Cationic colloidal gold (CCG) nanoparticles were used for labeling on the anioinic sites of living cells under two-photon fluorescence (TPF) microscope,and for delivering macromolecules into the target cells when irradiated by focused femtosecond laser pulses. 15 nm CCG nanoparticles which were made by conjugation with poly-L-Lysine,were attached on the anionic sites,especially on the membrane,of CHO-K1 cells because of their strong positive charge at physiological pH. Target cells labeled with cationic gold nanoparticles were imaged under TPF microscope,and lifetime images of the same targets were taken by time correlated single photon counting (TCSPC) technique in order to verify the fluorescence of the marker and the luminescence of the gold particles. The results shown that CCG nanoparticles first accumulated on the negatively charged sites of the membrane,then entered via endocytic pathway and attached anionic sites in plasma. A macromolecular 10 ku fluorescein isothiocyanate dextran (FITC-D) was added into the sample and the focused femtosecond laser of TPL microscope was employed to scan the target cells layer by layer. Typical laser power level used in biological imaging is about 3～5 mW. Here the laser power of scanning was below 5 mW in order to prevent photochemical damage of the fs-pulses alone and to localize effects to the nanoparticles on a nano-scale. After scanning the target cells under stack mode,macromolecular fluoresceins surrounding the cells was observed to cross the membrane and to diffuse in the cytoplasma. Comparing with the images before scanning,the two-photon fluorescence and fluorescence lifetime images revealed the delivery of FITC-D into target cells. Photothermal effects,which may be responsible for the permeabilisation,are highly localized in nanoscale and are not expected to cause damage exceeding the cell membrane. After extensive of laser scanning also cell death occurred. The ratio of the uptake of FITC-D and cellular death under different conditions were measured by flow cytometer. The results shown: with the increased scanning times or ratio of particles to cells,transfer efficiency increased first and decreased afterwards,but the ratio of cellular death went up all along.

Hepatitis B surface antigen (HBsAg) carrying preS sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. Here we report the success in achieving efficient and stable expression of hepatitis B virus S antigen and preS1 epitope fusion protein (S/preS1) in CHO cells. The HMRCHEF53u/Neo-S/preS1 expression vector carrying S/preS1 gene was constructed and transfected into CHO-S cells. A stable and high-expression CHO cell line, named 10G6, was selected by ELISA and limiting dilution analysis. Western blotting analysis showed S/preS1 expressed from 10G6 cells possessed both S and preS1 antigenicity. 10G6 cells displayed characters of favorable growth and stable S/preS1 expression in repeated batch cultures as evaluated by viable cell density, viability and S/preS1 concentration. And cultivation of 10G6 cells in fed-batch mode resulted in S/preS1 production at 17-20 mg/L with viable cell density at 7 x 10(6)-10 x 10(6) cells/mL.
Animals ; CHO Cells ; Cricetulus ; Epitopes ; biosynthesis ; genetics ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B Vaccines ; biosynthesis ; genetics ; Hepatitis B virus ; Protein Precursors ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

Objective To study the expression of eukaryotic translation initiation factor 4E(eIF4E)in the pathological scars and its probable role in the pathogenesis of pathological scars.Methods Immunohistochemiscal technique was performed to detect the expression and distribution of eIF4E protein in hypertrophic scars(14 cases),keloids(25 cases),mature scars(20 cases)and normal skins(20 cases).Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the eIF4E mRNA level in hypertrophic scars(7 cases),keloids(8 cases),mature scars(8 cases)and normal skins(8 cases).Results Thepositive rate of eIF4E protein expression was remarkably significant difference between normal scars and pathological scars(P＜0.05).The level of eIF4E mRNA in pathological scars 1.73±0.31was higher than that in control group 0.99±0.28.There was significant difference between two groups (P＜O.05).Conclusions The expression of eIF4E is increased in pathological scar.eIF4 E expression is closely associated with the development of pathological scar.Therefore,eIF4E overexpression may play an important role in the proliferation of fibroblasts and in the pathogenesis of pathological scar.

Cardiac reentry is one of the important factors to induce arrhythmias. It could lead to ventricular tachycardia (VT) or even fibrillation (VF), resulting in sudden cardiac death. With the wide use of computer in the quantitative study of electrophysiology, the three-dimensional virtual heart for simulations needs to be developed imminently in computer. In this paper, numerical algorithm of the model was studied. The three-dimensional model was constructed by integrating Luo-Rudy 1991 ventricular cell model and diffusion equation. The operator splitting method was employed to solve the model. The alternate direction iterative (ADI) format and seven-point centered difference method were used for the partial differential equation. And the discrete format with second-order accuracy was taken for the boundary conditions. The results showed that the ADI format and seven-point centered difference method both could successfully figure out the membrane potential and electrical activities with good numerical stability. However, computing consumption could be greatly reduced with the ADI format, implying that the ADI method with large time step was more powerful in numerical simulations.
Action Potentials ; Algorithms ; Heart ; physiology ; Heart Ventricles ; Humans ; Imaging, Three-Dimensional ; Models, Cardiovascular ; Myocardium

