Objective To evaluate the safety and feasibility of totally thoracoscopic anatomic pulmonary segmentectomy (TTAS) for the treatment of the peripheral stage ⅠA non small cell lung cancer(NSCLC).Methods The study involved 50 consecutive patients undergoing totally thoracoscopic anatomic segmentectomy (TTAS) from September 2010 to November 2012 in the First People's Hospital affiliatied to Nanjing Medical University.The diameter of the tumors were less than 2 cm [(mean diameter(1.35 ±0.48) cm].All lymph node sampling of N1 and N2 were neglive,All patients received symtematic lymph node dissection.The pulmonary vessels were individually ligated,and the bronchi were closed using an endoscopic stapler.The intersegmental plane was identified using the demarcation between the resected(inflated) and preserved(collapsed) lungs.Staplers were used for intersegmental dissection.Results The mean operative time and intraoperative bleeding were (191.5 ± 50.4) min and (49.2 ± 54.6) ml respectively.The chest tube drainage duration was (3 ± 1) days.The number of stapler cartridges used for intersegmental division was 3.9 ±0.8.The mean number of lymph nodes and nodal stations dissected were 12.6 ± 2.8 and 6.0 ± 1.5 respectively.No mortality and complications were observed 30 days after the surgery.Further,no local recurrence or metastases were observed during follow-up.Conclusion Totally thoracoscopic anatomic segmentectomy(TTAS) is a feasible and safe technique.With systematic lymph node dissection,TTAS can be a reasonable therapeutic option for stage ⅠA NSCLC.

Objective To evaluate the effects of different doses of lidocaine on suppressing fentanyl-induced cough and determine a safe suppressing dose.Methods Two hundred patients undergoing general anesthesia were randomized to four groups evenly.The following medications were given within ten seconds:normal saline 10ml (groupⅠ,control group),lidocaine 1 mg/kg (groupⅡ),lidoeaine 1.5 mg/kg(groupⅢ),lidocaine 2mg/kg (groupⅣ).Toxic symptoms of lidocaine were recorded within lmin after the administration of lidocaine,then fentanyl 3?g/ kg was given intravenously within 5 seconds.Cough incidence and cough grade were recorded within 2rain after the administration of fentanyl.Systolic blood pressure (SBP),diastolic blood pressure (DBP),heart rates (HR),and satu- ration of pulse oximeter(SpO2) were recorded during different time points of induction,all recorded data were anal- ysed by the statistical software,P value

Objective To evaluate the clinical application of CT-guided localization with combination of methylene blue and a Hookwire system for small pulmonary nodules (SPNs) before video-assisted thoracoscopic resection.Methods CTguided localization the SPNs before resection in 56 patients and 60 nodules,then underwent video-assisted thoracic surgery (VATS) resection.Among 56 patients,19 males and 37 females,aged from 35 to 81 years,mean age was (61.1 ±8.9)years.Results SPNs diameter (6.80 ±4.12) mm,distance from the parietal pleura (15.38 ±4.63) mm.CT-guided localization success rate was 100％,positioning time (10.76 ± 8.17) min,8.9％ (5/56) had micro pneumothorax aftet positioning,7.1％ (4/56) occurrence of needle tract bleeding,no conservative treatment.VATS resection rate was 100％.The pathology of 60 lesions were shown:Bronchiolo-alveolar carcinoma(BAC) were 33 lesions(55.0％),BAC and adenocarcinoma were 11 lesions(18.3％),Atypicaladenomatous hyperplasia (AAH) were 7 lesions (11.8％),Inflammation were 4 lesions (6.7％),Harmatoma were 3 lesions(5.0％),Tuberculoses were 2 lesions(3.3％).Conclusion CT-guided localization with combination of methylene blue and a Hookwire system before video-assisted thoracoscopic resection is a promising technique for small solitary pulmonary nodules.It could play an important role in accurate localization of small pulmonary nodules,and it is a safe technique with clinical application.

Objective To compare the infectivity between laboratory adapted human inununodefi- ciency virus(HIV-1) and primary HIV-1 isolates for different mucosal epithelial cell lines. Methods Mu-cosal epithelial cells Caco-2, T-84, HeLa and lymphocyte MT-4 were infected with laboratory adapted HIV-1 SF33 and 2 primary HIV-1 isolates (02010561, 02010141). Culture supernatant and cells were collected respectively on 3-4 days interval after virus inoculation. The former was tested for HIV-1 antigen P24 level and viral load, and the latter was tested for total viral DNA and integrated viral DNA. Results All 3 virus strains could infect MT-4 cells and integrate into their genome. Only HIV-1 SF33 could infect Caco-2 cells but could not integrate into their genomic DNA. Both HIV-1 SF33 and 02010561 infected HeLa cells but only integration of HIV-1 SF33 was detected. All the 3 HIV-1 strains infected T-84 cells but only the integra-tion of HIV-1 SF33 and 02010141 was observed. Conclusion Although laboratory adapted and primary HIV-1 strains are able to infect human mucosal epithelial cell lines, transient or productive infection estab-lished in different mucosal epithelial cells is dependent on the character of cells and virus strains.

