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Objective: To identify the best transfect conditions for lentiviral vector to transfect CD34+ stem cells from human cord blood., Methods: CD34+ hematopoietic stem cells from human cord blood were transduced with pTRIPdU3-RNAiTALh-EF1a-GFP plasmid expressing GFP by the second generation and third generation lentiviral vector system. The transfect conditions such as the concentration of the virus, polybrene, transfect volume and media, multiplicity of infection (MOI) values, incubating time and centrifugation in 12-well plate at 200 x g were tested to obtain optimal transfect conditions. The number of CFU were counted and the types of CFU were identified by light microscope after the transfected cells (non-infected stem cells served as control) were cultured for 14 days at a 37 degrees C, 5% CO2 incubator., Results: The second-generation lentiviral vector plasmid had higher infect rate than the third-generation. The optimal transfect conditions were determined as: fresh sorting CD34+ cells, 10(7) TU virus concentration, Polybrene 2 microg/mL in opti-MEM medium, centrifuged at 200 x g for 1 h and then co-culture 8 h for cells and virus mixture in one well in flat-bottomed 12-well plate (repeated once). Both infected and non-infected CD34+ stem cells developed CFUs with similar numbers and types of colonies after being cultured for 14 days in the cytokine-containing 1:1 liquid medium/semi-solid medium., Conclusion: The identified optimal conditions can enable effective lentiviral vector transduction of CD34+ without interrupting the differentiation potential of the hematopoietic stem cells.

Background: Type 2 Diabetes Mellitus (DM) is a common chronic disease associated with multiple clinical complications. Management guidelines have been established which recommend a risk-stratified approach to managing these patients in primary care. This study aims to evaluate the quality of care (QOC) and effectiveness of a multi-disciplinary risk assessment and management programme (RAMP) for type 2 diabetic patients attending government-funded primary care clinics in Hong Kong. The evaluation will be conducted using a structured and comprehensive evidence-based evaluation framework., Method/design: For evaluation of the quality of care, a longitudinal study will be conducted using the Action Learning and Audit Spiral methodologies to measure whether the pre-set target standards for criteria related to the structure and process of care are achieved. Each participating clinic will be invited to complete a Structure of Care Questionnaire evaluating pre-defined indicators which reflect the setting in which care is delivered, while process of care will be evaluated against the pre-defined indicators in the evaluation framework.Effectiveness of the programme will be evaluated in terms of clinical outcomes, service utilization outcomes, and patient-reported outcomes. A cohort study will be conducted on all eligible diabetic patients who have enrolled into RAMP for more than one year to compare their clinical and public service utilization outcomes of RAMP participants and non-participants. Clinical outcome measures will include HbA1c, blood pressure (both systolic and diastolic), lipids (low-density lipoprotein cholesterol) and future cardiovascular diseases risk prediction; and public health service utilization rate will include general and specialist outpatient, emergency department attendances, and hospital admissions annually within 5 years. For patient-reported outcomes, a total of 550 participants and another 550 non-participants will be followed by telephone to monitor quality of life, patient enablement, global rating of change in health and private health service utilization at baseline, 6, 12, 36 and 60 months., Discussion: The quality of care and effectiveness of the RAMP in enhancing the health for patients with type 2 diabetes will be determined. Possible areas for quality enhancement will be identified and standards of good practice can be established. The information will be useful in guiding service planning and policy decision making.

