Objective:To optimize the laboratory methods for isolation, culture, and preservation of Neisseria gonorrhoeae ( N. gonorrhoeae) . Methods:Five quality control bacterial strains ( N. gonorrhoeae strain 1, N. gonorrhoeae strain 2, Neisseria mucosa strain 3, Enterococcus faecalis strain 4, and mixed bacterial strain 5) were separately cultured using a self-made gonococcal selective medium and 7 commercialized gonococcal selective media (gonococcal isolation plates, gonococcal T-M selective medium, gonococcal chocolate agar plates, gonococcal selective medium, modified Thayer-Martin [MTM] agar plates, disposable gonococcus [GC] agar medium, and gonococcal agar plates), and their cultivation performance was evaluated. Sixteen strains of N. gonorrhoeae were cultured both inside and outside a candle jar to screen for CO 2-dependent strains. The preservation performance of 4 self-made gonococcal preservation solutions, including calf serum with 10% dimethyl sulfoxide, calf serum with 10% glycerol, brain-heart infusion broth with 20% glycerol, and trypticase soy broth with 30% glycerol, was evaluated. The survival of N. gonorrhoeae in freeze-dried and non-freeze-dried states was observed. Results:The growth performance of the 5 quality control strains varied across different commercialized gonococcal culture media. Concretely, N. gonorrhoeae strain 1 formed large and numerous colonies on both the self-made culture medium and MTM agar plates, which outperformed the other 6 culture media; the growth performance of N. gonorrhoeae strain 2 on the 7 commercialized culture media was inferior to that on the self-made culture medium; all 7 commercialized culture media had inhibitory effects on the growth of Neisseria mucosa strain 3, among which the self-made culture medium, gonococcal chocolate agar plates, MTM agar plates, disposable GC agar medium, and gonococcal agar plates could completely inhibit its growth; the gonococcal T-M selective medium could completely inhibit the growth of Enterococcus faecalis strain 4; mixed bacterial strain 5 showed better separation performance on the self-made culture medium, gonococcal isolation plates, gonococcal T-M selective medium, gonococcal chocolate agar plates, MTM agar plates, and disposable GC agar medium. Under CO 2-enriched conditions, all 16 strains of N. gonorrhoeae exhibited good growth performance; however, the growth of 11 strains was markedly inhibited without CO 2. No significant differences were observed in the preservation performance of the 4 preservation solutions at -70°C, and confluent colonies could be observed in all preservation solutions after 12 months of strain preservation; at -20 ℃, the trypticase soy broth with 30% glycerol showed the best preservation performance, with a few viable strains remaining after 6 months, while the calf serum with 10% dimethyl sulfoxide performed worst, with partial strains remaining viable after 2 weeks. Non-freeze-dried N. gonorrhoeae survived for varying duration at different temperatures (4 ℃, -18 ℃, -29 ℃, -70 ℃, and liquid nitrogen) ; no N. gonorrhoeae strains survived by day 4 when stored at 4°C; freeze-dried N. gonorrhoeae remained viable with the presence of confluent colonies for 6 months at 4 ℃. Conclusion:The self-made gonococcal selective medium demonstrated superior cultivation and isolation performance compared to commercialized gonococcal selective media; a small amount of CO 2 could promote the growth of N. gonorrhoeae; ultralow temperature and freeze-drying preservation could increase the survival time of N. gonorrhoeae, with freeze-drying at 4 ℃ being the most cost-effective long-term preservation method.

