The biotransformation, CYP reaction phenotyping, the impact of CYP inhibitors and enzyme kinetics of 3-cyanomethyl-4-methyl-DCK (CMDCK), a new anti-HIV preclinical candidate belonging to DCK analogs, were investigated in human intestinal microsomes and recombinant cytochrome P450 (CYP) enzymes. CMDCK (4 micromol L(-1)) was incubated with a panel of rCYP enzymes (CYP1A2, 2C9, 2C19, 2D6 and 3A4) in vitro. The remaining parent drug in incubates was quantitatively analyzed by a LC-MS method. CYP3A4 was identified as the principal CYP isoenzyme responsible for its metabolism in intestinal microsomes. The major metabolic pathway of CMDCK was oxidation and a number of oxidative metabolites were screened with LC-MS. The Km, Vmax, CLint and T1/2 of CMDCK obtained from human intestinal microsome were 45.6 micromol L(-1), 0.33 micromol L(-1) min(-1), 12.1 mL min(-1) kg(-1) and 25.7 min, respectively. Intestinal clearance of CMDCK was estimated from in vitro data to be 3.3 mL min(-1) kg(-1), and was almost equal to the intestinal blood flow rate (4.6 mL min(-1) kg(-1)). The selective CYP3A4 inhibitors, ketoconazole, troleandomycin and ritonavir demonstrated significant inhibitory effects on CMDCK intestinal metabolism, which suggested that co-administration of CMDCK with potent CYP3A inhibitors, such as ritonavir, might decrease its intestinal metabolic clearance and subsequently improve its bioavailability in body.

Objective To study the effect of bel-2 siRNA on apoptosis of HL-60 cells.Methods bcl-2 siRNA was synthesized in vitro transcription with silencer siRNA construction kit.The synthesized siRNA was transfected into HL-60 cells with Amine siPORT transfection.We used MTT flow cytometer and hoechst 33258 flourescence stainning t0 evaluate cell proliferation and apoptosis. Results.Bcl-2 siRNA could partially inhibit the growth of HL-60 cells.After incubated with bcl-2 siRNAl for 48 hours,HL-60 cells exhibited morphologic characteristic of apoptosis including chromatin condensation,crescents formation and nuclear fragmentation.Conclusion Effective bcl-2 siRNA can induce apoptosis and inhibit cell proliferation.

Objective To explore the treatment of tubal pregnancy, the use of ultrasound guided methotrexate local injection effect. Methods A total of 80 patients with tubal pregnancy who were enrolled in our hospital from January 2015 to July 2016 were randomly divided into control group and observation group (n=40). The patients in the control group were given methotrexate by conventional muscle, and the observation group was injected with methotrexate under the guidance of B ultrasound. Observe and compare the treatment of two groups of patients. Results The recovery time of HCG in the observation group was (22.35±1.05) days, the mean value of the control group was (34.26±1.32) days, the recovery time of HCG in the observation group was significantly faster than that in the control group (P<0.05). Tubal patency rate was higher than the control group, the difference between the two groups was statistically significant (P<0.05).Conclusion B-ultrasound guided methotrexate local injection treatment of tubal pregnancy can rapidly improve the patient's local drug concentration, it can rapidly improve The local drug concentration of the patient can improve the clinical situation of the patient, and has better therapeutic effect, so it is worthy of clinical reference.

OBJECTIVETo explore the mechanism of reactive oxygen species (ROS) in manganese chloride (MnCl(2))-induced apoptosis in PC12 cells.

METHODSThe model that MnCl(2) induced apoptosis in PC12 cells was established. The apoptotic effect of MnCl(2) on PC12 cells was analyzed with the MTT, the flow cytometry and the DNA fragmentation. The production of ROS and ATP in MnCl(2)-induced apoptosis of PC12 cells was examined. The influence of MnCl(2) on the expression of bcl-xl, bax and the activity of Caspase 3 was also analyzed.

RESULTSMnCl(2) triggered PC12 cells apoptosis in a dose-and time-dependent manner (P < 0.01). The rate of apoptosis was significantly increased (P < 0.01) when MnCl(2) of 2 mmol/L induced PC12 cells for 36 hours. The production of ROS was increased (P < 0.001) and the quantity of ATP was decreased (P < 0.01) in PC12 cells with the same inducement of MnCl(2). The expression of bcl-xl was inhibited and the bax was activated in this process (P < 0.01). Caspase 3 was also activated (P < 0.01).

CONCLUSIONMnCl(2) induces apoptosis of PC12 cells, which is related to the increase of ROS, the inhibition of the mitochondria and the activation of Caspase 3.

Adenosine Triphosphate ; biosynthesis ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Chlorides ; toxicity ; DNA Fragmentation ; drug effects ; Manganese Compounds ; PC12 Cells ; Rats ; Reactive Oxygen Species ; metabolism ; bcl-2-Associated X Protein ; biosynthesis ; bcl-X Protein ; biosynthesis

AIMTo study the effect of curcumin on the expression of prostate specific antigen (PSA).

