ObjectiveTo investigate the association between the Clock T3111C and T257G gene polymorphisms and sleep epilepsy patients in Han population of Hunan province.MethodsThree hundred and eleven subjects with epilepsy ( sleep epilepsy group ( n =112 ),aperiodic group ( n =95 ),awakening epilepsy group ( n =104 ) ) and 300 sex- and age-matched healthy controls were enrolled in the study.Genomic DNAs were extracted from peripheral blood leucocytes by phenol-chloroform methods.The Clock T3111C and T257G polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).Results(1) Two genotypes(TT and TC) were detected in Clock T3111C.The frequency of Clock site T3111C genotypes in all of the people was 86.09％ (TT,526/611),13.91％(TC,85/611),0( CC),T allele gene frequency was 93.04％ (1134/1222) and C allele gene frequency was 6.96％ (85/1222).There was no significant difference in genotype and gene distribution of Clock gene T3111C polymorphism between sleep epilepsy group,aperiodic group,awakening epilepsy group and control group.(2)Two genotypes(TT and TG) were detected in Clock T257G.The frequency of Clock site T257G genotypes in all of the people was 85.92％ (TT,525/611 ),14.08％ (TG,86/611 ),0( GG),T allele gene frequency was 92.96％ (1136/1222) and G allele gene frequency was 7.04％ (86/1222).There was no significant difference in genotype and gene distribution of Clock gene T257G polymorphisms between sleep epilepsy group,aperiodic epilepsy group,awakening epilepsy group and control group.(3)There was an almost complete correspondence (complete linkage disequilibrium) of bases between the positions 257 and 3111.ConclusionClock gene T3111C and T257G polymorphisms are not associated with sleep epilepsy in Han population of Hunan province.

Objective To explore the diagnosis and treatment of five children with tuberculosis with arthritis as the initial manifestation. Methods The clinical features, laboratory tests and imaging manifestation of 5 children with joint tuberculosis were retrospectively analyzed. Results The course of disease was different. All the five patients were males (mean age 8.5 ±2.9 years old) and suffered from articular symptoms as initial feature. Four of them were diagnosed and treated as rheumatoid arthritis by other hospitals for up to three years, two patients have tuberculosis contact history, and another two patients were found with bone destruction, and one patient has pathologic fracture. Conclusions Tuberculosis is easily misdiagnosed as juvenile idiopathic arthritis , which deserves attention from a pediatric rheumatology physician.

Objective To compare the clinical features, diagnosis, treatment and prognosis between macrophages activation syndrome (MAS) and other hemophagocytic syndrome (HPS). Methods Thirty-six children with HPS were identified at Nanjing Children's Hospital during January 2006 to March 2009. They could be classi-fied into MAS group (13 patients) and other HPS group (23 patients). All relevant clinical features, laboratory data, treatments and outcomes were analyzed with t test,χ2 test and Fisher's exact test.Results Patients with MAS tended to be elder than those with other HPSs [(7.7±1.3) years vs (2.6±0.5)years, t=3.899, P=0.004]. There was no difference in gender distribution. In MAS cases, the central nervous system (69% vs 13%, P=0.001), circulatory system (23% vs 9%, P=0.047) and the urinary system (38% vs 9%, P=0.033) were usually involved. The clinical symptoms of MAS were more sever than other HPS. Serum ferritin [(9703±9819) μg/L vs (4569±1396) μg/L, t=2.854, P=0.015] and erythrocyte sedimentation rate (ESR) [(53±32) mm/1 h vs (20±14) mm/1 h, t=2.708, P=0.020] changed more obviously in MAS cases compared with other HPS. Conclusion, Childhood MAS is different from other HPS in terms of age, etiology, clinical manifestations, laboratory tests and treatments.