Objective:To optimize the laboratory methods for isolation, culture, and preservation of Neisseria gonorrhoeae ( N. gonorrhoeae) . Methods:Five quality control bacterial strains ( N. gonorrhoeae strain 1, N. gonorrhoeae strain 2, Neisseria mucosa strain 3, Enterococcus faecalis strain 4, and mixed bacterial strain 5) were separately cultured using a self-made gonococcal selective medium and 7 commercialized gonococcal selective media (gonococcal isolation plates, gonococcal T-M selective medium, gonococcal chocolate agar plates, gonococcal selective medium, modified Thayer-Martin [MTM] agar plates, disposable gonococcus [GC] agar medium, and gonococcal agar plates), and their cultivation performance was evaluated. Sixteen strains of N. gonorrhoeae were cultured both inside and outside a candle jar to screen for CO 2-dependent strains. The preservation performance of 4 self-made gonococcal preservation solutions, including calf serum with 10% dimethyl sulfoxide, calf serum with 10% glycerol, brain-heart infusion broth with 20% glycerol, and trypticase soy broth with 30% glycerol, was evaluated. The survival of N. gonorrhoeae in freeze-dried and non-freeze-dried states was observed. Results:The growth performance of the 5 quality control strains varied across different commercialized gonococcal culture media. Concretely, N. gonorrhoeae strain 1 formed large and numerous colonies on both the self-made culture medium and MTM agar plates, which outperformed the other 6 culture media; the growth performance of N. gonorrhoeae strain 2 on the 7 commercialized culture media was inferior to that on the self-made culture medium; all 7 commercialized culture media had inhibitory effects on the growth of Neisseria mucosa strain 3, among which the self-made culture medium, gonococcal chocolate agar plates, MTM agar plates, disposable GC agar medium, and gonococcal agar plates could completely inhibit its growth; the gonococcal T-M selective medium could completely inhibit the growth of Enterococcus faecalis strain 4; mixed bacterial strain 5 showed better separation performance on the self-made culture medium, gonococcal isolation plates, gonococcal T-M selective medium, gonococcal chocolate agar plates, MTM agar plates, and disposable GC agar medium. Under CO 2-enriched conditions, all 16 strains of N. gonorrhoeae exhibited good growth performance; however, the growth of 11 strains was markedly inhibited without CO 2. No significant differences were observed in the preservation performance of the 4 preservation solutions at -70°C, and confluent colonies could be observed in all preservation solutions after 12 months of strain preservation; at -20 ℃, the trypticase soy broth with 30% glycerol showed the best preservation performance, with a few viable strains remaining after 6 months, while the calf serum with 10% dimethyl sulfoxide performed worst, with partial strains remaining viable after 2 weeks. Non-freeze-dried N. gonorrhoeae survived for varying duration at different temperatures (4 ℃, -18 ℃, -29 ℃, -70 ℃, and liquid nitrogen) ; no N. gonorrhoeae strains survived by day 4 when stored at 4°C; freeze-dried N. gonorrhoeae remained viable with the presence of confluent colonies for 6 months at 4 ℃. Conclusion:The self-made gonococcal selective medium demonstrated superior cultivation and isolation performance compared to commercialized gonococcal selective media; a small amount of CO 2 could promote the growth of N. gonorrhoeae; ultralow temperature and freeze-drying preservation could increase the survival time of N. gonorrhoeae, with freeze-drying at 4 ℃ being the most cost-effective long-term preservation method.

Objective To compare the effects of the left and right stellate ganglion resection on myocardial injury in rats with acute myocardial infarction(AMI).Methods According to the random number table method,30 healthy male Sprague Dawley rats were divided into the AMI group,left stellate ganglionectomy group,and right stellate ganglionectomy group,with 10 rats in each group.AMI models were prepared by ligation of the left anterior descending branch of the coronary artery in all the three groups.In the AMI group,the stellate ganglion was isolated(randomly left or right)without excision.The rats in the left and right stellate ganglionectomy groups underwent the left and right stellate ganglionectomy,respectively.At 24 hours after modeling,2 mL of subclavian venous blood was extracted from the three groups of rats.The serum levels of cardiac troponin Ⅰ(cTnⅠ),noradrenaline(NE),interleukin(IL)-1β,IL-6,tumor necrosis factor-α(TNF-α),malondialdehyde(MDA),and superoxide dismutase(SOD)were detected by enzyme-linked immunosorbent assay.The left ventricular fractional shortening(LVFS),left ventricular ejection fraction(LVEF)and cardiac output(CO)of rats in the three groups were measured by echocardiography one week after modeling.Results The serum levels of NE,cTnⅠ and MDA in the left and right stellate ganglionectomy groups were significantly lower than those in the AMI group,and SOD level was significantly higher than that in the AMI group(P＜0.05);the serum levels of NE,cTnⅠ and MDA in the right stellate ganglionectomy group were significantly lower than those in the left stellate ganglionectomy group,and SOD level was significantly higher than that in the left stellate ganglionectomy group(P＜0.05).The levels of serum IL-1β,IL-6 and TNF-α in the left and right stellate ganglionectomy groups were significantly lower than those in the AMI group(P＜0.05);the levels of serum IL-1β,IL-6 and TNF-αin the right stellate ganglionectomy group were significantly lower than those in the left stellate ganglionectomy group(P＜0.05).LVEF,LVFS and CO in the left and right stellate ganglionectomy groups were significantly higher than those in the AMI group,and LVEF and LVFS in the right stellate ganglionectomy group were significantly higher than those in the left stellate ganglionectomy group(P＜0.05);there was no significant difference in CO between the left and right stellate ganglionectomy groups(P＞0.05).Conclusion Stellate ganglionectomy has a protective effect on AMI induced by ligation of the left anterior descending branch of the coronary artery,which may be related to reducing inflammatory reaction and oxidative stress damage.The right stellate ganglion resection has more protective effects on cardiac function than the left stellate ganglion resection.