Objective To summarize the technique and clinical experience of hybrid procedure under cardiopulmonary bypass (CPB) in children with muscular ventricular septal defect (mVSD). Methods From January 2006 to June 2010, 45 cases of mVSD underwent hybrid procedure with CPB. mVSDs were closed with devices under direct vision in 45 cases. Of them, there were 20 males and 25 females. They ranged from 52 days to 12 years [mean (2.05 ±2.48) year] in age and from 3 to 30 kg [(11.93 ±7.70)kg] in body weight. Preoperatively, most of children were highly susceptible to respiratory tract infections. The hybrid approach was used in all patients with CPB under the guidance of transesophageal echocardiography (TEE). The diameter of mVSDs ranged from 2 to 7 mm under TEE. Of 45 cases, 40 patients had increased rates of pulmonary blood flow. 29 patients had left axis deviation and 12 cases had sinus arrhythmia on electrocardiography (ECG). 19 had other congenital heart lesions, including transposition of great arteries in 1 case, tetralogy of Fallot in 2, pulmonary artery stenosis in 3, patent ductus arteriosus in 6, atrial septal defects in 6) and aorta coactation in 1. The quantity of VSDs were from 1 to 7 (single, in 7; two, in 24 case; three, in 8 case; four, in 5 case and seven, in lease. 37 patients were combined with pulmonary hypertension in our cohort. 38 patients with another large VSD and 19 with other congenital heart lesions were required surgical repair at sometime. Results The hybrid procedures were undertaken in all 45 cases of this cohort. All cases were successful and no deaths occurred during operation. A total of 48 devices were implanted in 45 patients, including single devices in 42 cases (device size ranged from 4 to 10 mm) and two devices in 3 cases (device size ranged from 4 -7 mm). The average time on CPB was (58.28 ±20.70) min , while aortic crossclamp time was(34. 94 ± 14.75) min. In addition, the time on mechanical ventilation postoperatively ranged from 2 hours to 6 days. Compared to the older children, 20 infante aged less than 8 monhad a significant difference in cardiac function in the early postoperative period. One infant was given up treatment because of serious infection. Anather cases recovered with the use of supportive treatment, such as using vasoactive agents, digoxin, inhaling nitric oxide, diuresis, and so on. The enteric-coated aspirin was given at dose of 5 mg ? kg -1. day -1 for a period of 3 to 6 months as usual postoperatively. All patients attended follow-up at 1 week, 1 month, 3 months, 6 months, 1 year and 2 years post-procedure. No major complications were encountered during this period. All cases were no instance of migration of any of the devices, residual shunt, aortic regurgitation, atrioventricular valve dysfunction, Ⅲo atrial-ventricular conduction block, new arrhythmia, and so on. There are no death in long-term follow-up. Conclusion Hybrid procedure is safe and effective for the closure of congenital heart defects in children.

Objective To analyze the prognostic factors in patients with initially diagnosed bone-only metastatic nasopharyngeal carcinoma (NPC). Methods We collected the data of 68 patients with initially diagnosed bone-only metastatic NPC admitted to The Affiliated Tumor Hospital of Shantou University Medical College from 1997 to 2015. Forty-nine patients received chemoradiotherapy. The Kaplan-Meier method was used to calculate the overall survival rate;the log-rank test was used for univariate prognostic analysis;the Cox model was used for multivariate prognostic analysis. Results The median follow-up was 953 months. The 1-, 2-, 3-, and 5-year overall survival ( OS) rates were 53%, 38%, 21%, and 15%, respectively. The median OS time was 134 months. The univariate prognostic analysis showed that spinal metastases, the number of bone metastases, lactic dehydrogenase level before treatment, the radiotherapy technology and dose for primary tumor, and the short-term outcome of primary tumor were associated with OS ( P=002, 001, 000, 002, 002, 001 ) . The multivariate prognostic analysis showed that ≤3 bone metastases, dose to primary tumor>65 Gy, and intensity-modulated radiotherapy ( IMRT) were favorable prognostic factors for OS ( P=003,002,004) . Conclusions For patients with initially diagnosed bone-only metastatic NPC, active treatment ( IMRT, dose to primary tumor>65 Gy) should be considered for those with ≤3 bone metastases to achieve a complete response of primary tumor.

