Toxicity assessment of modified Cry1Ac1 proteins and genetically modified insect-resistant Agb0101 rice

Insect-resistant Agb0101 rice was recently developed by modifying the cry1ac1 gene by changing codon usage changes relative to the native truncated cry1ac1 gene. To assess the toxicity of genetically modified Agb0101 rice, we conducted bioinfomational comparisons of the amino acid sequences that are not similar to known toxic proteins. Sufficient quantities of mCry1Ac1 protein were produced in Escherichia coli for in vitro evaluation and animal study. We compared the amino acid sequences and molecular mass. There have the same amino acid sequences and molecular masses after purifying the modified Cry1Ac1 (mCry1Ac1) protein from highly expressed bacteria and genetically modified rice were identical. We also investigated the acute and 90-days oral toxicities. No adverse effects were observed in mice following acute oral exposure to 2,000 mg/ kg body weight mCry1Ac1 protein of body weight and 90 days oral exposure to Agb0101. These results indicate that mCry1Ac1 proteins and Agb0101 rice demonstrate no adverse effects in these tests when applied via gavage and feed, respectively.Key words: Modified Cry1Ac1, food safety assessment, toxicity, insect- resistant rice Agb0101

Effects of pharmacological gap junction and sodium channel blockade on S1S2 restitution properties in Langendorff-perfused mouse hearts

Gap junctions and sodium channels are the major molecular determinants of normal and abnormal electrical conduction through the myocardium, however, their exact contributions to arrhythmogenesis are unclear. We examined conduction and recovery properties of regular (S1) and extrasystolic (S2) action potentials (APs), S1S2 restitution and ventricular arrhythmogenicity using the gap junction and sodium channel inhibitor heptanol (2 mM) in Langendorff-perfused mouse hearts (n=10). Monophasic action potential recordings obtained during S1S2 pacing showed that heptanol increased the proportion of hearts showing inducible ventricular tachycardia (0/10 vs. 5/8 hearts (Fisher’s exact test, P < 0.05), prolonged activation latencies of S1 and S2 APs, thereby decreasing S2/S1 activation latency ratio (ANOVA, P < 0.05) despite prolonged ventricular effective refractory period (VERP). It did not alter S1 action potential duration at 90% repolarization (APD90) but prolonged S2 APD90 (P < 0.05), thereby increasing S2/S1 APD90 ratio (P < 0.05). It did not alter maximum conduction velocity (CV) restitution gradient or maximum CV reductions but decreased the restitution time constant (P < 0.05). It increased maximal APD90 restitution gradient (P < 0.05) without altering critical diastolic interval or maximum APD90 reductions. Pro-arrhythmic effects of 2 mM heptanol are explicable by delayed conduction and abnormal electrical restitution. We concluded that gap junctions modulated via heptanol (0.05 mM) increased arrhythmogenicity through a delay in conduction, while sodium channel inhibition by a higher concentration of heptanol (2 mM) increased arrhythmogenicity via additional mechanisms, such as abnormalities in APDs and CV restitution.GT received research funding from the BBSRC for this research and is currently supported by a Clinical Assistant Professorship from the Croucher Foundation of Hong Kong. WTW is supported by the Direct Grant for Research from the Research Committee of the Chinese University of Hong Kong, China

Effects of pharmacological gap junction and sodium channel blockade on S1S2 restitution properties in Langendorff-perfused mouse hearts