Background and Aims: Altered levels of circulating cell-free mitochondrial DNA (ccf mtDNA) have been recently detected in several cancer types and have been proposed as a promising noninvasive diagnostic or prognostic biomarker. There has been no report regarding quantification of ccf mtDNA in patients with Ewing's sarcoma (EWS)., Methods: Ccf mtDNA copy number in serum samples obtained from 25 patients with EWS as well as 20 age-matched individuals were detected by quantitative real-time PCR assays. A receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic applicability of serum ccf mtDNA as a noninvasive biomarker for discriminating between patients and healthy cohorts. The potential connection between ccf mtDNA levels and various clinicopathological factors of EWS was also determined., Results: We found that levels of ccf mtDNA in the serum of EWS patients were significantly lower than in healthy controls. The receiver operating curve analysis demonstrated that using serum ccf mtDNA content as a molecular marker allowed distinguishing between EWS patients and healthy subjects with a sensitivity of 76.1 and a specificity of 68.4%. In addition, serum levels of ccf mtDNA were associated with the status of tumor metastasis., Conclusions: These results suggest that serum ccf mtDNA has the potential capacity as a novel and convenient noninvasive biomarker for molecular diagnosis of EWS. Scrutinizing quantitative changes of serum ccf mtDNA may provide valuable clues for better management of EWS patients., (Copyright © 2012 IMSS. Published by Elsevier Inc. All rights reserved.)

Clinically effective cardioprotection under acute myocardial infarction (AMI) can only be achieved by establishing the mechanisms of reperfusion-induced cardiac cell death. In spite of the numerous earlier studies on the prevention of ischemia-reperfusion injury of myocardium, the problem of cardiac cell death upon reperfusion is not yet resolved. Even though animal models provide an immense opportunity in the understanding of the mechanisms of ischemia-reperfusion injury, clinically relevant animal models through which translation of this knowledge into clinic are lacking. In this work, we have established a reperfusion model in rabbits with induced AMI by obstructing and releasing the left anterior ventricular branch of left circumflex coronary artery, which is clinically more relevant. This was achieved by cutting the two left ribs of the rabbit followed by obstructing and releasing the artery unlike the traditional approach, which involves incision through sternum and blocking the anterior descending coronary artery. This animal model of ischemia-reperfusion more closely mimics the physiological condition and also the trauma the animal suffers is much smaller with higher survival rate and thus is a potentially better model for studying the pathology related to ischemia-reperfusion injury.

Two non-CO2 greenhouse gas emissions (methane and nitrous oxide) and related environmental factors were measured within rice growing season under five treatments including non-fertilization (CK), balanced fertilization (BF), decreased nitrogen and phosphate 1 (DNP1), decreased nitrogen and phosphate 2 (DNP2) and increased nitrogen and phosphate 1 (INP) in double rice fields of red clay soil in 2009, using the method of static chamber-gas chromatograph techniques. The results showed that the average CH4 emission fluxes for treatments of BF, DNP1, DNP2 and INP were 4.57, 5.42, 4.70 and 4.65 mg x (m2 x h)(-1) during early rice growing period, which increased by 39%, 49%, 41% and 40% compared with non-fertilizer treatment, respectively. The average CH4 emission fluxes in late rice growing season was higher than preseason's. Compared to CK, CH4 emission increased by 11%, 1%, 26% and - 4% in treatments of BF, DNP1, DNP2 and INP within late rice growing season. Applying nitrogen and phosphate enhanced CH4 emission in turning green period for early and late rice. No significant difference was observed between the CH4 emissions of five treatments during early and late rice growing season (p > 0.05). N2O emission was very little during mid-seasonal drainage period. In contrast, N2O emission peaks were observed in period of alternation of wetting and drying after mid-seasonal drainage in this experiment. N2O emission was, on average, equivalent to 0.18% of the nitrogen applied in double rice growing season. Statistically, air temperature, soil Eh and soil moisture (water-filled pore space, WFPS) at 0-10cm depth significantly affected the fluctuations of the seasonal CH4 flux, but no significant correlationship has been found between N2O flux and related environmental factors. CH4 was the dominated greenhouse gas in double rice fields which contributed approximately 90% for the integrated global warming potential of CH4 and N2O released during the rice growing season. Therefore, the mitigation options should focus on how to reduce CH4 emission in local area. The result indicates that BF is a recommended fertilization method for early rice production, and a optimum fertilization for late season can increase rates of nitrogen and phosphate fertilizers on the basis of BF treatment slightly by considering total global warming potential and grain yield. The rates of BF treatment were 150-90-90 kg x hm(-2) N-P2O5-K2O for early rice, and 180-90-135 kg x hm(-2) N-P2O5-K2O for late rice, respectively.