Objective To investigate the efficacy and safety of tocilizumab inpatients with refractory systemic'onset juvenile idiopathic arthritis (SoJIA),and to provide a new option for the treatment of this severe disease.Methods We retrospectively studied 25 cases of hospitalized patients with refractory SoJIA treated withtocilizumab,of whom 22 had data that fit for analysis,from May 2005 to February 2016.Data of 22 cases were collected retrospectively from physicians in charge of the patients.Children with SoJIA were treated with nonsteroidal antiinflammatory drugs (NSAIDs),Glucocorticoid (GC),methotrexate,cyclosporin A,etanerceptetc before,but still in high disease activity due to inadequate response were involved.Weretrospective analyzedthe laboratory test results like C'reactive protein (CRP),Erythrocyte sedimentation rate (ESR),Ferritin and other inflammatory index.Improvement of pain,fever,rash,hepatosplenomegaly and lymphadenectasis of active SoJIA (disease course ≥6 months,and inadequate response to NSAIDs and GC) after tocilizumab treatment (Body weight ≥30 kg,8 mg/kg;Body weight＜30 kg,12 mg/kg,per 4 weeks) were analyzed.Safety data of 22 cases were collected throughout the treatment period including neutropenia,infections,anaphylaxis and elevated liver enzymes etc.We also retrospectively analyzedthe dose change of GC and the long'term effect.Dichtomous paramenters were compared teween groups using thex2 test.Continuous parameters were compared using the analysis of uariance.Results In comparison to the indices before the treatment,the level of CRP [(8.7±2.2) mg/L vs (111.6±74.4) mg/L,F=5.192,P=0.002],ESR [(6.4±6.3) mm/1 h) vs (65.6±24.3) mm/1 h,F=50.393,P=0.000],white blood cell (WBC) [(8.4±2.5)×109/L vs (17.6±8.6)×109/L,F=9.321,P=0.000],Neutrophil count [(4.9±2.4)×109/L vs.(14.4±8.7)×109/L,F=10.541,P=0.000],blood platelet (PLT) [(269.5±79.2)×109/L vs (405.4± 145.3)×109/L,F=5.704,P=0.000] and globulin [(19.2±4.1) g/L vs (30.1±3.8) g/L,F=22.896,P=0.000] decreased rapidly and hemoglobin [(118.3±9.0) g/L vs (108.5±9.8) g/L,F=4.693,P=0.002] increased significantly at 24 weeks after Tocilizumab (TCZ) treatment.Clinical manifestationssuch as fever,rash,hepatosplenomegaly,joint swelling and pain were significantly improved.GC dose [(1.25±3.8) mg·kg-1·d-1 vs (16.2±12.8) mg·kg-1·d-1,F=8.21,P=0.000] were significantly reduced after TCZ treatment (P＜0.05);American College of Rheumatology (ACR) Pedi 30/50/70/90 was improved after TCZ treatment.Adverse events occurred in 3 cases of 25 children,who were not included in the statistical analysis group.Conclusion This retrospective case series has demonstrated the efficacy of tocilizumab in SoJIA,low incidence of adverse reactions.Further studies are needed to be developed because this case series haslimited sample size.

Objective Study and analyze the benefits and risks of unilateral thyroidectomy for 1 to 4 cm thyroid cancer in primary surgery.Methods The patients who underwent thyroidectomy for 1 to 4 centimeters WDTC (May 2014 to June 2015) were retrospectively reviewed,and the patients with preoperatively known high-risk characteristics were excluded.One hundred and seventy-one patients would have been eligible for lobectomy as the initial operation based on current American Thyroid Association guidelines.The proportion of patients who need secondary surgery was estimated basing on pathological characteristics.Result In this group,49％ patients would have to undergo a secondary surgery if they took lobectomy as the initial operation.Conclusions Patients with 1 to 4 centimeters WDTC who accept unilateral thyroidectomy may take the risk of secondary surgery.Surgeons and patients need to balance the relative benefits and risks of initial TT versus lobectomy before the operation.