OBJECTIVEThis study aimed at exploring the feasibility of using dried blood spots (DBS) to detect HIV drug resistance genotyping in China by comparing the results of drug resistance from DBS, plasma and whole blood samples.

METHODSBlood samples were collected from 39 AIDS patients from Anhui (10), Yunnan (13), Hunan (6) and Xinjiang (10) provinces and autonomous regions. The HIV strains that infected these patients covered all the major HIV-1 subtypes prevailing in China (B, CRF01_AE, CRF07_BC). HIV drug resistance genotyping assay was performed on DBS as well as on the whole blood and plasma samples from the same patients simultaneously by using an in-house nest RT-PCR method. Drug resistance levels were determined based on Stanford University HIV drug resistance database, and the results from these three types of samples were compared.

RESULTSThe percentages of successful amplification of protease and reverse transcriptase regions in the pol gene were 95% (37/39) from DBS, 92% (36/39) from whole blood and 100% (39/39) from plasma samples. The sequences from the three types of samples showed more than 99% identity.86% (31/36) of the DBS samples had the same set of drug resistance mutations as those which were detected from plasma samples. The differences probably resulted from mixed bases.

CONCLUSIONSThere was no major difference in detecting HIV drug resistance genotyping among DBS, plasma and whole blood samples. Therefore, DBS is useful for detection of HIV drug resistance genotyping and is particularly valuable in developing countries like China, especially in remote rural regions.

Dried Blood Spot Testing ; Drug Resistance, Viral ; genetics ; Feasibility Studies ; Genotype ; HIV Infections ; blood ; genetics ; virology ; HIV Seropositivity ; blood ; genetics ; virology ; HIV-1 ; drug effects ; genetics ; Humans ; Reverse Transcriptase Polymerase Chain Reaction ; Viral Load

Objective To explore the clinical application of percutaneous endoscopic gastrostomy in gastroin- testinal nutrition among critically ill patients.Methods Twenty-two patients were undertaken percutaneous endo- scopic gastrostomy under the lead of gastroscope.The puncturatio site was located in anterior wall of stomach.Results All the procedures were performed successfully for one time.Intraoperational blood pressure was very steady.At the same time,intraoperational SpO_2 of the patients all exceeced 97%.The bleeding amount and operation time were respectively (3.8?1.9) ml and (15.5?2.3) min.Severe complications such as gastrostoma,gastrocolic fistula, pneumoperitoneum,refluxing or aspiration of gastric juice,inhalant pneumonia didn't occur after the operation.Con- clusion Percutaneous endoscopic gastrostomy was safe and feasible among critically ill patients who needed gastroin- testinal nutrition because of its slight injury,little bleeding and shorter operational time.

Purpose To explore the accuracy of ALK fused gene expression by immunohistochemistry ( IHC) in non-small cell lung cancer ( NSCLC) patients, and to investigate the clinical and pathological features of ALK-positive NSCLC patients. Methods By u-sing rabbit monoclonal D5F3 antibody, ALK IHC was performed on 234 NSCLC patients. ALK positive cases were confirmed by reverse transcription-polymerase chain reaction ( RT-PCR) . Results The positive incidence of ALK by IHC in 234 NSCLC specimens was 8. 97% (21/234), the positive rate of ALK fused gene verificated by RT-PCR was 5. 98% (14/234). There was significant difference with histological type, age, stage (P<0. 05), but no significant difference with gender, smoking history, tumor differentiation. Of 21 cases of ALK-positive NSCLC patients, the consistency of IHC and RT-PCR was 0 when IHC was ( +) , however, when IHC was or immunohistochemical score was >120, the consistency rate was 100%. Conclusion Although immunohistochemical expres-sion of ALK fused gene may have a certain false positive, IHC or immunohistochemical score> 120 show very high value for ALK fused gene RT-PCR followed by ALK immunohistochemistry in lung cancer is a economical and feasible method for the valuation of ALK fused gene.