Objective: To investigate the protective effects of sodium selenite and aloin against ultraviolet A (UVA) radiation on the dermal fibroblasts including the suppression of proliferation, oxidative damage and collagen synthesis., Methods: Human dermal fibroblasts were divided into the negative control group, the positive control UVA radiation group, the sodium selenite treatment plus UVA radiation group and the aloin treatment plus UVA radiation group, which were incubated with sodium selenite or aloin and irradiated with 5 J/cm2 UVA respectively. The MTT spectrophotometry and biochemical assay were used to determine the proliferation, collagen level and the SOD and GSH-Px activity of the fibroblast cells after radiation., Results: 5 J/cm2 UVA affect the proliferation, collage protein, SOD and GSH-Px activity in the fibroblast cells (P < 0.05). Sodium selenite (10-100 microg/L) and aloin (1-100 mg/L) could enhance the proliferation and the SOD and GSH-Px activity. A increased collagen synthesis in dose dependant manner was also observed (P < 0.05)., Conclusion: Sodium selenite and aloin in a certain range of concentration have protective effects on ultraviolet radiation induced fibroblast proliferation inhibition, oxidative injury, and decreased collagen synthesis.

Objectives: In order to investigate the relationship among the toll-like receptor 2 (TLR2), hepatitis B e antigen and HBV DNA, the expression levels of TLR2 on peripheral blood monocytes of chronic hepatitis B (CHB) patients as well as on their monocytes stimulated by ligand of TLR2 (Pam3CSK4) and HBeAg were analyzed., Methods: Sixty-eight adults with CHB were enrolled, including 37 HBeAg-positive patients, 17 HBeAg-negative and HBV DNA negative patients, and 14 HBeAg-negative and HBV DNA positive patients. Sixteen healthy volunteers were also studied as controls. TLR2 expression levels on their peripheral blood monocytes stimulated with Pam3CSK4 or not stimulated were analyzed by FACS Caliber. The relationship of the expression levels of TLR2, HBeAg and HBV DNA were also analyzed. The level of TLR2 on peripheral blood monocytes of healthy volunteers and HBeAg-negative CHB patients stimulated by HBeAg was examined for six hours., Results: The TLR2 expression levels on CD14+ cells were significantly reduced in HBeAg-positive patients (47.57%+/-21.40 %) compared to both healthy volunteers (76.51%+/-7.46%) and HBeAg-negative patients (HBV DNA positive group 73.2%+/-14.2%, HBV DNA negative group 75.2%+/-11.3%); but there was no difference between those of the HBeAg-negative patients and the healthy volunteers. Expression levels of TLR2 on monocytes stimulated by TLR2 ligand in HBeAg-positive patients were obviously increased, and reached the basic levels of the healthy volunteers and the HBeAg-negative patients. The expression levels of TLR2 on monocytes stimulated by HBeAg of the healthy volunteers and the HBeAg-negative patients were markedly reduced., Conclusions: In the presence of HBeAg, HBV down-regulates the expressions of TLR2 on CD14+ cells from peripheral blood, and there is no correlation between HBV-DNA and TLR2. Pam3CSK4 can boost the TLR2 expression in HBeAg-positive patients. The proposed interaction between HBV and TLR2 may provide an important clue to explain the reasons of the establishment of persistent HBV infection.

Based on the remote sensing data, meteorological data and other related data from 1981 to 2004, the grassland net primary productivity (NPP) and its spatiotemporal distribution in Northern Tibet were analyzed by CASA (Carnegie-Ames-Stanford Approach) model. The results indicated that in the study area, the spatial distribution of grassland NPP was affected by the local water and heat conditions, and represented a horizontal zonality. From southeast to northwest, the grassland NPP reduced from 230 g C x m(-2) x a(-1) to near 0 g C x m(-2) x a(-1). The overall level of grassland NPP in Northern Tibet was rather low, with the multi-years average value of total NPP being 21.3 x 10(12) g C x a(-1) and the mean value of NPP being 48.1 g C x m(-2) x a(-1), which were obviously lower than those in Qinghai-Tibetan Plateau and other grassland areas of China. The mean values of NPP on flat land (slope gradient <1 degree) and south slope were relatively lower. On the main alpine grasslands in Northern Tibet, the NPP from July to September occupied 64.0%-70.0% of the whole year. From 1981 to 2004, the grassland NPP within the whole Northern Tibet had a greater annual fluctuation, and tended to further reduce.