Objective To investigate the application of intraoperative neuromonitoring (IONM) during thyroidectomy for non-recurrent laryngeal nerve (NRLN).Methods From Oct.2013 to Apr.2016,2846 patients underwent thyroidectomy with the application of IONM,and 11 patients with non-recurrent laryngeal nerve were analyzed.Results 11 cases of NRLN were all accurately identified by IONM,and no injury of NRLN occurred during thyroid surgery.Conclusions NRLN is uncommon in clinical and it is difficult to be predicted before surgery and easy to be injured.The application of IONM can reduce the possibility of NRLN injury remarkably.

Objective:To explore the pathogenesis of coronary artery lesions in Kawasaki disease(KD) by detecting expression of CD40L on T-cells from patients with KD.Methods:Blood samples were collected from 26 patients with KD before and after intravenous immunoglobulin(IVIG) treatment. Age-matched febrile 16 children with various diseases were studied in parallel as controls. Age-matched normal control 15 children were studied as controls. CD40L expression on T-cells was detected by flow cytometry, soluble E-selection and soluble CD40L levels were measured by enzyme-linked immunosorbent assay.Results:CD40L expression on CD4+T-cells and soluble E-selection levels were significantly higher in patients with KD than that in the febrile control(FC) group and normal group(P

Objective:To evaluate the role of miR-146a in hippocampal inflammatory responses in postoperative cognitive dysfunction (POCD) in mice.Methods:One hundred and sixty clean-grade male C57BL/6 mice, aged 12-16 weeks, weighing 22-28 g, were divided into 5 groups ( n=32 each) using a random number table method: control group (group C), group POCD, miR-146a agomir group (group Ag), miR-146a antagomir group (group At) and negative control group (group NC). The mice were subjected to an intramedullary fixation for tibial fracture under 1.5% isoflurane anesthesia to establish POCD model.At 2 days before operation, miR-146a agomir 0.5 nmol (0.1 nmol/μl) was injected into bilateral hippocampi in group Ag, miR-146a antagomir 2.5 nmol (0.5 nmol/μl) was injected in group At, miR-146a negative control solution 2.5 nmol (0.5 nmol/μl) was given in group NC, and the animals in group C did not receive any treatment.At 1 day before operation and at 1, 3 and 7 days after operation, open-field test was performed to evaluate spontaneous motor activity, and contextual fear conditioning test was performed to evaluate cognitive ability 15 min later.At 1 and 3 days after operation, the animals were sacrificed and hippocampi was removed for determination of expression of CD11b (a marker for activation of microglia) in hippocampal CA1 region by immunofluorescence staining.At 6, 12 and 24 h after operation, the expression of miR-146a was detected by quantitative real-time polymerase chain reaction, the expression of interleukin-1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), nuclear factor kappa B p65 (NF-κB p65) and tumor necrosis factor-alpha (TNF-α) was determined by Western blot and interleukin-1beta (IL-1β) and IL-6 contents were determined by enzyme-linked immunosorbent assay. Results:There was no significant difference in the total exploring distance in the open-field test or percentage of freezing time in tone-fear conditioning test at each time point among the five groups( P>0.05). Compared with group C, the percentage of freezing time in the contextual fear conditioning test was significantly decreased at 1, 3 and 7 days after operation, the expression of CD11b at 1 and 3 days after surgery and expression of miR-146a, IRAK1, TRAF6, NF-κB p65 and TNF-α were up-regulated and the contents of IL-1 β and IL-6 were increased at 6, 12 and 24 h after operation in group POCD ( P<0.05). Compared with group NC, the percentage of freezing time in the contextual fear conditioning test was significantly increased at 1, 3 and 7 days after operation, and the expression of CD11b was down-regulated at 1 and 3 days after surgery, and the expression of miR-146a, IRAK1, TRAF6, NF-κB p65 and TNF-α was up-regulated and IL-1β and IL-6 contents were decreased at 6, 12 and 24 h after operation in group Ag, and the percentage of freezing time in the contextual fear conditioning test was decreased at 1, 3 and 7 days after operation, the expression of CD11b at 1 and 3 days after surgery was up-regulated, the expression of miR-146a was down-regulated and IRAK1, TRAF6, NF-κB p65 expression was up-regulated at 6, 12 and 24 h after operation, TNF-α expression was up-regulated and IL-1β and IL-6 contents were increased at 12 and 24 h after operation in group At ( P<0.05). Conclusion:miR-146a is involved in the process of hippocampal inflammatory responses, and the mechanism may be related to the inhibition of IRAK1-TRAF6-NF-κB signaling pathway in mice.

