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*

Infertility has become a serious threat to the physical and mental health of women of childbearing age. With the development of modern medicine, stem cell has been widely concerned and gradually applied in medical research. As an emerging research focus in the field of reproductive medicine, stem cell therapy has shown good therapeutic effects in the treatment of infertility. This article reviewed the research progress of stem cell therapy in infertility diseases, aiming to provide new ideas for clinical treatment.

N6-methyladenine (m6A) modification is an RNA post-transcriptional level modification regulated by m6A methyltransferase, demethylated transferase and reading protein, which is the most widespread in messenger RNA. And it plays an important role in the reproductive development of mammals. On the one hand,m6A regulates RNA metabolism during oocyte maturation and early embryonic development, and on the other hand, m6A is closely associated with ovarian and uterine diseases, especially in gynecological tumors, m6A affects the proliferation and migration of tumor cells. This article reviews the research progress of m6A modification in female reproductive physiology and related diseases in order to provide new ideas for clinical treatment.

Infertility has become a serious threat to the physical and mental health of women of childbearing age. With the development of modern medicine, stem cell has been widely concerned and gradually applied in medical research. As an emerging research focus in the field of reproductive medicine, stem cell therapy has shown good therapeutic effects in the treatment of infertility. This article reviewed the research progress of stem cell therapy in infertility diseases, aiming to provide new ideas for clinical treatment.

N6-methyladenine (m6A) modification is an RNA post-transcriptional level modification regulated by m6A methyltransferase, demethylated transferase and reading protein, which is the most widespread in messenger RNA. And it plays an important role in the reproductive development of mammals. On the one hand,m6A regulates RNA metabolism during oocyte maturation and early embryonic development, and on the other hand, m6A is closely associated with ovarian and uterine diseases, especially in gynecological tumors, m6A affects the proliferation and migration of tumor cells. This article reviews the research progress of m6A modification in female reproductive physiology and related diseases in order to provide new ideas for clinical treatment.

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.