METHODSAXSYM system-chemical luciferase method was used to examine the content of PSA in prostate cancer cell lines, LNCap after treated with different doses of curcumin. pGL3-PSA luciferase expression vector, containing 640 bp DNA of PSA gene 5' promoter region was constructed and transfected into LNCap cell with lipofectin. Through detecting the activity of luciferase, the effect of curcumin on the promoter of PSA was studied. Western blotting was used to detect expression of androgen receptor (AR) in LNCap cell with different concentrations of curcumin.

RESULTSThe expression of PSA was inhibited and activity of luciferase was reduced by curcumin. There was also significant difference in AR expression as shown by Western blotting experiment after treatment of different doses of curcumin.

CONCLUSIONThrough inhibiting AR expression, curcumin reduced the function of PSA promoter and inhibited PSA protein expression.

Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Curcumin ; pharmacology ; Humans ; Luciferases ; metabolism ; Male ; Promoter Regions, Genetic ; drug effects ; Prostate-Specific Antigen ; genetics ; metabolism ; Prostatic Neoplasms ; immunology ; metabolism ; pathology ; Receptors, Androgen ; metabolism

Objective· To establish an integrative method for the gene-panel sequencing data to automatically complete quality control, detection of gene mutation and visualization. Methods · Integrate several methods, e.g. FastQC, preprocessing and information of sequences (Prinseq) to develop an R package that can be used to visualize and control the quality of the raw sequencing reads and final mutations result. The sequencing reads mapped against to the reference genome using Burrows-Wheeler Alignment Tool (BWA)/Torrent Mapping Alignment Program (TMAP). Lofreq, Varscan2, the Genome Analysis Toolkit (GATK) and Torrent Variant Caller (TVC) were used to detect gene mutation and get the variant call format (VCF) format file. Annotate the gene mutation sites using Annovar. Results · Thirty-six cases of acute myeloid leukemia sequencing from Ion Torrent Personal Genome Machine (PGM) platform were passed by this analysis tool.Ten mutation sites of 2 demo data were found in DNMT3A,TET2,JAK2,PHF6,ASXL1,NPM1 and CEBPA which were validated by sanger sequencing. Conclusion · The analysis method that integrated and developed several tools for gene-panel sequencing data analysis can accomplish the gene-panel sequencing data analysis effectively. Besides, it can reduce the false positive ratio and improve the sensitivity of gene mutation detection that provides support for the analysis of gene-panel sequencing data.

The biotransformation, CYP reaction phenotyping, the impact of CYP inhibitors and enzyme kinetics of 3-cyanomethyl-4-methyl-DCK (CMDCK), a new anti-HIV preclinical candidate belonging to DCK analogs, were investigated in human intestinal microsomes and recombinant cytochrome P450 (CYP) enzymes. CMDCK (4 micromol L(-1)) was incubated with a panel of rCYP enzymes (CYP1A2, 2C9, 2C19, 2D6 and 3A4) in vitro. The remaining parent drug in incubates was quantitatively analyzed by a LC-MS method. CYP3A4 was identified as the principal CYP isoenzyme responsible for its metabolism in intestinal microsomes. The major metabolic pathway of CMDCK was oxidation and a number of oxidative metabolites were screened with LC-MS. The Km, Vmax, CLint and T1/2 of CMDCK obtained from human intestinal microsome were 45.6 micromol L(-1), 0.33 micromol L(-1) min(-1), 12.1 mL min(-1) kg(-1) and 25.7 min, respectively. Intestinal clearance of CMDCK was estimated from in vitro data to be 3.3 mL min(-1) kg(-1), and was almost equal to the intestinal blood flow rate (4.6 mL min(-1) kg(-1)). The selective CYP3A4 inhibitors, ketoconazole, troleandomycin and ritonavir demonstrated significant inhibitory effects on CMDCK intestinal metabolism, which suggested that co-administration of CMDCK with potent CYP3A inhibitors, such as ritonavir, might decrease its intestinal metabolic clearance and subsequently improve its bioavailability in body.
Anti-HIV Agents ; metabolism ; pharmacokinetics ; Biological Availability ; Bridged Bicyclo Compounds, Heterocyclic ; metabolism ; pharmacokinetics ; Coumarins ; metabolism ; pharmacokinetics ; Cytochrome P-450 CYP3A ; Cytochrome P-450 CYP3A Inhibitors ; Humans ; Intestines ; metabolism ; Ketoconazole ; pharmacology ; Metabolic Clearance Rate ; Microsomes ; metabolism ; Ritonavir ; pharmacology ; Troleandomycin ; pharmacology