Recently, we have reported a new gfp gene isolated from Aequorea macrodactyla. The protein purified from expressed E. coli exhibited an excitation peak at 476 nm and an emission peak at 496 nm. However, the drawback of only maturing to fluorescence at low temperature limited its applications. In this paper, we further describe twelve mutants of GFPxm. Seven mutants produced enhanced fluorescence when expressed in E. coli at higher temperature (37 degrees C). After six hours of induction at 25 degrees C, 32 degrees C and 37 degrees C respectively, the relative fluorescent intensities of GFPxm16, GFPxm18 and GFPxm19 were higher than that of EGFP, moreover GFPxm16 and GFPxm163 could preserve high fluorescent intensity even expressed at 42 degrees C. Four mutants of the seven could reach high expression level in three kind of mammalian cells. Another 6 mutants had red-shift of excitation-emission maxima, and longest excitation-emission maxima were 514nm and 525nm. Another three mutants had two excitation peaks, and one mutant had only one UV-excitation peak. The most exciting result is the mutant of OFPxm with orange color. The mutant has an excitation peak at 509 nm and an emission peak at 523nm. 523nm is yellowish green but the protein is orange observed by eyes. The mutant could reach high expression level and matured at higher temperature but the fluorescent intensity was comparatively low because of low quantum yield.
Animals ; Electrophoresis, Polyacrylamide Gel ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Mutation ; Temperature

AIMTo prepare the liposomes which protect antisense oligodeoxynucleotides (ASON) against nuclease degradation and delivery ASON into cytoplasmic efficiently.

METHODSA cationic derivative of cholesterol, 3 beta-[N-(N',N'-dimethylaminoethan)-carbamoyl] cholesterol (DC-Chol) was synthesized and used to prepare cationic liposome. The characteristics of liposomes/ASON complexes including size, drug loaded efficiency and structure were investigated. Cellular uptake of fluorescence labled ASON (FAM-ASON) under different condition was determined by flow cytometric analysis. Denatured polyacryamide gel electrophoresis (DPGE) was used to analyze the role of liposomes in protecting ASON.

RESULTSThe mean values of preliposomes and liposomes/ASON complexes size were 185.7 and 228.2 nm, respectively. Cationic liposomes showed a high adsorption capacity for ASON. When the +/- charge ratio exceeded 2:1, more than 90% of the ASON was loaded into liposomes. Agarose gel electrophoresis showed three different existence of ASON in liposomes formulation: free, absorbed and encapsulated types. Concerning cellular uptake, DC-Chol liposomes indicated high efficient effect of increasing cellular uptake of ASON. Compared with free ASON, the total fluorescence intensity in cytoplasma was significantly enhanced. The level of increasing was largely depended on +/- charge ratio. The cellular uptake of FAM-ASON decreased in the presence of serum. The cellular total fluorescence intensity in 10% and 30% fetal bovine serum of cultured medium were only 22.3% and 15.5% as that of serum-free media, respectively. DPGE confirmed that free ASON was rapidly degraded by DNase I while ASON encapsulated into liposomes was efficiently protected.

CONCLUSIONThe cationic DC-Chol liposomes are shown to be promising carriers to deliver ASON into cytoplasma.

Cholesterol ; analogs & derivatives ; pharmacology ; Drug Carriers ; Drug Delivery Systems ; HeLa Cells ; Humans ; Liposomes ; pharmacology ; Multiple Myeloma ; pathology ; Oligonucleotides, Antisense ; administration & dosage ; blood ; metabolism ; Tumor Cells, Cultured

Mammalian cells are prone to apoptosis when cultured in large scale for production of biopharmaceuticals. And this will reduce production duration and result in high cost of production. Apoptosis is triggered by various factors, and delicately regulated by a set of genes. Bcl-2, a component integrated in mitochondria membrane, is an important member of these genes. By maintaining the integrity of mitochondria membrane, Bcl-2 keeps cytochrome C from releasing into cytoplasm, and thus blocks the activation of caspases, and subsequent onset of apoptosis. Over-expression of Bcl-2 has proven to be useful in blocking apoptosis in various cell lines, including CHO, hybridoma, myeloma, lymphoma and insect cells. Ammonia, a metabolite of cultured cells, however, showed apparent pro-apoptosis activity. In living cells, ammonia can be utilized by glutamine synthetase (GS) to synthesize glutamine, and thus lower the concentration of ammonia in medium, and its negative effects. Glutamine is essential to living cells. If not added into medium, glutamine can only be synthesized by GS, which makes GS a qualified selection marker. This marker can be used for gene amplification by adding into medium increased concentration of MSX, an inhibitor of GS. In this study, we over-expressed Bcl-2 using GS amplification in a recombinant CHO cell line stably expressing human interferon-beta. The modified cell line, with higher expression of Bcl-2 and lower production of ammonia, exhibited good anti-apoptosis quality and higher interferon-beta production in continuous culture.
Animals ; Apoptosis ; genetics ; physiology ; Biopharmaceutics ; CHO Cells ; cytology ; metabolism ; Cricetinae ; Cricetulus ; Glutamate-Ammonia Ligase ; genetics ; metabolism ; Interferon-beta ; metabolism ; Models, Genetic ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism