by morphological identification, VITEK automatic microorganism identification and 16s rRNA gene sequencing.

RESULTSVITEK automatic microorganism identification and 16s rRNA gene sequencing showed an agreement rate of 22.39% in identifying the bacteria in the samples. We identified altogether 63 bacterial genus (175 species), among which Streptococcus, Actinomyces and Staphylococcus were the most common bacterial genus, and Streptococcus anginosus, Actinomyces oris, Streptococcus mutans and Streptococcus mitis were the most common species. Streptococcus anginosus was commonly found in patients with chronic periapical periodontitis. Streptococcus intermedius and Staphylococcus aureus were common in patients with radiation caries, and in patients with rampant caries, Streptococcus mutans was found at considerably higher rate than other species.

CONCLUSIONAerobic and facultative anaerobic bacteria are commonly found in the oral cavity, and most of them are gram-positive. 16s rRNA gene sequencing is more accurate than VITEK automatic microorganism identification in identifying the bacteria.

Actinomyces ; isolation & purification ; Dental Caries ; Dental Plaque ; microbiology ; Humans ; Mouth ; microbiology ; RNA, Ribosomal, 16S ; genetics ; Saliva ; microbiology ; Staphylococcus aureus ; isolation & purification ; Streptococcus ; isolation & purification

Objective ①Observing urinary neutrophil gelatinase-associated lipocalin (uNGAL)'s concentration variation under the intervention of sepsis; ②Evaluatingu NGAL' s diagnostic value for early acute kidney injury (AKI).Method Fifty-six SD (Sprague Dawley) rats were randomly (random number) divided into four groups,including 16 rats in model group (CLG),16 rats in Xuebijing group (XBG),16 rats in Huangqi and Chaihu injection jointly applied group (HCG),and 8 rats in sham operation group (SOG).The septic models in CLG group,HCG group and XBG group were established by cecal ligation and puncture (CLP).Then,the rats in HCG group was treated with intraperitoneal injectionby Huangqi and Chaihu injections; the XBG group was treated with intravenous injection by Xuebijing injection; the SOG group was treated with open surgery without CLP.After the CLP,serial urine and serum samples were obtained at baseline (just prior to operation),6 h,12 h,18 h,24 h,36 h,48 h,and 72 h,and were measured by sCr,uCr,uNa,and uNGAL.The line graph of uNGAL' s concentration variation was plotted,based on the time.Diagnostic characteristics of urinary NGAL in predicting AKI were assessed by calculating the area under the receiver operating characteristic curve (AUC).Results After the CLP,the uNGAL of sepsis model rats increased quickly within 6 hours.The time points of each group model reaching their peak were 6 hours after CLP in CLG groupand 24 hours after CLP in HCG group and XBG group.These groups' uNGAL all decreased quickly after the peak.The cuNGAL of sepsis model rats was increased quickly within 6 hours after CLP,reached its peak at 24 hours after CLP.In CLG group,the line graphs of uNGAL or cuNGAL were almost overlapped.There is little difference in the concentration of uNGAL or cuNGAL at each time point (uNGAL:6h,t=0.691; 12h,t=1.627; 18 h,t=0.511,cuNGAL:6h,t =0.371 ; 12 h,t =0.474; 18 h,t =-1.187.Statistical significance of all above value was P ＞0.05).InXBG group,the line graph of uNGAL and cuNGAL were not overlapped,but difference between uNGAL and cuNGAL concentration at each time point was not significant (uNGAL:6 h,t =1.222 ; 12 h,t =1.178 ; 18h,t=1.272; 24h,t=0.918; 36h,t =0.442.cuNGAL:6 h,t =1.482; 12 h,t =1.314; 18 h,t=1.280; 24 h,t =0.280; 36 h,t =0.467.Statistical significance of all above value was P ＞ 0.05).In HCG group,uNGAL of AKI rats were higher than non-AKI rats at each time points since 6 hours later (6 h,t =2.351,P＜0.05; 12h,t=3.086,P＜0.01; 18h,t=2.535,P＜0.05;24h,t=2.150,P＜0.05;36h,t =2.485,P ＜ 0.05),The average cuNGAL of AKI rats and non-AKI rats have statistical significance at 6h,18 h,and 24 h (6 h,t=3.013.P＜0.01; 18 h,t =4.804,P＜0.01; 24 h,t=2.682,P＜0.05).At 6 h,Uout can increase cuNGAL' s ability of predicting AKI' s occurrence in 24 hours (AUC increased from 0.839 to 0.900,P ＜ 0.05).Conclusions The intervention to the sepsis rats have influence on the secretion volume and secretion sequence of NGAL in rat urine.uNGAL and cuNGAL are good predictor of AKI occurrence in sepsis rats.

Objective To establish a systematic method for isolation and identification of aerobic and facultative anaerobic bacteria in the oral cavity. Methods Samples of the saliva, dental plaque and periapical granulation tissue were collected from 20 subjects with healthy oral condition and from 8 patients with different oral diseases. The bacteria in the samples were identified by morphological identification, VITEK automatic microorganism identification and 16s rRNA gene sequencing. Results VITEK automatic microorganism identification and 16s rRNA gene sequencing showed an agreement rate of 22.39%in identifying the bacteria in the samples. We identified altogether 63 bacterial genus (175 species), among which Streptococcus, Actinomyces and Staphylococcus were the most common bacterial genus, and Streptococcus anginosus, Actinomyces oris, Streptococcus mutans and Streptococcus mitis were the most common species. Streptococcus anginosus was commonly found in patients with chronic periapical periodontitis. Streptococcus intermedius and Staphylococcus aureus were common in patients with radiation caries, and in patients with rampant caries, Streptococcus mutans was found at considerably higher rate than other species. Conclusion Aerobic and facultative anaerobic bacteria are commonly found in the oral cavity, and most of them are gram-positive. 16s rRNA gene sequencing is more accurate than VITEK automatic microorganism identification in identifying the bacteria.