Objective To explore the influencing factors of the onset of autism spectrum disorder in male children.Methods Totally 151 male children with autism spectrum disorder were selected as case group and 119 healthy male children matched with the age of the case group in the same administrative region were taken as the control group.All children were assessed with the questionnaire for children's autism etiology and risk factors.Results (1) The differences in children having anorexia and partial eclipse (x2 =50.763,P＜0.01),father's age during pregnancy (x2 =11.441,P=0.043),place of pregnancy (x2 =50.763,P＜0.01),hypertension of pregnancy (x2 =5.693,P=0.026),intrauterine hypoxia (x2 =9.332,P=0.002),umbilical cord around the neck(x2 =18.483,P＜0.01),parents smoking and drinking history during pregnancy (x2 =13.660,P=0.008),parental smoking (x2 =12.901,P=0.005) and alcohol consumption (x2 =8.386,P=0.039) during pregnancy,birth height of child (x2 =8.870,P=0.031),amniotic fluid pollution (x2 =4.561,P=0.043),participation time of artificial feeding,major caregivers,delayed development indicators in infants and young children and whether or not the harmonious parent-child relationship were statistically significant(P＜0.05).(2) Children with anorexia and partial diet (OR =12.284,95％ CI =2.768-54.507),living in rural areas during pregnancy (OR =17.251,95％ CI =1.899-1 56.745),parents' history of smoking and drinking (OR =6.191,95％ CI =1.678-22.838),and intrauterine hypoxia during pregnancy (OR=38.859,95％CI=2.944-512.930) may be risk factors for male autism spectrum disorder.Conclusion To correct children's anorexia bias,improve the living environment in pregnancy,reduce pregnancy complications and avoid exposure to tobacco and alcohol pollution during maternal pregnancy can be an effective entry point for the prevention and control of autism spectrum disorders in male children.

Objective: Through the study of angiotensinⅡ- type 2 receptor agonist (AT2R) after pretreatment of mechanical ventilation lung injury (VILI) in rats model, to clarify the role of angiotensin Ⅱ - type 2 receptor agonist (C21) in alleviating VILI inflammation and the damage of immune function and its possible mechanism. Methods: In this experiment, the acute lung injury model was established by mechanical spring-volume ventilation in SD rats, and C21 pretreatment was performed to observe the pathological condition of lung tissue in rats with different ventilation duration, and to detect the inflammatory changes of BALF lavage fluid. Flow cytometry was used to detect the CD68+/iNOS+ labeled M1 type AMφ and the CD68+/Arg-1+ labeled M2 type AMφ in alveolar lavage fluid. Results: The mechanical VILI rat model was successfully established. The pathological injury score of the mechanical ventilation 4 h model, the wet/dry weight of lung tissue, the number of cells and protein in BALF lavage fluid were increased significantly, the levels of TNF-α and IL - 1 were increased significantly, the levels of IL-4 and IL-10 were decreased significantly, and the level of inflammatory reaction decreased with the increase of ventilation time. The M1/M2 ratio in the 4 h ventilation model group was the highest, which was significantly different from the control group (P<0.05). Compared to the model group, the C21 pretreatment significantly reduced the levels of proinflammatory factors TNF-αand IL-1, and increased the levels of anti-inflammatory factors IL-4 and IL-10 in BALF (P<0.01). After the intervention of AT2R agonist at 4 h, 6 h and 8 h, the M1/M2 ratio of the model was lower than that of the model without AT2R agonist at 4 h, 6 h and 8 h. Conclusion Mechanical ventilation for 4 h in SD rats can establish a mechanical ventilation lung injury model. AT2R agonist C21 can promote the polarization of macrophages to m2-type, and C21 pretreatment may alleviate VILI inflammation and immune damage by altering the polarization of macrophages.

In contrast to the well-established genomic 5-methylcytosine (5mC), the existence of N⁶-methyladenine (6 mA) in eukaryotic genomes was discovered only recently. Initial studies found that it was actively regulated in cancer cells, suggesting its involvement in the process of carcinogenesis. However, the contribution of 6 mA in tongue squamous cell carcinoma (TSCC) still remains uncharacterized. In this study, a pan-cancer type analysis was first performed, which revealed enhanced 6 mA metabolism in diverse cancer types. The study was then focused on the regulation of 6 mA metabolism, as well as its effects on TSCC cells. To these aspects, genome 6 mA level was found greatly increased in TSCC tissues and cultured cells. By knocking down 6 mA methylases N6AMT1 and METTL4, the level of genomic 6 mA was decreased in TSCC cells. This led to suppressed colony formation and cell migration. By contrast, knockdown of 6 mA demethylase ALKBH1 resulted in an increased 6 mA level, enhanced colony formation, and cell migration. Further study suggested that regulation of the NF-κB pathway might contribute to the enhanced migration of TSCC cells. Therefore, in the case of TSCC, we have shown that genomic 6 mA modification is involved in the proliferation and migration of cancer cells.
AlkB Homolog 1, Histone H2a Dioxygenase/metabolism* ; Carcinoma, Squamous Cell/pathology* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism* ; Tongue Neoplasms/metabolism*