Objective: To identify and clone the genes related to breast cancer metastasis through comparing the mRNA expression profiling between primary breast cancer and paired lymph node metastasis., Methods: First, primary breast cancer cells and paired lymph node metastasis tissues were used to identify their differentially expressed genes by using mRNA differential display, and fragments of differentially expressed genes were obtained by gel cutting and cloning to pGEM-T vector, then sequencing and blasting with the GenBank for their homologous genes. Then, the obtained genes were validated by using reverse dot blot hybridization and gene microarray. Finally, the mRNA expression levels in the 7 breast cancer cell lines with different metastasis behaviors were detected by real-time RT-PCR., Results: The mRNA expression profiling of the metastatic breast cancers in lymph node was similar to that of their primary cancers. 16 differentially expressed gene fragments were obtained from mRNA differential display; four of them were found to be unknown genes, and had been accepted by the GenBank, with the accession numbers BG518428, BG518429, BM005520, and BM005521. Of the other 12 genes, 8 were known genes, and 4 were genes with known sequences but unknown functions. The kinesin-like DNA binding protein (KNSL4) and dihydropyrimidine dehydrogenase (DYPD) were down-regulated in the metastatic tissues in comparison with the paired primary tissues, which was identified by both radioactivity-labeled reverse dot blot hybridization and fluorescence-labeled gene microarray hybridization. KNSL4 mRNA expression had a down-regulation trend with increasing metastatic ability of breast cancer cell lines., Conclusions: The gene expression profiling of the lymph node metastasis is almost similar to that of their primary breast cancer, indicating that the metastatic cancer cells in lymph node are the subclone with higher metastasis ability of the primary cancer cells, and that some differentially expressed genes between them may be involved in the change of metastasis phenotype. KNSL4 and DYPD are potential metastasis-related genes, and KNSL4 mRNA expression is correlated with metastasis behaviors of the breast cancer cell lines, suggesting that KNSL4 may play an important role in the metastasis process of breast cancer.

Background & Objective: Previous screening of breast cancer metastasis-related genes found that the mRNA level of kinesin-like 4 (KNSL4) gene is down-regulated in metastatic lymph nodes as compared with the paired primary breast cancer. This study was to clarify the correlations of KNSL4 mRNA expression to metastasis and prognosis of breast cancer, and explore the correlation of KNSL4 expression to c-erbB-2 expression to explore potential mechanisms of promoting metastasis by KNSL4., Methods: Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the mRNA level of KNSL4 in 108 specimens of primary breast cancer. The correlations of KNSL4 mRNA level to metastasis and prognosis of the 108 cases were analyzed. Immunohistochemistry was used to assess c-erbB-2 protien expression in 76 out of the 108 cases, and the correlation of KNSL4 expression to c-erbB-2 expression was analyzed., Results: The mRNA level of KNSL4 was significantly lower in the cases at stages iii-iv than in the cases at stages iii-iv (P<0.001), significantly lower in the cases with more than 3 metastatic lymph nodes than in the cases with 0-3 metastatic positive lymph nodes (P<0.01), slightly lower in the cases with negative estrogen receptor or prognesterone receptor than in the cases with positive receptors (P>0.05), lower in the 6 cases with distant metastasis than in the rest cases without distant metastasis within 24 month follow up, lower in the 3 cases with bilateral breast cancer than in other cases with unilateral breast cancer, and significantly lower in c-erbB-2-positive group than in c-erB-2-negative group (P<0.01)., Conclusions: The down-regulation of KNSL4 mRNA level is correlated to prognosis of primary breast cancer. It may enhance metastatic ability of breast cancer cells through promoting c-erbB-2 transcription and translation.

