Development of a System for Directed Evolution of \u3cem\u3eArabidopsis\u3c/em\u3e Formate Dehydrogenase to Utilize NADP as a Cofactor

Formate dehydrogenase (FDH) is a NAD-dependent enzyme found in methylotrophic bacteria, yeast and plants. This enzyme catalyzes the reversible oxidation of formate to carbon dioxide. The goal of this research was to determine the feasibility of using a directed evolution approach to generate an altered Arabidopsis FDH with a high affinity for NADP as a cofactor. A PCR procedure that induced approximately 1.5 mutations in the wild-type Arabidopsis FDH sequence per thousand base pairs was developed and the amplified products were transformed into E. coli cells. Approximately 1300 cell lines were assayed in 96-well microplates for activity with NADP+ and 100 putative mutants were selected for further study. One particular mutant line, pFDH-18, possessed reproducible NADP+-FDH activity. Sequence analysis showed that a single T in the wild-type DNA sequence had been changed to a G. The result of this mutation was that an isoleucine (Ile) residue at position 188 in the wild-type enzyme was converted to a methionine. This particular Ile residue is conserved in the known FDH sequences from higher plants and is located in the region of the enzyme that contains the binding domain for the NAD cofactor

GaAs on Si epitaxy by aspect ratio trapping: analysis and reduction of defects propagating along the trench direction

The Aspect Ratio Trapping technique has been extensively evaluated for improving the quality of III-V heteroepitaxial films grown on Si, due to the potential for terminating defects at the sidewalls of SiO2 patterned trenches that enclose the growth region. However, defects propagating along the trench direction cannot be effectively confined with this technique. We studied the effect of the trench bottom geometry on the density of defects of GaAs fins, grown by metal-organic chemical vapor deposition on 300 mm Si (001) wafers inside narrow (<90 nm wide) trenches. Plan view and cross sectional Scanning Electron Microscopy and Transmission Electron Microscopy, together with High Resolution X-Ray Diffraction, were used to evaluate the crystal quality of GaAs. The prevalent defects that reach the top surface of GaAs fins are {111} twin planes propagating along the trench direction. The lowest density of twin planes, 8 108 cm 2, was achieved on “V” shaped bottom trenches, where GaAs nucleation occurs only on {111} Si planes, minimizing the interfacial energy and preventing the formation of antiphase boundaries

The Loss of ATRX Increases Susceptibility to Pancreatic Injury and Oncogenic KRAS in Female But Not Male Mice

Female mice lacking ATRX in the pancreas have increased sensitivity to pancreatic cancer, whereas male mice without ATRX are protected. This study identifies such susceptibility in pancreatic cancer and highlights the need for sex-specific approaches in cancer treatment. BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in North America, accounting for \u3e30,000 deaths annually. Although somatic activating mutations in KRAS appear in 97% of PDAC patients, additional factors are required to initiate PDAC. Because mutations in genes encoding chromatin remodelling proteins have been implicated in KRAS-mediated PDAC, we investigated whether loss of chromatin remodeler.-thalassemia, mental-retardation, X-linked (ATRX) affects oncogenic KRAS\u27s ability to promote PDAC. ATRX affects DNA replication, repair, and gene expression and is implicated in other cancers including glioblastomas and pancreatic neuroendocrine tumors. The hypothesis was that deletion of Atrx in pancreatic acinar cells will increase susceptibility to injury and oncogenic METHODS: Mice allowing conditional loss of Atrx within pancreatic acinar cells were examined after induction of recurrent cerulein-induced pancreatitis or oncogenic KRAS (KRASG12D). Histologic, biochemical, and molecular analysis examined pancreatic pathologies up to 2 months after induction of Atrx deletion. RESULTS: Mice lacking Atrx showed more progressive damage, inflammation, and acinar-to-duct cell metaplasia in response to injury relative to wild-type mice. In combination with KRASG12D, Atrx-deficient acinar cells showed increased fibrosis, inflammation, progression to acinar-to-duct cell metaplasia, and pre-cancerous lesions relative to mice expressing only KRASG12D. This sensitivity appears only in female mice, mimicking a significant prevalence of ATRX mutations in human female PDAC patients. CONCLUSIONS: Our results indicate the absence of ATRX increases sensitivity to injury and oncogenic KRAS only in female mice. This is an instance of a sex-specific mutation that enhances oncogenic KRAS\u27s ability to promote pancreatic intraepithelial lesion formation

