In this study,the L.dumoffii TEX-KL (ATCC 33343),L.dumoffii NY23 (ATCC 33279) and L.pneumophila philadelphila-1 (ATCC 33155) strains were used to explore the adhesion,invasion and intracellular growth ability in the epithelial cells.Approximately 1× 108 bacteria were pelleted,resuspended,and diluted (1 ∶ 10) in RPMI 1640 tissue culture medium.The bacteria were then added to A549 cells (1 × 105 per well) in 24-well plates to give a multiplicity of infection (MOI) of about 100.The Gimenez staining and colony counting methods were used for the determination of the strain adhesion,invasion and intracellular growth ability.It was found that in vitro growth ability of L.pneumophila philadelphila-1,L.dumoffii TEX-KL and L.dumoffii NY23 strains had no significant difference.For in vivo assay,there was also no significant difference in adhesion ability of these strains.However,the CFU counts of L.dumoffii TEX-KL strain invaded into A549 cells was 1 000 times higher than that of the other two strains.Compared with L.pneumophila philadelphila-1 and L.dumoffii NY23 strains,L.dumoffii TEX-KL strain has higher invasion ability and,therefore,higher intracellular growth ability.

Accumulating evidence suggests that the small G protein Rho and its downstream effector Rho kinase may play important roles in kidney biology. The present study examined the effects of a Rho-kinase inhibitor, fasudil, on daunorubicin-induced progressive glomerulosclerosis and explored the underlying mechanism by which fasudil ameliorates glomerulosclerosis. Thirty-six male SD rats were randomly allocated into sham-operation group (sham group, n=12), unilateral nephrectomy (UNX)+daunorubicin (DRB) group (model group, n=12), UNX+DRB+Fasudil group (treatment group, n=12). Two to four weeks after the establishment of the animal model, 6 rats in each group were taken randomly for the detection of 24-h urine protein excretion. Kidney sections were examined by HE and PAS staining, immunohistochemistry and transmission electric microscopy (TEM). The expression of Rho-kinase mRNA and P27 mRNA in kidney were detected by RT-PCR. It was found that the 24-h urine protein excretion in model group was increased significantly as compared with sham group (P<0.01). But this increase was significantly suppressed by fasudil (P<0.05). At 4 week, the foot process effacement in podocytes, mesangial proliferation and ECM accumulation were observed in model group, presenting as focal segmental glomerulosclerosis. But in the treatment group, the fasudil alleviated glomerular injury, with proliferating cell nuclear antigen (PCNA)-positive cell infiltration ameliorated and the expression of P27 increased. The expression of Rho-kinase mRNA was significantly enhanced in model group and was suppressed in treatment group. Moreover, fasudil up-regulated the mRNA expression of P27. Our study demonstrated that the glomerulosclerosis was substantially ameliorated by inhibiting the expression of Rho-kinase. It is suggested that Rho-kinase pathway is involved in the renal injury and the inhibition of Rho-kinase may constitute a therapeutic strategy for the treatment of renal injury.

Objective To establish TaqMan real-time PCR method for detection and identification of Haemophilus influenzae and Streptococcus pneumonia. Methods Two sets of primers and FAM-labeled probes targeting different genes of Haemophilus influenzae and Streptococcus pneumoniae were designed and synthesized. The bexA gene was used for identification of Haemophilus influenzae and lytA for Streptococcus pneumoniae. The sensitivity and specificity of real-time PCR were assessed for different primers and probes. Cut-off values of cycle threshold (Ct) were determined. Two hundred and seventy-eight cerebrospinal fluid (CSF) specimens from suspected bacterial meningitis cases were detected by real-time PCR assay, latex agglutination test and bacteria culture simultaneously. Results Haemophilus influenzae isolates of serotype a to d could be detected and identified by bexA primers and probe. All Streptococcus pneumoniae isolates of different serotypes could be detected and identified by lytA primers and probe. The respective sensitivities for Haemophilus influenzae and Streptococcus pneumoniae were 10 and 90 genome DNA copies in each PCR reaction. Of the 278 CSF specimens, four were positive by Haemophilus influenzae and seven positive by Streptococcus pneumoniae when detected by real-time PCR. Of the four Haemophilus influenzae positive specimens, two were positive by culture and one positive hy latex. Of the seven Streptococcus pneumonia positive specimens, two were positive by culture and two positive by latex. Conclusions Real-time PCR could rapidly detect and identify Haemophilus influenzae of serotype a to d and Streptococcus pneumoniae of different serotypes with high sensitivity. TaqMan real-time PCR could be widely used for the diagnosis of invasive meningitis caused by Haemophilus influenzae and Streptococcus pneumoniae. It can improve the rate of positivity for diagnosis of suspicious bacterial meningitis cases.