Objective To investigate the effects of lumbar epidural block of renal sympathetic nerve on cardiac function and serum inflammatory factors in rats with myocardial infarction.Methods Thirty healthy male Sprague Dawley rats were divided into asham operation group,myocardial infarction group and sympathetic nerve block group according to the random number table method,with 10 rats in each group.Lumbar epidural catheterization was applied in all rats in the 3 groups.After catheter insertion,myocardial infarction was induced by ligation of the anterior descending branch of the left coronary artery of rats in the myocardial infarction group and the sympathetic nerve block group,while the left anterior descending branch of rats in the sham operation group was not ligated.After myocardial infarction,the rats in the sympathetic nerve block group were injected with 50 μL of ropivacaine(volume fraction:0.2％)via a lumbar epidural catheteronce every 24 hours,and the injection lasted until 14 days after surgery.In the myocardial infarction group,50 μL of 9 g·L-1sodium chloride was injected into the rats through a lumbar epidural catheteronce every 24 hours,and the injection lasted until 14 days after surgery.Rats in the sham group did not receive the injection of any drugs.At 14 days after operation,echocardiography was performed on rats in the 3 groups to measure ejection fraction(EF),left ventricular short-axis shortening rate(FS)and cardiac output(CO).A total of 1 mL of cervical venous blood was extracted from each rat in the 3 groups at 24 h after surgery,and 5 mL of abdominal aortic blood was extracted from rats in the 3 groups at 14 days after surgery.Serum levels of norepinephrine(NE),cardiac troponin I(cTnl),interleukin-18(IL-18),interleukin-1β(IL-1 β)and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay.At 14 days after surgery,the rats in the 3 groups were anesthetized by intraperitoneal injection of 100 g·L-1 chloral hydrate(300 mg·kg-1),and the thoracic cavity of the rats was opened to remove the heart.The myocardial infarction area of the rats was detected by 2,3,5-triphenyltetrazolium chloride staining,and the proportion of the myocardial infarction area was calculated.Results On the 14th day after surgery,EF,FS and CO of rats in the myocardial infarction group and sympathetic nerve block group were significantly lower than those in the sham operation group,while EF,FS and CO of rats in the sympathetic nerve block group were significantly higher than those in the myocardial infarction group(P＜0.05).The serum cTnI level of rats in the myocardial infarction group and sympathetic nerve block group was significantly higher than that in the sham operation group(t=68.260,15.110;P＜0.05),and the serum cTnI level of rats in the myocardial infarction group was significantly higher than that in the sympathetic nerve block group(t=27.920,P＜0.05).The proportion of the myocardial infarction area of rats in the sympathetic nerve block group was significantly lower than that in the myocardial infarction group(t=14.182,P＜0.001).There was no significant difference in the serum NE,IL-18,IL-1 β and TNF-α levels of rats between 24 hours and 14 days after surgery in the sham operation group(P＞0.05).The serum NE,IL-1 β and TNF-αlevels of rats at 14 days after surgery were significantly higher than those at 24 hours after surgery,and the IL-18 level of rats was significantly lower than that at 24 hours after surgery in the myocardial infarction group(P＜0.05).The serum NE,IL-18 and TNF-α levels of rats at 14 days after surgery were significantly lower than those at 24 hours after surgery,and the IL-1 βlevel of rats was significantly higher than that at 24 hours after surgery in the sympathetic nerve block group(P＜0.05).At 24 hours and 14 days after surgery,the serum NE,IL-18,1L-1 β and TNF-α levels of rats in the myocardial infarction group and sympathetic nerve block group were significantly higher than those in the sham operation group,while the serum NE,IL-18,IL-1 β and TNF-α levels of rats in the sympathetic nerve block group were significantly lower than those in the myocardial infarction group(P＜0.05).Conclusion Lumbar epidural block of renal sympathetic nerve in rats can significantly reduce the levels of serum inflammatory factors and NE,and improve cardiac function.