G-Quadruplex Dynamics Contribute To Regulation Of Mitochondrial Gene Expression

Single-stranded DNA or RNA sequences rich in guanine (G) can adopt non-canonical structures known as G-quadruplexes (G4). Mitochondrial DNA (mtDNA) sequences that are predicted to form G4 are enriched on the heavy-strand and have been associated with formation of deletion breakpoints. Increasing evidence supports the ability of mtDNA to form G4 in cancer cells; however, the functional roles of G4 structures in regulating mitochondrial nucleic acid homeostasis in non-cancerous cells remain unclear. Here, we demonstrate by live cell imaging that the G4-ligand RHPS4 localizes primarily to mitochondria at low doses. We find that low doses of RHPS4 do not induce a nuclear DNA damage response but do cause an acute inhibition of mitochondrial transcript elongation, leading to respiratory complex depletion. We also observe that RHPS4 interferes with mtDNA levels or synthesis both in cells and isolated mitochondria. Importantly, a mtDNA variant that increases G4 stability and anti-parallel G4-forming character shows a stronger respiratory defect in response to RHPS4, supporting the conclusion that mitochondrial sensitivity to RHPS4 is G4-mediated. Taken together, our results indicate a direct role for G4 perturbation in mitochondrial genome replication, transcription processivity, and respiratory function in normal cells

Erosion Processes of the Discharge Cathode Assembly of Ring-Cusp Gridded Ion Thrusters

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/77019/1/AIAA-2006-3558-252.pd

Drug-gene interactions of antihypertensive medications and risk of incident cardiovascular disease: a pharmacogenomics study from the CHARGE consortium

Background Hypertension is a major risk factor for a spectrum of cardiovascular diseases (CVD), including myocardial infarction, sudden death, and stroke. In the US, over 65 million people have high blood pressure and a large proportion of these individuals are prescribed antihypertensive medications. Although large long-term clinical trials conducted in the last several decades have identified a number of effective antihypertensive treatments that reduce the risk of future clinical complications, responses to therapy and protection from cardiovascular events vary among individuals. Methods Using a genome-wide association study among 21,267 participants with pharmaceutically treated hypertension, we explored the hypothesis that genetic variants might influence or modify the effectiveness of common antihypertensive therapies on the risk of major cardiovascular outcomes. The classes of drug treatments included angiotensin-converting enzyme inhibitors, beta-blockers, calcium channel blockers, and diuretics. In the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium, each study performed array-based genome-wide genotyping, imputed to HapMap Phase II reference panels, and used additive genetic models in proportional hazards or logistic regression models to evaluate drug-gene interactions for each of four therapeutic drug classes. We used meta-analysis to combine study-specific interaction estimates for approximately 2 million single nucleotide polymorphisms (SNPs) in a discovery analysis among 15,375 European Ancestry participants (3,527 CVD cases) with targeted follow-up in a case-only study of 1,751 European Ancestry GenHAT participants as well as among 4,141 African-Americans (1,267 CVD cases). Results Although drug-SNP interactions were biologically plausible, exposures and outcomes were well measured, and power was sufficient to detect modest interactions, we did not identify any statistically significant interactions from the four antihypertensive therapy meta-analyses (Pinteraction &gt; 5.0×10−8). Similarly, findings were null for meta-analyses restricted to 66 SNPs with significant main effects on coronary artery disease or blood pressure from large published genome-wide association studies (Pinteraction ≥ 0.01). Our results suggest that there are no major pharmacogenetic influences of common SNPs on the relationship between blood pressure medications and the risk of incident CVD