Genetic engineering of transitory starch accumulation by knockdown of OsSEX4 in rice plants for enhanced bioethanol production.

Rice straw, a common agricultural waste, is used as a potential feedstock for bioethanol production. Currently, bioethanol is made mostly from the microbial fermentation of starch-containing raw materials. Therefore, genetically engineered starch-excess rice straw through interference of starch degradation as a potential strategy to enhance bioethanol production was evaluated in this study. Arabidopsis Starch Excess 4 (SEX4) encodes a chloroplast-localized glucan phosphatase and plays a role in transitory starch degradation. Despite the identification of a SEX4 homolog in rice, OsSEX4, its biological function remains uncertain. Ectopic expression of OsSEX4 complementary DNA complemented the leaf starch-excess phenotype of the Arabidopsis sex4-4 mutant. OsSEX4-knockdown transgenic rice plants were generated using the RNA interference approach. Starch accumulation was higher in OsSEX4-knockdown suspension-cultured cells, leaves, and rice straw compared with the wild type, suggesting that OsSEX4 plays an important role in degradation of transitory starch. The OsSEX4-knockdown rice plants showed normal plant growth and no yield penalty. Starch-excess OsSEX4-knockdown rice straw used as feedstock for fermentation resulted in improved bioethanol yield, with a 50% increase in ethanol production in a vertical mass-flow type bioreactor, compared with that of the wild-type straw.

Rapid and label-free detection of the troponin in human serum by a TiN-based extended-gate field-effect transistor biosensor.

In this article, the TiN sensitive film as a sensing membrane was deposited onto n+-type Si substrate by a DC sputtering technique for extended-gate field-effect transistor (EGFET) pH sensors and detection of cardiac troponin-I (cTn-I) in the patient sera for the first time. The crystal structure, Raman spectrum, element profile, surface roughness, and surface morphology of the TiN sensitive film were characterized by X-ray diffraction, Raman spectroscopy, secondary ion mass spectroscopy, atomic force microscopy, and scanning electron microscopy, respectively. The sensing performance of the TiN sensitive film is correlated with its relative structural feature. A high sensitivity of 57.49 mV/pH, a small hysteresis voltage of ∼1 mV, and a low drift rate of 0.31 mV/h were obtained in the TiN sensitive film. In addition, the pH sensitivity of this TiN EGFET sensor was preserved approximately 57 mV/pH after operation time of 180 days. Subsequently, the cTn-I antibodies with carboxyl groups activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) along with N-hydroxysuccinimide (NHS) were immobilized on the TiN sensitive film functionalizing with 3-aminopropyl triethoxysilane (APTES). After obtaining the successful immobilization of cTn-I antibodies on the TiN EGFET biosensor, the cTn-I antigen specifically binds with its relative antibody. The cTn-I EGFET biosensor showed a high sensitivity of 21.88 mV/pCcTn-I in a wide dynamic range of 0.01-100 ng/mL. Furthermore, the concentrations of cTn-I in patient sera measured by our TiN EGFET biosensors are comparable to those determined by commercial enzyme-linked immuno-sorbent assay kits.

Epstein-Barr virus nuclear antigen 2 disrupts mitotic checkpoint and causes chromosomal instability.

The Epstein-Barr virus nuclear antigen 2 (EBNA2) plays a key role in transformation of B-lymphocytes mediated by Epstein-Barr virus (EBV) and can induce tumor formation in transgenic mice. However, the precise mechanism underlying EBNA2-mediated tumorigenesis remains elusive. Here, we report that EBNA2 can compromise mitotic spindle checkpoint (MSC) induced by the spindle inhibitor nocodazole and cause chromosomal instability (CIN) in HEp-2, U2-OS and BJAB cells. When EBNA2-expressing cells were treated with nocodazole, they exited mitosis prematurely and initiated another round of DNA synthesis. Nucleolocalization of EBNA2 was essential for EBNA2 to compromise MSC and to cause CIN. The metaphase chromosome spread data indicated that the EBNA2-expressing U2-OS cells showed a more heterogenous chromosome number distribution than the vector-transfected and parental cells. The median chromosome number for EBNA2-expressing, vector-transfected and parental U2-OS cells is 75, 65 and 64, respectively. EBNA2 was shown to be able to downregulate mitotic arrest deficient 2 (MAD2) approximately 2- to 3-fold and upregulate polo-like kinase 1 (PLK1) approximately 2-fold. The dysregulation of MAD2 and PLK1 may lead to activation of anaphase promoting complex/cyclosome and premature degradation of securin. Indeed, we found that when MSC was induced by nocodazole, securin was prematurely degraded in EBNA2-expressing cells. Finally, we show that EBNA2 could induce micronuclei and multinuclei formation in HEp-2 and U2-OS cells. Together, these studies reveal a new function of EBNA2 in cell-cycle regulation and may shed light on the role of EBNA2 in EBV-mediated tumorigenesis.