BACKGROUND: It is unclear whether autophagy mediates the long-term exercise adaptation of the skeletal muscle induced by high-intensity interval training (HIIT) . OBJECTIVE: To identify the influence of HIIT on skeletal muscle autophagy of middle-aged rats over time, and to understand the potential regulatory effects of autophagy on maintaining the muscle mass and improving aerobic capacity by HIIT. METHODS: Rats were randomly divided into quiet, moderate training group (50-minute running at the intensity of 60% VO2 max) and HIIT group (6 times of 3-minute running at 80% VO2 maxand 3-minute active recovery at 50% VO2 maxwith a 7-minute warm-up and a 7-minute cool-down at 60% VO2 max). All rats were tested for VO2 max, exhaustive running time and distance at baseline and after 4, 8 and 12 weeks of exercise, respectively. The soleus muscle were collected and weighted, and then the expression levels of autophagy-related protein Beclin 1, LC3 and P62 were detected by western blot assay. RESULTS AND CONCLUSION: The soleus muscle mass in the quiet group decreased at the 12thweek (P < 0.01), while the moderate training and HIIT groups improved this decline (P < 0.05). The HIIT group dramatically improved the VO2 maxfrom the 4thweek when compared with the pre-experiment and the quiet group, until the 12thweek. The expression levels of Beclin1 and LC3II in the quiet group declined at the 12thweek (P < 0.05, P < 0.001), but the expression level of LC3II and ratio of LC3II/LC3I raised from the 4thand 8thweeks till the 12thweek compared with the pre-experiment and the quiet group, and the expression level of LC3II and ratio of LC3II/LC3I point time significantly raised in the moderate training group at the 12thweek (P < 0.001, P < 0.01). In the quiet group, the content of P62 significantly increased at the 4th and 8thweeks (P < 0.05), and decreased at the 12thweek (P < 0.001), but the content of P62 remained at the low level in the moderate exercise and HIIT groups. These results imply that an equivalent or better effects on improving basic autophagy level, skeletal muscle mass and aerobic capacity of the middle aged rats,by HIIT versus moderate training,providing the theory and empirical data that highlight the role of HIIT in health promotion.

Objective:To investigate the efficacy and safety of acute stent implantation during endovascular treatment for patients with emergent large vessel occlusion due to intracranial atherosclerotic stenosis.Methods:A retrospective analysis was carried out on 46 patients with emergent large vessel occlusion due to intracranial atherosclerotic stenosis who received endovascular treatment at the Strategic Support Force Medical Center from January 2015 to August 2022. Twenty-seven patients underwent balloon angioplasty alone and 19 patients underwent acute stent implantation. The baseline characteristics, modified thrombolysis in cerebral infarction (mTICI) score of the responsible vessels, modified Rankin scale (mRS) score 90 days after operation, incidence of symptomatic intracranial hemorrhage and mortality of the two groups were evaluated.Results:The proportion of effective recanalization of the offending vessels (mTICI≥2b) in the acute stenting group was slightly higher than that in the balloon angioplasty group (16/19 vs. 81.5%), but the difference was not statistically significant ( P>0.05). Besides, there was no significant difference in the median of mRS between the acute stenting group [3.0(0, 4.0)] and the balloon angioplasty group [4.0(1.0, 5.0)] 90 days after operation ( P>0.05). In terms of safety, the incidence of symptomatic intracranial hemorrhage and mortality were comparable between the two groups ( P>0.05). Conclusions:The effect of acute stent implantation during endovascular treatment for patients with emergent large vessel occlusion due to intracranial atherosclerotic stenosis is not inferior to that of balloon angioplasty, and it does not increase the risk of intracranial bleeding complications.

The emergence of SARS-CoV-2 variants of concern and repeated outbreaks of coronavirus epidemics in the past two decades emphasize the need for next-generation pan-coronaviral therapeutics. Drugging the multi-functional papain-like protease (PLpro) domain of the viral nsp3 holds promise. However, none of the known coronavirus PLpro inhibitors has been shown to be in vivo active. Herein, we screened a structurally diverse library of 50,080 compounds for potential coronavirus PLpro inhibitors and identified a noncovalent lead inhibitor F0213 that has broad-spectrum anti-coronaviral activity, including against the Sarbecoviruses (SARS-CoV-1 and SARS-CoV-2), Merbecovirus (MERS-CoV), as well as the Alphacoronavirus (hCoV-229E and hCoV-OC43). Importantly, F0213 confers protection in both SARS-CoV-2-infected hamsters and MERS-CoV-infected human DPP4-knockin mice. F0213 possesses a dual therapeutic functionality that suppresses coronavirus replication via blocking viral polyprotein cleavage, as well as promoting antiviral immunity by antagonizing the PLpro deubiquitinase activity. Despite the significant difference of substrate recognition, mode of inhibition studies suggest that F0213 is a competitive inhibitor against SARS2-PLpro via binding with the 157K amino acid residue, whereas an allosteric inhibitor of MERS-PLpro interacting with its 271E position. Our proof-of-concept findings demonstrated that PLpro is a valid target for the development of broad-spectrum anti-coronavirus agents. The orally administered F0213 may serve as a promising lead compound for combating the ongoing COVID-19 pandemic and future coronavirus outbreaks.
Animals ; Coronavirus Papain-Like Proteases/antagonists & inhibitors* ; Cricetinae ; Humans ; Mice ; Pandemics ; SARS-CoV-2/enzymology* ; COVID-19 Drug Treatment