Objective：To explore the characteristics of behavioral problems and self-concept level in children with attention deficit hyperactivity disorder(ADHD)accompanying anxiety disorder.Methods：With stratified random sampling method,a sampling survey was made in 9495 children(aged 5～17 years)from 6 regions of Hunan province together.Using the two stage epidemiologic research method,three groups,including control group(214 cases),pure ADHD(219 cases)and ADHD accompanying anxiety disorder(111 cases)were determined as the subjects.All the subjects completed the Children's Self-Concept Scale(CSCS),and the Achenbach Child Behavior Checklist(CBCL)was completed by their parents.Results：(1)Compared to the control group,the pure ADHD group scored higher in CBCL [e.g.,the total score:(30.35±24.32)vs.(16.97±14.86)],and scored lower in CSCS [e.g.,total score:(50.85±9.98)vs.(57.46±10.80),P<0.05].(2)Compared to the control group,children with ADHD accompanying anxiety disorder scored higher in CBCL [e.g.,total score:(41.66±28.07),P<0.01],and in CSCS except physical activities(Ps<0.05).(3)Compared to the pure ADHD group,ADHD accompanying anxiety disorder group scored higher in CBCL and lower in CSCS[behavior:(10.44±3.17)vs.(11.55±2.80);anxiety:(8.06±2.61)vs.(9.21±2.61),gregariousness:(7.42±2.22)vs.(8.16±1.89);total score:(47.25±9.93)vs.(50.85±9.98);Ps<0.05].Conclusion：ADHD children have more behavioral problems and lower level of self-concept than normal children,and children with ADHD accompanying anxiety disorder have more prominent presence of behavioral problems and lower self-concept rating than children with pure ADHD.

Objective To evaluate the relationship between endoplasmic reticulum stress and diabetes mellitus (DM)-caused influence on cardioprotection induced by remifentanil postconditioning in rats.Methods Adult male Sprague-Dawley rats,weighing 250-300 g,were used in the study.A model for type 1 DM was established by intraperitoneal streptozotocin 50 mg/kg and confirmed by blood glucose ≥ 16.7 mmol/L.Thirty six rats with type 1 DM were randomly divided into 3 groups (n =12 each) using a random number table:sham operation group (DM-S group),myocardial ischemia-reperfusion (I/R) group (DM-I/R group) and remifentanil postconditioning group (DM-R group).Another 36 normal rats were exposed to single intraperitoneal injection of sodium citrate-hydrochloric acid buffer solution and served as control group.Two weeks later 36 normal rats with nondiabetes mellitus were also randomly divided into 3 groups (n =12 each) using a random number table:sham operation group (NDM-S group),myocardial I/ R group (NDM-I/R group) and remifentanil postconditioning group (NDM-R group).Myocardial I/R was produced by 30 min occlusion of left anterior descending branch of coronary artery followed by 120 min reperfusion.Remifentanil postconditioning was induced by 10 min infusion of remifentanil 10 μg · kg-1 · min 1 via the femoral vein starting from 5 min before reperfusion.Before ischemia and at 30 and 120 min of ischemia,MAP,SP and HR were recorded and rate-pressure product (RPP) was calculated.At 120 min of reperfusion,arterial blood samples were collected for measurement of plasma cardiac troponin I (cTnI) concentration.The animals were then sacrificed and hearts were removed for determination of myocardial infarct size (IS).The left 6 rats from each group were sacrificed at 120 min of reperfusion,the specimens from their left ventricular apex were obtained to detect the expression of endoplasmic reticulum stress marker glucose-regulated protein 78 (GRP78),C/EBP homologous protein (CHOP) and caspase-12 by Western blot.Results MAP and RPP were significantly decreased,the plasma concentration of cTnI was increased,changes of cardiac infarction were found,and the expression of GRP78,CHOP and caspase-12 was up-regulated in diabetic and nondiabetic rats.Remifentanil postconditioning could inhibit the expression of GRP78,CHOP and caspase-12,increase MAP and RPP,decrease the plasma concentration of cTnI,and reduce myocardial infarct size in nondiabetic rats,but it had no such effects in the diabetic rats.The expression of GRP78,CHOP and caspase-12 was significantly higher after remifentanil postconditioning in diabetic rats than in nondiabetic rats.Conclusion Enhanced endoplasmic reticulum stress is involved in DM-caused loss of cardioprotection induced by remifentanil postconditioning in rats.

