Small interfering RNA and gene expression analysis using a multiplex branched DNA assay without RNA purification

The authors have developed a novel multiplex detection system that quantitatively measures the expression level of 11 messenger RNAs (mRNAs) directly from cell lysates or tissue homogenates without RNA purification. The system incorporates branched DNA (bDNA) technology from Bayer and a multiplex bead array platform from Luminex. In this study, a 21-nt synthetic small interfering RNA (siRNA; specifically designed to knockdown interleukin-8 [IL-8] expression) was delivered into HeLa cells. Using the multiplex bDNA assay, gene expression levels were measured simultaneously from cell lysates for 11 genes. After treating the HeLa cells for 20 h with phorbol myristate acetate (PMA), IL-8 mRNA levels were induced by almost 50-fold; transfection with 30 nM IL-8-specific siRNA reduced the PMA-induced IL-8 mRNA by 80%. In addition, PMA induced mRNA expression in IL-1alpha (3-fold) and IL-6 (4-fold); however, the IL-8 siRNA did not affect the expression of either of these 2 cytokine genes, indicating that the siRNA was selective for IL-8 mRNA expression. Three housekeeping genes' expression levels were measured under all conditions tested. The multiplex bDNA assay provides a powerful tool for quantitative multiplex gene expression analysis directly from cell lysates, which could be extremely valuable for conservation of rare or difficult-to-obtain samples.     

Astrocyte influences on ischemic neuronal death

Glutamate excitotoxicity, oxidative stress, and acidosis are primary mediators of neuronal death during ischemia and reperfusion. Astrocytes influence these processes in several ways. Glutamate uptake by astrocytes normally prevents excitotoxic glutamate elevations in brain extracellular space, and this process appears to be a critical determinant of neuronal survival in the ischemic penumbra. Conversely, glutamate efflux from astrocytes by reversal of glutamate uptake, volume sensitive organic ion channels, and other routes may contribute to extracellular glutamate elevations. Glutamate activation of neuronal N-methyl-D-aspartate (NMDA) receptors is modulated by glycine and D-serine: both of these neuromodulators are transported by astrocytes, and D-serine production is localized exclusively to astrocytes. Astrocytes influence neuronal antioxidant status through release of ascorbate and uptake of its oxidized form, dehydroascorbate, and by indirectly supporting neuronal glutathione metabolism. In addition, glutathione in astrocytes can serve as a sink for nitric oxide and thereby reduce neuronal oxidant stress during ischemia. Astrocytes probably also influence neuronal survival in the post-ischemic period. Reactive astrocytes secrete nitric oxide, TNFalpha, matrix metalloproteinases, and other factors that can contribute to delayed neuronal death, and facilitate brain edema via aquaporin-4 channels localized to the astrocyte endfoot-endothelial interface. On the other hand erythropoietin, a paracrine messenger in brain, is produced by astrocytes and upregulated after ischemia. Erythropoietin stimulates the Janus kinase-2 (JAK-2) and nuclear factor-kappaB (NF-kB) signaling pathways in neurons to prevent programmed cell death after ischemic or excitotoxic stress. Astrocytes also secrete several angiogenic and neurotrophic factors that are important for vascular and neuronal regeneration after stroke.     

P2X7 receptors mediate NADH transport across the plasma membranes of astrocytes

NADH plays critical roles in mitochondrial functions and energy metabolism. There has been no study demonstrating that NADH can be transported across the plasma membranes of cells. In this study we tested our hypothesis that NADH can be transported across the plasma membranes of astrocytes by a P2X7 receptor (P2X7R)-mediated mechanism. We found that treatment of astrocytes with NADH led to increases in both intracellular NADH and NAD+. Three lines of studies suggest that P2X7R mediates the NADH transport into astrocytes: the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) blocked the NADH transport; RNAi knockdown of P2X7R led to decreased NADH transport; and transfection of HEK293 cells with mouse P2X7R cDNA led to increased NADH transport. Collectively, our study provides the first direct evidence demonstrating that NADH can be transported across the plasma membranes of astrocytes by a P2X7R-mediated mechanism. Our study also suggests a novel approach for manipulating intracellular NADH and NAD+ levels.     

寒温带岛状林沼泽土壤呼吸速率和季节变化

2011年生长季内利用静态箱-气相色谱法,研究了寒温带典型湿地白桦(Betula platyphylla)岛状林沼泽、兴安落叶松(Larix gmelinii)岛状林沼泽土壤呼吸速率的季节动态及其主要环境因子,利用壕沟隔断法对土壤呼吸各组分间的差异进行研究。结果表明:生长季白桦和兴安落叶松岛状林沼泽土壤呼吸速率具有明显的季节性规律,土壤呼吸总速率分别为368.60和312.46 mg m-2h-1,异养呼吸速率分别为300.57和215.70 mg m-2h-1,占土壤呼吸总速率的81.5%和69.0%;自养呼吸速率为68.03和96.76 mg m-2h-1,占土壤呼吸总速率的18.5%和31.0%。不同处理条件下的土壤呼吸在季节变化上表现基本一致,高峰期都发生在夏季;土壤呼吸与温度呈极显著相关性,但与土壤湿度的相关性较差。生长季白桦和兴安落叶松岛状林沼泽土壤呼吸总量分别为12.64和10.61 t/hm2。     

TMEM158 promotes pancreatic cancer aggressiveness by activation of TGFβ1 and PI3K/AKT signaling pathway

Pancreatic cancer (PC) is one of the most deadly digestive cancers world-wide, with a dismal five-year survival rate of <8%. Upregulation of transmembrane protein 158 (TMEM158) is known to facilitate the progression of several carcinomas. However, little is known concerning the potential roles of TMEM158 in PC. Herein, we first found that TMEM158 was significantly upregulated in PC samples as well as PC cell lines. The overexpression of TMEM158 was significantly correlated with advanced clinicopathologic features (including tumor size, TNM stage, and blood vessel invasion) and poorer prognosis of patients with PC in clinic. Evidenced based on a series of loss- and gain-of-function assays uncovered that TMEM158 enhanced PC cell proliferation, migration, and invasion by stimulating the progression of cell cycle, epithelial–mesenchymal transition, and MMP-2/9 production. Furthermore, mechanism-related investigations disclosed that activation of TGFβ1 and PI3K/AKT signal might be responsible for TMEM158-triggered PC aggressiveness. Collectively, TMEM158 was upregulated in PC and promoted PC cell proliferation, migration, and invasion through the activation of TGFβ1 and PI3K/AKT signaling pathways, highlighting its potential as a tumor promoter and a therapeutic target for PC.     

Cage opening and fragmentation of C60 fullerene induced by an ultrashort laser pulse

Response of C₆₀ fullerene to a 40 fs full-width at half-maximum laser pulse with a photon energy of 2.0 eV and different laser intensities is studied by semiclassical dynamics simulation technique. The simulation results show that soon after the irradiation with a strong laser pulse, many C–C bonds abruptly break but no fragments are produced. The breaking of multiple C–C bonds induces a quick increase in the kinetic energy and potential energy and a decrease in electronic energy. These results suggest that the opening of the C₆₀ cage is an effective channel for the conversion of electronic energy to kinetic energy for the electronically excited C₆₀ fullerene.     

Laboratory Investigation of Ecological Factors Influencing the Environmental Presence of Burkholderia pseudomallei

Factors like temperature, pH value, water content in soil, and ultraviolet rays that might have influence on the survival of environmental Burkholderia pseudomallei strains were evaluated. Data showed that the optimal temperature and pH value for B. pseudomallei were 24 C to 32 C and 5 to 8, respectively. Water content in soil of less than 10% brought about the death of the bacteria within 70 days, while water content of more than 40% maintained bacteria life for 726 days. The bacteria were easily killed by ultraviolet rays at 465 μW/cm² for 7.75 min while other permanent soil bacteria were killed at 1,860 μW/cm² for 31 min. From these results, it could be concluded that proper temperature, enough water in soil, and suitable soil pH might be the three major ecological conditions governing the environmental presence of B. pseudomallei.