Objective To investigate the effectiveness of three different anesthetic techniques in intraventricular catheterization and its effect on the survival rate of rats. Methods Thirty Wistar rats were equally allocated into 3 groups:chloral hydrate group,pentobarbital sodium group and isoflurane group. Intraventricular catheterization was performed in the rats after anesthesia with i. p. injection of chloral hydrate and pentobarbital sodium, and isoflurane inhalation, respectively. Levels of blood glucose were detected before and at 15 and 30 minutes and 1, 3, 7, 14, 28 days after anesthesia. Body mass and 24-hour food intake were recorded before and at 1, 3, 7 days after anesthesia. The onset time and effective time of anesthesia, operation time and the survival rates on 30 days of the rats were compared and analyzed. Results The onset time and effective time of anesthesia, and the operation time in the isoflurane group were shorter than that in the chloral hydrate group, while these parameters in this group were shorter than that in the pentobarbital sodium group. Blood glucose in the chloral hydrate group was apparently increased during the surgical operation, while the body mass, 24-hour food intake and blood glucose were decreasing since one day after operation, and all the rats in this group died during the 30-day observation, mainly, due to enteroplegia. Blood glucose in the pentobarbital sodium group was mildly increased after anesthesia, while the body mass, 24-hour food intake and blood glucose were mildly decreased at one day after operation and recovered within one week. In this group, 3 rats died of respiratory distress due to overdose anesthesia and one rat died during the 30 day-observation. The blood glucose in the isoflurane group was mildly increased after operation, while the 24-hour food intake and blood glucose did not markedly changed, the body mass was stably increased, and no rat died during the 30-day-observation. Conclusions Intraperitoneal injection of chloral hydrate is not suitable for intraventricular catheterization in rats. Intraperitoneal injection of pentobarbital sodium can be only carefully applied for intraventricular catheterization under poorly-limited conditions. Isoflurane inhalation anesthesia is recommended for intraventricular catheterization in rats.

Lactoferrin in milk is a multifunctional protein. In addition, lactoferrin has antiviral, antifungal and antiparasitic activity. In this study, the N-terminus from porcine lactoferrin (PLF-N) was designed to express the antimicrobial action of recombinant porcine lactoferrin. We cloned a 1077 bp fragment of the PLF gene from mammary gland tissue of the lactating sow at the third day. Comparing nucleotide sequence with four strains of PLF gene published on GenBank, the homology was more than 99%. With the reference template of the cloned fragment of PLF-N and optimizing codon bias, we synthesized the gene of N-terminus encoding porcine lactoferrin (PLF-NS). The high expression gene of PLF-NS was cloned into the fusion expression vector pET30b and expressed in E. coli BL21 (DE3). After induced with Isopropyl beta-D-1-Thiogalactopyranoside (IPTG), the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. The protein had a molecular weight of 42 kDa and accounted for 32% of the total cellular protein. After purification and renaturation, the purity of the expressed protein was 98%. The expressed PLF-NS protein showed obviously antibacterial activity. This method provides an excellent way for high expression of antimicrobial proteins when optimizing codon bias.
Amino Acid Sequence ; Animals ; Anti-Infective Agents ; metabolism ; pharmacology ; Base Sequence ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Lactoferrin ; biosynthesis ; genetics ; pharmacology ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Swine