Objective To observe the effect of flexible laryngeal mask (FLMA) in transnaso-sphenoidal microsurgery for pituitary adenoma on airway management and variation of stress response.Methods One hundred patients (71 males, 29 females, aged 18-65 years, BMI 21-28 kg/m2, ASA physical status Ⅰ or Ⅱ) undergoing transnaso-sphenoidal microsurgery for pituitary adenoma were randomly divided into two groups: the FLMA group (group F) and the reinforced endotracheal tube group (group T) using a random number table, 50 cases in each group.The plasma concentration of epinephrine and norepinephrine were measured before anesthesia induction (T0), at the time of inserting the FLMA or reinforced endotracheal tube (T1), 1 min (T2) and 5 min (T3) after insertion.The Berry scores of the preoperative and postoperative airway exposure by branchofiberoscope in group F were assessed.The time of removal of FLMA (endotracheal tube) and the occurrence of choking, laryngeal spasm, sore throat, hoarseness and other adverse reactions were recorded.Results The levels of epinephrine and norepinephrine were were significantly lower at T2 and T3 in group F than those in group T (P<0.05).There was no significant difference in airway Berry scores.The time of extubation was shorter in group F than that in group T [(9±3) min vs (17±6) min] (P<0.05).The incidence of choking (2% vs 22%) and sore throat (4% vs 30%) were significantly lower in group F than those in group T (P<0.05).Conclusion Compared with the reinforced endotracheal tube, FLMA can be applied safely and effectively to transnaso-sphenoidal microsurgery for pituitary adenoma, reduces stress respond associated with anesthesia and post-extubation complications, improves the recovery of patients.

Objective To evaluate the effect of the acupressure wristbands combined with tro-pisetron on the effect in preventing postoperative nausea and vomiting (PONV)and the quality of re-covery during early period after radical mastectomy.Methods Seventy-five patients,aged 35-68 years,ASA physical status Ⅰ or Ⅱ,scheduled for elective radical mastectomy,were randomly as-signed into three groups (n=25):acupressure wristband group (group A),tropisetron group (group B)and acupressure wristbands combined with tropisetron group (group C).At 30 min before the sur-gery,the acupuncture pins were placed on Nei Guan point till 24 h after the surgery in group A.At 30 min before the end of the surgery,tropisetron 6 mg was intravenously injected in group B.At 30 min before the surgery,the acupuncture pins were placed on Nei Guan point till 24 h after the surgery;at 30 min before the end of the surgery,tropisetron 6 mg was intravenousy injected in group C.The three groups received total intravenous anesthesia.The occurrence of nausea and vomiting was recor-ded immediately after extubation,6 h after surgery and 6-24 h after surgery.The global QoR-40 ag-gregate scores were recorded between the three groups on the day before surgery and 1 day after sur-gery.Results Compared with groups A and B,the incidence of PONV was significantly decreased in group C between the time immediately after extubation-6 h after surgery (P<0.05);and the severity of PONV was significantly decreased in group C (P<0.05 ).Compared with groups A and B, patients in group C produced higher physical comfort scores,emotional state scores,and pain scores (P<0.05 or P<0.01);the global QoR-40 scores of group C were significantly increased on 1 day after surgery (P<0.01).Conclusion The acupressure wristbands combined with tropisetron can re-duce the incidence and the severity of PONV,improve the quality of recovery during the early period after radical mastectomy.