High glucose-induced apoptosis in human vascular endothelial cells is mediated through NF-kappaB and c-Jun NH2-terminal kinase pathway and prevented by PI3K/Akt/eNOS pathway.

Our previous studies demonstrated that high glucose-induced apoptosis in human umbilical vein endothelial cells (HUVECs) is mediated by sequential activation of c-Jun N-terminal kinase (JNK) and caspase, and prevented by exogenous nitric oxide (NO). In this study we further elucidated the roles of the transcriptional factor NF-kappaB, phosphatidylinositol 3'-kinase (PI3K), Akt and endothelial nitric oxide synthase (eNOS) in the apoptosis of HUVECs induced by high glucose. The results showed that high glucose-induced apoptosis was significantly enhanced by PI3K inhibitors (wortmannin and LY294002), NOS inhibitor (NG-nitro-arginine methyl ester) and eNOS antisense oligonucleotide. In contrast, apoptosis was markedly reduced by NF-kappaB inhibitor (pyrrolidine dithiocarbamate, PDTC), NF-kappaB antisense oligonucleotide, NO donor (sodium nitroprusside, SNP), and overexpression of Akt. The high glucose-induced NF-kappaB activation and transient Akt phosphorylation were prevented by the presence of vitamin C. Moreover, high glucose-induced increase in eNOS expression was attenuated by PI3K inhibitors and the negative mutant of PI3K. The activity of JNK induced by high glucose was suppressed by NF-kappaB-specific antisense oligonucleotide. Taken together our results demonstrated that high glucose-induced HUVECs apoptosis is through NF-kappaB-dependent JNK activation and reactive oxygen species (ROS)-dependent Akt dephosphorylation. Activation of the ROS/PI3K/Akt/eNOS signaling pathway in early phase exerts protective effects against the induction of apoptosis by high glucose.

The anti-inflammatory carbazole, LCY-2-CHO, inhibits lipopolysaccharide-induced inflammatory mediator expression through inhibition of the p38 mitogen-activated protein kinase signaling pathway in macrophages.

1. The present study was undertaken to investigate the anti-inflammatory effects of a synthetic compound, LCY-2-CHO, on the expression of inducible nitric oxide synthase (iNOS), COX-2, and TNF-alpha in murine RAW264.7 macrophages. 2. Within 1-30 microm, LCY-2-CHO concentration-dependently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E(2) (PGE(2)), and tumor necrosis factor-alpha (TNF-alpha) formation, with IC(50) values of 2.3, 1, and 0.8 microm, respectively. Accompanying inhibition of LPS-induced iNOS, cyclooxygenase-2 (COX-2), and pro-TNF-alpha proteins was observed. 3. Reverse transcription-polymerase chain reaction (RT-PCR) and promoter analyses indicated that iNOS expression was inhibited at the transcriptional level (IC(50)=2.3 microm), that inhibition of COX-2 expression only partially depended on gene transcription (IC(50)=7.6 microm), and that TNF-alpha transcription was unaffected. 4. Transcriptional assays revealed that activation of AP-1, but not NF-kappaB, was concomitantly blocked by LCY-2-CHO. Our results showed that LCY-2-CHO was capable of interfering with post-transcriptional regulation, altering the stability of COX-2 and TNF-alpha mRNAs. 5. Since the 3'-untranslated region (3' UTR) of both COX-2 and TNF-alpha mRNA contains a p38 mitogen-activated protein kinase (MAPK)-regulated element involved in mRNA stability, we assessed the effect of LCY-2-CHO on p38 MAPK. Our data clearly indicated an inhibition (IC(50)=1.7 microm) of LPS-mediated p38 MAPK activity, but not of extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) activity. However, kinase assays ruled out a direct inhibition of p38 MAPK action. The selective p38 MAPK inhibitor, SB203580, inhibited the promoter activities of iNOS and COX-2 rather than that of TNF-alpha. 6. In conclusion, LCY-2-CHO downregulates inflammatory iNOS, COX-2, and TNF-alpha gene expression in macrophages through interfering with p38 MAPK and AP-1 activation.