Gap junctions and sodium channels are the major molecular determinants of normal and abnormal electrical conduction through the myocardium, however, their exact contributions to arrhythmogenesis are unclear. We examined conduction and recovery properties of regular (S1) and extrasystolic (S2) action potentials (APs), S1S2 restitution and ventricular arrhythmogenicity using the gap junction and sodium channel inhibitor heptanol (2 mM) in Langendorff-perfused mouse hearts (n=10). Monophasic action potential recordings obtained during S1S2 pacing showed that heptanol increased the proportion of hearts showing inducible ventricular tachycardia (0/10 vs. 5/8 hearts (Fisher’s exact test, P < 0.05), prolonged activation latencies of S1 and S2 APs, thereby decreasing S2/S1 activation latency ratio (ANOVA, P < 0.05) despite prolonged ventricular effective refractory period (VERP). It did not alter S1 action potential duration at 90% repolarization (APD90) but prolonged S2 APD90 (P < 0.05), thereby increasing S2/S1 APD90 ratio (P < 0.05). It did not alter maximum conduction velocity (CV) restitution gradient or maximum CV reductions but decreased the restitution time constant (P < 0.05). It increased maximal APD90 restitution gradient (P < 0.05) without altering critical diastolic interval or maximum APD90 reductions. Pro-arrhythmic effects of 2 mM heptanol are explicable by delayed conduction and abnormal electrical restitution. We concluded that gap junctions modulated via heptanol (0.05 mM) increased arrhythmogenicity through a delay in conduction, while sodium channel inhibition by a higher concentration of heptanol (2 mM) increased arrhythmogenicity via additional mechanisms, such as abnormalities in APDs and CV restitution.GT received research funding from the BBSRC for this research and is currently supported by a Clinical Assistant Professorship from the Croucher Foundation of Hong Kong. WTW is supported by the Direct Grant for Research from the Research Committee of the Chinese University of Hong Kong, China

Eastern asian expert panel opinion: designing clinical trials of molecular targeted therapy for hepatocellular carcinoma

The largest burden of hepatocellular carcinoma (HCC) lies in Asia, secondary to hepatitis B virus (HBV) infection. Improved survival with sorafenib has fostered new research but many challenges remain in designing clinical trials. The disease, its management, and populations affected by it are heterogeneous worldwide and within Asia. An expert conference of Eastern Asian oncologists and hepatologists was convened to foster consensus in clinical trial design. The panel identified key areas that need to be addressed to facilitate clinical trials in Asia. Stratification by viral etiology is desirable within Asia and by region in global trials. Antiviral therapy should also be considered as a stratification factor and incorporated into HCC management in trials. The panel agreed that histological diagnosis is not required for trial entry and that Barcelona-Clinic Liver Cancer (BCLC) staging is acceptable for trials as long as portal hypertension can be better defined with standardized methodology. Consensus in treatment must be sought to allow multi-national trials and it must be recognized that first-line sorafenib is not largely feasible in Asia. Finally, Asian nations must be urged to participate in clinical trials, many of which are ongoing, to advance new treatment options in this challenging disease

Glucuronidation by UGT1A1 Is the Dominant Pathway of the Metabolic Disposition of Belinostat in Liver Cancer Patients

10.1371/journal.pone.0054522PLoS ONE81

Differential Effects of HIF-1 Inhibition by YC-1 on the Overall Outcome and Blood-Brain Barrier Damage in a Rat Model of Ischemic Stroke

Hypoxia-inducible factor 1 (HIF-1) is a master regulator of cellular adaptation to hypoxia and has been suggested as a potent therapeutic target in cerebral ischemia. Here we show in an ischemic stroke model of rats that inhibiting HIF-1 and its downstream genes by 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) significantly increases mortality and enlarges infarct volume evaluated by MRI and histological staining. Interestingly, the HIF-1 inhibition remarkably ameliorates ischemia-induced blood-brain barrier (BBB) disruption determined by Evans blue leakage although it does not affect brain edema. The result demonstrates that HIF-1 inhibition has differential effects on ischemic outcomes and BBB permeability. It indicates that HIF-1 may have different functions in different brain cells. Further analyses show that ischemia upregulates HIF-1 and its downstream genes erythropoietin (EPO), vascular endothelial growth factor (VEGF), and glucose transporter (Glut) in neurons and brain endothelial cells and that YC-1 inhibits their expression. We postulate that HIF-1-induced VEGF increases BBB permeability while certain other proteins coded by HIF-1's downstream genes such as epo and glut provide neuroprotection in an ischemic brain. The results indicate that YC-1 lacks the potential as a cerebral ischemic treatment although it confers certain protection to the cerebral vascular system

Epigenetic modification of histone 3 at lysine 9 in sheep zygotes and its relationship with DNA methylation