Objective:To evaluate the effect of superior cervical ganglion block (SCGB) on cardiac function and nucleotide like receptor protein 3 (NLRP3) signaling pathway in a rat model of myocardial ischemia-reperfusion (I/R).Methods:Sixty healthy SPF male Sprague-Dawley rats, weighing 250-300 g, aged 2-3 months, were divided into 4 groups ( n=15 each) using a random number table method: sham operation group (sham group), myocardial I/R group (IR group), myocardial I/R + normal saline group (IR+ NS group), and myocardial I/R + SCGB group (IR+ SCGB group). Myocardial I/R model was developed by ligation of the left anterior descending branch of the coronary artery for 45 min followed by restoration of blood flow in anesthetized aninals. IR+ SCGB group received SCGB (0.25% ropivacaine 0.1 ml) at 10 min before reperfusion once a day for 2 consecutive weeks, while 0.9% sodium chloride was given instead of ropivacaine in IR+ NS group. Blood samples were collected at 24 h and 14 days of reperfusion for determination of serum concentrations of norepinephrine (NE), troponin T (TnT), tumor necrosis factor-alpha (TNF-α), interleukin-18 (IL-18) and IL-1β by enzyme-linked immunosorbent assay. Echocardiography was performed before ischemia and at 14 days of reperfusion, and left ventricular short axis shortening rate (FS), ejection fraction (EF), and cardiac output (CO) were measured. The rats were sacrificed at 14 days of reperfusion and the hearts were taken for determination of the contents of norepinephrine (NE) in myocardial tissues in the infarction area (by enzyme-linked immunosorbent assay), percentage of myocardial fibrosis area (by Masson staining), M1 macrophage marker CD68 + cell count in the infarction area (by immunohistochemical method), and expression of NLRP3 and gasdermin D (GSDMD) in myocardial tissues (by Western blot). Results:Compared with Sham group, the serum concentrations of TnT, TNF-α, IL-18 and IL-1β, percentage of myocardial fibrosis area, and NE levels in serum and myocardial tissues were significantly increased, the expression of NLRP3 and GSDMD in myocardial tissues was up-regulated, CD68 + cell count was increased, and EF, CO and FS were decreased in IR group ( P<0.05). Compared with IR group, the serum concentrations of TnT, TNF-α, IL-18 and IL-1β, percentage of myocardial fibrosis area, and NE levels in serum and myocardial tissues were significantly decreased, the expression of NLRP3 and GSDMD in myocardial tissues was down-regulated, CD68 + cell count was decreased, and EF, CO and FS were increased in IR+ SCGB group ( P<0.05), and no statistically significant changes were found in the parameters mentioned above in IR+ NS group ( P>0.05). Conclusions:SCGB can improve the cardiac function in a rat model of myocardial I/R, and the mechanism may be related to the inhibition of NLRP3 signaling pathway.