Aim: To investigate the regulatory effect of paclitaxel on proliferation and apoptosis in human acute leukemia HL-60 cells., Methods: HL-60 cell growth was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tertrazolium bromide (MTT) colorimetric assay. Cell cycle kinetics and apoptosis were analyzed by flow cytometry and microscopic examination. In addition, DNA microarrays containing 14,400 EST elements were used to investigate the gene expression pattern of HL-60 cells exposed to paclitaxel 1 micromol/L., Results: Paclitaxel inhibited HL-60 cell growth significantly in a dose-dependent and time-dependent manner (P<0.01). Marked cell accumulation in G2/M phase and multinucleated cells were also observed after treatment with paclitaxel 0.1 and 1 micromol/L. Among 14400 EST elements, 277 genes were found to be markedly up- or down-expressed in the HL-60 cells treated with paclitaxel 1 micromol/L for 0.5 h, comprising 210 known genes and 67 unknown genes., Conclusion: Paclitaxel suppresses the growth of HL-60 cells in vitro by causing cell-cycle arrest and apoptosis. The results of microarray suggest that paclitaxel initiates apoptosis through multiple mechanisms.

The gene encoding high-molecular-weight (HMW) subunit 1Bx20 was isolated from durum wheat cv. Lira. It encodes a mature protein of 774 amino acid residues with an M(r) of 83,913. Comparison with the sequence of subunit 1Bx7 showed over 96% identity, the main difference being the substitution of two cysteine residues in the N-terminal domain of subunit 1Bx7 with tyrosine residues in 1Bx20. Comparison of the structures and stabilities of the two subunits purified from wheat using Fourier-transform infra-red and circular dichroism spectroscopy showed no significant differences. However, incorporation of subunit 1Bx7 into a base flour gave increased dough strength and stability measured by Mixograph analysis, while incorporation of subunit 1Bx20 resulted in small positive or negative effects on the parameters measured. It is concluded that the different effects of the two subunits could relate to the differences in their cysteine contents, thereby affecting the cross-linking and hence properties of the glutenin polymers.

Background and Objective: Recently, it has been demonstrated that topoisomerase I(Topo I) inhibitor is a useful alternative for treatment of the patients with chronic myelogenous leukemia(CML) in accelerated or blast crisis. In order to get insight into pharmalogic activity of Topo I inhibitor, this study was designed to investigate the killing and proapoptotic activity of Topo I inhibitor(Topotecan, TPT) using K562 cell line, which carry abnormal chromosome[t(9;22)] of characteristics of CML., Methods: The cytotoxic effect of TPT on K562 cells was determined using MTT assay; TPT-induced apoptosis in K562 cells was identified by morphological analysis and Annexin V FITC staining; correlation between TPT-mediated killing or apoptosis and caspase-8 activation was investigated using caspase-8 inhibitor IETD-fmk., Results: After incubation of K562 cells with 0.15 mumol/L TPT for 12, 24, 48, and 72 h, the survival rates, compared with control, progressively reduced to (92 +/- 36)% [P > 0.05 vs (94 +/- 27)%], (68 +/- 21)% [P < 0.05 vs (119 +/- 13)%], (54 +/- 15)% [P < 0.05 vs (132 +/- 31)%], and (21 +/- 10)% [P < 0.01 vs (114 +/- 19)%]. Targeted cells meanwhile demonstrated phosphatidylserine externalization, cell shrinkage, chromatin margination, karyorrehxis and eventually disintegrate into numerous apoptotic bodies. After exposure to TPT in combination with IETD-fmk for 24 h and 48 h, K562 cells were still survival at viability of (95 +/- 29)% and (87 +/- 11)%, the latter was significantly higher than that of to TPT only [P < 0.05 vs (54 +/- 15)%]., Conclusion: Topoisomerase I inhibitor TPT possess killing activity and was induce apoptosis to K562 cells, which could be dependent on activation of caspase-8.