<p>Abstract</p> <p>Background</p> <p>Previous studies indicated that, unlike mouse zygotes, sheep zygotes lacked the paternal DNA demethylation event. Another epigenetic mark, histone modification, especially at lysine 9 of histone 3 (H3K9), has been suggested to be mechanically linked to DNA methylation. In mouse zygotes, the absence of methylated H3K9 from the paternal pronucleus has been thought to attribute to the paternal DNA demethylation.</p> <p>Results</p> <p>By using the immunofluorescence staining approach, we show that, despite the difference in DNA methylation, modification of H3K9 is similar between the sheep and mouse zygotes. In both species, H3K9 is hyperacetylated or hypomethylated in paternal pronucleus relative to maternal pronucleus. In fact, sheep zygotes can also undergo paternal DNA demethylation, although to a less extent than the mouse. Further examinations of individual zygotes by double immunostaining revealed that, the paternal levels of DNA methylation were not closely associated with that of H3K9 acetylation or tri-methylation. Treatment of either 5-azacytidine or Trichostatin A did not induce a significant decrease of paternal DNA methylation levels.</p> <p>Conclusion</p> <p>Our results suggest that in sheep lower DNA demethylation of paternal genomes is not due to the H3K9 modification and the methylated DNA sustaining in paternal pronucleus does not come from DNA <it>de novo </it>methylation.</p

Induction of Bcl-2 Expression by Hepatitis B Virus Pre-S2 Mutant Large Surface Protein Resistance to 5-Fluorouracil Treatment in Huh-7 Cells

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Our previous studies have indicated that expression of Hepatitis B virus pre-S2 large mutant surface antigen (HBV pre-S2Δ) is associated with a significant risk of developing HCC. However, the relationship between HBV pre-S2Δ protein and the resistance of chemotherapeutic drug treatment is still unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that the expression of HBV pre-S2Δ mutant surface protein in Huh-7 cell significantly promoted cell growth and colony formation. Furthermore, HBV pre-S2Δ protein increased both mRNA (2.7±0.5-fold vs. vehicle, p=0.05) and protein (3.2±0.3-fold vs. vehicle, p=0.01) levels of Bcl-2 in Huh-7 cells. HBV pre-S2Δ protein also enhances Bcl-2 family, Bcl-xL and Mcl-1, expression in Huh-7 cells. Meanwhile, induction of NF-κB p65, ERK, and Akt phosphorylation, and GRP78 expression, an unfolded protein response chaperone, were observed in HBV pre-S2Δ and HBV pre-S-expressing cells. Induction of Bcl-2 expression by HBV pre-S2Δ protein resulted in resistance to 5-fluorouracil treatment in colony formation, caspase-3 assay, and cell apoptosis, and can enhance cell death by co-incubation with Bcl-2 inhibitor. Similarly, transgenic mice showed higher expression of Bcl-2 in liver tissue expressing HBV pre-S2Δ large surface protein in vivo. CONCLUSION/SIGNIFICANCE: Our result demonstrates that HBV pre-S2Δ increased Bcl-2 expression which plays an important role in resistance to 5-fluorouracil-caused cell death. Therefore, these data provide an important chemotherapeutic strategy in HBV pre-S2Δ-associated tumor

A Transcriptional “Scream” Early Response of E. coli Prey to Predatory Invasion by Bdellovibrio

We have transcriptionally profiled the genes differentially expressed in E. coli prey cells when predatorily attacked by Bdellovibrio bacteriovorus just prior to prey cell killing. This is a brief, approximately 20–25 min period when the prey cell is still alive but contains a Bdellovibrio cell in its periplasm or attached to and penetrating its outer membrane. Total RNA was harvested and labelled 15 min after initiating a semi-synchronous infection with an excess of Bdellovibrio preying upon E. coli and hybridised to a macroarray spotted with all predicted ORFs of E. coli. SAM analysis and t-tests were performed on the resulting data and 126 E. coli genes were found to be significantly differentially regulated by the prey upon attack by Bdellovibrio. The results were confirmed by QRT-PCR. Amongst the prey genes upregulated were a variety of general stress response genes, potentially “selfish” genes within or near prophages and transposable elements, and genes responding to damage in the periplasm and osmotic stress. Essentially, the presence of the invading Bdellovibrio and the resulting damage to the prey cell elicited a small “transcriptional scream”, but seemingly no specific defensive mechanism with which to counter the Bdellovibrio attack. This supports other studies which do not find Bdellovibrio resistance responses in prey, and bodes well for its use as a “living antibiotic”