Objective:To investigate the regulation of transcription factor CCCTC binding factor (CTCF) on the expression of B-cell lymphoma 2 ( Bcl-2) gene in pterygium and its molecular mechanism. Methods:Pterygium tissue samples from 22 primary pterygium patients who underwent pterygium excision combined with autologous limbal stem cell transplantation in The First Hospital of Changsha from June 2017 to February 2019 were collected during the operation as pterygium group.Normal conjunctival tissue from 20 patients with ocular trauma due to conjunctiva rupture, eyeball rupture or eyeball perforation in the same period were collected during the repair of ocular trauma as control group.Real-time PCR and Western blot were used to detect the expression levels of CTCF and Bcl-2 in the two groups.The DNA methylation level of the Bcl-2 promoter in the samples of the two groups was detected by bisulfite sequencing PCR (BSP). Pterygium fibroblasts were isolated and cultured.Fibroblasts were identified by immunohistochemistry using vimentin antibody.The cultured pterygium fibroblasts were divided into a CTCF interference group transfected with CTCF interference plasmid, and a control group transfected with control plasmid.The expression levels of CTCF and Bcl-2 in pterygium fibroblasts in CTCF interference and control groups were detected by real-time PCR and Western blot.The cell vitality was detected with cell counting kit-8 at 12, 24, and 48 hours after transfection.The DNA methylation level of the Bcl-2 promoter in the cells of the CTCF interference and control groups after transfection was determined by BSP.Differences of the indexes among groups were analyzed.Correlation between Bcl-2 mRNA and Bcl-2 gene promoter methylation level of CTCF protein in pterygium tissue was analyzed by Pearson linear correlation analysis.This study protocol was approved by the Ethics Committee of The First Hospital of Changsha (No.KL-2017021). Written informed consent was obtained from the patients from whom the specimens were collected.Results:The relative expression levels of CTCF mRNA and protein in pterygium group were 7.23±3.34 and 0.92±0.21, respectively, which were significantly higher than 1.10±0.44 and 0.28±0.07 in normal conjunctiva group ( t=-8.136, -13.025; both at P<0.01). The relative expression levels of Bcl-2 mRNA and protein in pterygium group were 10.27±4.64 and 0.95±0.27, which were higher than 1.10±0.41 and 0.32±0.14 in normal conjunctiva group, showing statistically significant differences ( t=-8.789, -10.782; both at P<0.01). The CTCF protein expression was significantly positively correlated with the Bcl-2 mRNA expression in pterygium group ( r=0.746, P<0.01). The DNA methylation level of the Bcl-2 promoter in pterygium group was 0.65±0.09, which was lower than 0.83±0.06 in normal conjunctiva group, with a statistically significant difference ( t=7.408, P<0.01). The DNA methylation level was significantly negatively correlated with the Bcl-2 mRNA expression in pterygium group ( r=-0.635, P<0.01). After the interference of CTCF expression in pterygium fibroblasts, the relative expression levels of CTCF and Bcl-2 mRNA in CTCF interference group were 0.37±0.03 and 0.53±0.06, which were significantly lower than 1.02±0.06 and 0.99±0.07 in control group ( t=20.035, 9.029; both at P<0.01). The relative expression levels of CTCF and Bcl-2 proteins in CTCF interference group were 0.23±0.06 and 0.56±0.07, which were lower than 0.52±0.05 and 0.92±0.12 in control group, showing statistically significant differences ( t=6.914, 4.719; both at P<0.01). The cell viability of pterygium fibroblasts in CTCF interference group was 0.10±0.01, 0.17±0.01, 0.38±0.04 at 12, 24, and 48 hours after interference, respectively, which were lower than 0.12±0.01, 0.29±0.01 and 0.85±0.06 in control group, and the differences were statistically significant ( t=3.718, 18.350, 15.621; all at P<0.01). The DNA methylation level of Bcl-2 promoter in CTCF interference group was 0.75±0.04, which was significantly higher than 0.61±0.03 in control group ( t=-4.472, P<0.05). Conclusions:CTCF is excessively expressed in pterygium, which may mediate the overexpression of Bcl-2 through down-regulating DNA methylation level.

We aimed to evaluate the effectiveness and safety of single-course initial regimens in patients with low-risk gestational trophoblastic neoplasia (GTN). In this trial (NCT01823315), 276 patients were analyzed. Patients were allocated to three initiated regimens: single-course methotrexate (MTX), single-course MTX + dactinomycin (ACTD), and multi-course MTX (control arm). The primary endpoint was the complete remission (CR) rate by initial drug(s). The primary CR rate was 64.4% with multi-course MTX in the control arm. For the single-course MTX arm, the CR rate was 35.8% by one course; it increased to 59.3% after subsequent multi-course MTX, with non-inferiority to the control (difference -5.1%,95% confidence interval (CI) -19.4% to 9.2%, P = 0.014). After further treatment with multi-course ACTD, the CR rate (93.3%) was similar to that of the control (95.2%, P = 0.577). For the single-course MTX + ACTD arm, the CR rate was 46.7% by one course, which increased to 89.1% after subsequent multi-course, with non-inferiority (difference 24.7%, 95% CI 12.8%-36.6%, P < 0.001) to the control. It was similar to the CR rate by MTX and further ACTD in the control arm (89.1% vs. 95.2%, P =0.135). Four patients experienced recurrence, with no death, during the 2-year follow-up. We demonstrated that chemotherapy initiation with single-course MTX may be an alternative regimen for patients with low-risk GTN.
Antineoplastic Combined Chemotherapy Protocols/adverse effects* ; Dactinomycin/adverse effects* ; Female ; Gestational Trophoblastic Disease/drug therapy* ; Humans ; Methotrexate/therapeutic use* ; Pregnancy ; Retrospective Studies

Objective: To establish and evaluate a multiplex PCR method for rapid identification of Mycobacterium species in order to provide an approach for rapid diagnosis of pathogens causing tuberculosis. Methods: Four genes including 16S rRNA, Rv0577, RD9 and mtbk_20680 were selected to establish the multiplex PCR method. Specific primers were designed and the reaction system and conditions were optimized. The sensitivity and specificity of the multiplex PCR method were evaluated through identifying Mycobacterium africanum (M.africanum), Mycobacterium bovis (M.bovis), M. bovis BCG, seven common non-tuberculosis Mycobacterium (NTM), seven reference species of common respiratory bacteria and 93 clinical strains of Mycobacterium tuberculosis (MTB) isolated from patients with tuberculosis in Gansu Province of China. Results: The fragments of 16S rRNA, Rv0577, RD9 and mtbk_20680 genes were 543 bp, 786 bp, 369 bp and 231 bp in length, respectively. MTB strains of the Beijing genotype were positive for all of the four genes, while the non-Beijing genotype strains were negative for mtbk_20680. M. africanum, M. bovis and M. bovis BCG strains were negative for 16S rRNA and Rv0577. NTM strains only carried 16S rRNA and none of four genes were detected in the seven species of respiratory bacteria. Among the 93 clinical MTB strains, 80 (86.02%) belonged to the Beijing genotype and the other 13 (13.98%) were non-Beijing genotype strains. The specificity of the multiplex PCR method was 100%. Conclusions The established multiplex PCR method could accurately distinguish Mycobacterium, Mycobacterium tuberculosis complex (MTBC), NTM, MTB, and Beijing and non-Beijing genotype MTB with high sensitivity and specificity.

Objective: To investigate a poisonous mushroom poisoning incident and analyze its clinical data. Methods: Investigate a poisonous mushroom poisoning incident in a place in Shandong in July 2018, at the same time, the clinical data of 2 cases of mushroom poisoning were analyzed and summarized. Results: The incident was caused by a poisoning incident caused by residents eating poisonous mushrooms. The poisonous mushroom in this incident was identified as a scaled white goose cream. Two patients with poisoning developed gastrointestinal symptoms such as nausea and vomiting, abdominal pain and diarrhea, and liver damage occurred later. After active rescue and treatment, one patient was discharged from hospital, and the other patient developed acute pulmonary embolism during the treatment. He was discharged after interventional thrombolysis and follow-up treatment. Conclusion After investigation, the incident was caused by the ingestion of poisonous mushrooms mainly based on the scalloped white goose cream. After active treatment, they were cured and discharged.

Objective To investigate in vitro combined effect of ceftriaxone and azithromycin against clinical isolates of Neisseria gonorrhoeae(NG). Methods A total of 25 NG clinical isolates were collected from the STD clinic of Dalian Dermatosis Hospital in 2012. Epsilometer test(Etest)method was used to determine the minimum inhibitory concentrations(MICs)of ceftriaxone and azithromycin against NG isolates. Fractional inhibitory concentration index (FICI) was calculated to evaluate the in vitro combined effect of ceftriaxone and azithromycin against NG isolates. Results The mean MICs of ceftriaxone and azithromycin were 0.032 mg/L (range, 0.008- 0.064 mg/L) and 0.834 mg/L (range, 0.064-4.000 mg/L), respectively. The FICI ranged from 0.724 to 2.696, and ceftriaxone and azithromycin showed an additive effect against the above NG isolates. Conclusion Ceftriaxone and azithromycin show an additive effect against NG in vitro, but further studies with large sample size are needed to confirm their effects.