Accumulating evidence suggests that the small G protein Rho and its downstream effector Rho kinase may play important roles in kidney biology. The present study examined the effects of a Rho-kinase inhibitor,fasudil,on daunorubicin-induced progressive glomerulosclerosis and explored the underlying mechanism by which fasudil ameliorates glomerulosclerosis. Thirty-six male SD rats were randomly allocated into sham-operation group (sham group,n=12),unilateral ncphrectomy (UNX)+daunorubicin (DRB) group (model group,n=12),UNX+DRB+Fasudil group (treatment group,n=12). Two to four weeks after the establishment of the animal model,6 rats in each group were taken randomly for the detection of 24-h urine protein excretion. Kidney sections were examined by HE and PAS staining,immunohistochemistry and transmission electric microscopy (TEM).The expression of Rho-kinase mRNA and P27 mRNA in kidney were detected by RT-PCR. It was found that the 24-h urine protein excretion in model group was increased significantly as compared with sham group (P＜0.01). But this increase was significantly suppressed by fasudil (P＜0.05). At 4 week,the foot process effacement in podocytes,mesangial proliferation and ECM accumulation were observed in model group,presenting as focal segmental glomerulosclerosis. But in the treatment group, the fasudil alleviated glomerular injury, with proliferating cell nuclear antigen (PCNA)-positive cell infiltration ameliorated and the expression of P27 increased. The expression of Rho-kinase mRNA was significantly enhanced in model group and was suppressed in treatment group. Moreover,fasudil up-regulated the mRNA expression of P27. Our study demonstrated that the glomerulosclerosis was substantially ameliorated by inhibiting the expression of Rho-kinase. It is suggested that Rho-kinase pathway is involved in the renal injury and the inhibition of Rho-kinase may constitute a therapeutic strategy for the treatment of renal injury.

Objective To investigate the molecular characteristics of serogroup B neisseria meningitidis in China. Methods Total of 485 (100 strains isolated from cerebrospinal fluid or blood samples of encephalomyelitis cases, and 385 strains isolated from nasopharynx of healthy carriers) Meningococcal serogroup B (MenB) strains, isolated from 29 provinces of China between 1968 and 2016, were analyzed by multilocus sequence typing (MLST) and PorA typing methods. Further, the genetic diversity of three MenB vaccine proteins, FHbp, NadA and NHBA, were analyzed. Results The 485 study strains belonged to 270 sequence types (STs), 107 of which (representing 211 strains) could be grouped into ten clonal complexes (CC). CC4821 has been the predominant lineage in China since 2005 (28.7%, n=139). The most common PorA types of MenB strains from invasive meningococcal cases were P1.5-2,2?2 (10.0%, n=10), P1.5-1,2?2 (9.0%, n=9) and P1.5-1,10?4 (9.0%, n=9). Four hundred and twenty one strains had intact fhbp gene; variant 1, 2 and 3 accounted for 12.8% (54 strains), 85.0% (358 strains) and 2.2% (9 strains) respevtively. Ten out of 432 strains (2.3%) contained complete nadA gene. All the 172 strains for which the nhba gene was sequenced had intact gene sequence which corresponded to 68 peptide types. Conclusion CC4821 was the predominant CC of MenB strains in China; the vaccine proteins were diverse about the sequences. The vaccine proteins should be carefully selected when developing MenB vaccines in China.

Objective To investigate the molecular characteristics of serogroup B neisseria meningitidis in China. Methods Total of 485 (100 strains isolated from cerebrospinal fluid or blood samples of encephalomyelitis cases, and 385 strains isolated from nasopharynx of healthy carriers) Meningococcal serogroup B (MenB) strains, isolated from 29 provinces of China between 1968 and 2016, were analyzed by multilocus sequence typing (MLST) and PorA typing methods. Further, the genetic diversity of three MenB vaccine proteins, FHbp, NadA and NHBA, were analyzed. Results The 485 study strains belonged to 270 sequence types (STs), 107 of which (representing 211 strains) could be grouped into ten clonal complexes (CC). CC4821 has been the predominant lineage in China since 2005 (28.7%, n=139). The most common PorA types of MenB strains from invasive meningococcal cases were P1.5-2,2?2 (10.0%, n=10), P1.5-1,2?2 (9.0%, n=9) and P1.5-1,10?4 (9.0%, n=9). Four hundred and twenty one strains had intact fhbp gene; variant 1, 2 and 3 accounted for 12.8% (54 strains), 85.0% (358 strains) and 2.2% (9 strains) respevtively. Ten out of 432 strains (2.3%) contained complete nadA gene. All the 172 strains for which the nhba gene was sequenced had intact gene sequence which corresponded to 68 peptide types. Conclusion CC4821 was the predominant CC of MenB strains in China; the vaccine proteins were diverse about the sequences. The vaccine proteins should be carefully selected when developing MenB vaccines in China.