Tyrosine, Cysteine, and S-Adenosyl Methionine Stimulate In Vitro [FeFe] Hydrogenase Activation

Background: [FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H2. These proteins are distinguished by an active site cofactor known as the H-cluster. This unique [6Fe–6S] complex contains multiple non-protein moieties and requires several maturation enzymes for its assembly. The pathways and biochemical precursors for H-cluster biosynthesis have yet to be elucidated. Principal Findings: We report an in vitro maturation system in which, for the first time, chemical additives enhance [FeFe] hydrogenase activation, thus signifying in situ H-cluster biosynthesis. The maturation system is comprised of purified hydrogenase apoprotein; a dialyzed Escherichia coli cell lysate containing heterologous HydE, HydF, and HydG maturases; and exogenous small molecules. Following anaerobic incubation of the Chlamydomonas reinhardtii HydA1 apohydrogenase with S-adenosyl methionine (SAM), cysteine, tyrosine, iron, sulfide, and the non-purified maturases, hydrogenase activity increased 5-fold relative to incubations without the exogenous substrates. No conditions were identified in which addition of guanosine triphosphate (GTP) improved hydrogenase maturation. Significance: The in vitro system allows for direct investigation of [FeFe] hydrogenase activation. This work also provides a foundation for studying the biosynthetic mechanisms of H-cluster biosynthesis using solely purified enzymes and chemical additives

Institutions and policies to implement the Tanzania livestock master plan

Bill & Melinda Gates Foundatio

Senior Theses: Department of Physical Sciences

1993 Fall Semester Senior Theses for the class Physical Science 471: Petrologic Classification of Igneous and Metamorphic Rocks by Frank Baldridge X -Ray Analysis of Cave Sediments From Pigeon Water Cave of Northeastern Pine Mountain by Billy B. Stapleton The Correlation of Stream-deposited Breccias In Bat Cave, Carter Caves, Kentucky by James Bond Jointing and Faulting in Selected Areas of Eastern Kentucky by Mark A. Blai

Hypercholesterolemia and microvascular dysfunction: interventional strategies

Hypercholesterolemia is defined as excessively high plasma cholesterol levels, and is a strong risk factor for many negative cardiovascular events. Total cholesterol levels above 200 mg/dl have repeatedly been correlated as an independent risk factor for development of peripheral vascular (PVD) and coronary artery disease (CAD), and considerable attention has been directed toward evaluating mechanisms by which hypercholesterolemia may impact vascular outcomes; these include both results of direct cholesterol lowering therapies and alternative interventions for improving vascular function. With specific relevance to the microcirculation, it has been clearly demonstrated that evolution of hypercholesterolemia is associated with endothelial cell dysfunction, a near-complete abrogation in vascular nitric oxide bioavailability, elevated oxidant stress, and the creation of a strongly pro-inflammatory condition; symptoms which can culminate in profound impairments/alterations to vascular reactivity. Effective interventional treatments can be challenging as certain genetic risk factors simply cannot be ignored. However, some hypercholesterolemia treatment options that have become widely used, including pharmaceutical therapies which can decrease circulating cholesterol by preventing either its formation in the liver or its absorption in the intestine, also have pleiotropic effects with can directly improve peripheral vascular outcomes. While physical activity is known to decrease PVD/CAD risk factors, including obesity, psychological stress, impaired glycemic control, and hypertension, this will also increase circulating levels of high density lipoprotein and improving both cardiac and vascular function. This review will provide an overview of the mechanistic consequences of the predominant pharmaceutical interventions and chronic exercise to treat hypercholesterolemia through their impacts on chronic sub-acute inflammation, oxidative stress, and microvascular structure/function relationships

DNA recognition by the Salmonella enterica serovar Typhimurium transcription factor SlyA

The Salmonella regulatory protein, SlyA is implicated in virulence, survival in macrophages and resistance to oxidative stress and anti-microbial peptides.  SlyA is a member of the MarR family of winged-helix transcription factors. Systematic mutational analysis of the SlyA operator sequence and of the predicted DNA-binding region of SlyA shows that no single base pair in the palindromic SlyA operator sequence is essential for DNA binding, and identifies amino acid residues required to allow SlyA to recognise DNA. Combining the structure-function studies described here and elsewhere with the structures of MarR family proteins suggests a possible model for regulation of SlyA binding to DNA

Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach

Hepatitis A virus (HAV) is one of the most common causes of acute viral hepatitis in humans. Although HAV has a relatively small genome, there are several factors limiting whole genome sequencing such as PCR amplification artefacts and ambiguities in de novo assembly. The recently developed Oxford Nanopore technologies (ONT) allows single-molecule sequencing of long-size fragments of DNA or RNA using PCR-free strategies. We have sequenced the whole genome of HAV using a PCR-free approach by direct reverse-transcribed sequencing. We were able to sequence HAV cDNA and obtain reads over 7 kilobases in length containing almost the whole genome of the virus. The comparison of these raw long nanopore reads with the HAV reference wild type revealed a nucleotide sequence identity between 81.1 and 96.6%. By de novo assembly of all HAV reads we obtained a consensus sequence of 7362 bases, with a nucleotide sequence identity of 99.0% with the genome of the HAV strain pHM175/18f. When the assembly was performed using as reference the HAV strain pHM175/18f a consensus with a sequence similarity of 99.8 % was obtained. We have also used an ONT amplicon-based assay to sequence two fragments of the VP3 and VP1 regions which showed a sequence similarity of 100% with matching regions of the consensus sequence obtained using the direct cDNA sequencing approach. This study showed the applicability of ONT sequencing technologies to obtain the whole genome of HAV by direct cDNA nanopore sequencing, highlighting the utility of this PCR-free approach for HAV characterization and potentially other viruses of the Picornaviridae family

Impairment of Coronary Arteriolar Endothelium-Dependent Dilation after Multi-Walled Carbon Nanotube Inhalation: A Time-Course Study

Engineered nanomaterials have been developed for widespread applications due to many highly unique and desirable characteristics. The purpose of this study was to assess pulmonary inflammation and subepicardial arteriolar reactivity in response to multi-walled carbon nanotube (MWCNT) inhalation and evaluate the time course of vascular alterations. Rats were exposed to MWCNT aerosols producing pulmonary deposition. Pulmonary inflammation via bronchoalveolar lavage and MWCNT translocation from the lungs to systemic organs was evident 24 h post-inhalation. Coronary arterioles were evaluated 24–168 h post-exposure to determine microvascular response to changes in transmural pressure, endothelium-dependent and -independent reactivity. Myogenic responsiveness, vascular smooth muscle reactivity to nitric oxide, and α-adrenergic responses all remained intact. However, a severe impact on endothelium-dependent dilation was observed within 24 h after MWCNT inhalation, a condition which improved, but did not fully return to control after 168 h. In conclusion, results indicate that MWCNT inhalation not only leads to pulmonary inflammation and cytotoxicity at low lung burdens, but also a low level of particle translocation to systemic organs. MWCNT inhalation also leads to impairments of endothelium-dependent dilation in the coronary microcirculation within 24 h, a condition which does not fully dissipate within 168 h. The innovations within the field of nanotechnology, while exciting and novel, can only reach their full potential if toxicity is first properly assessed

Establishing Foundation Archives: A Reader and Guide to First Steps

This publication is an anthology of papers presented at a conference held at the Rockefeller Archive Center in January 1990, and sponsored by the Council on Foundations. This collaboration of the Archive Center and the Council provided a rare opponunity for foundations to learn both why preserving documents is imponant and how several foundations have approached finding a repository or setting up and managing an archives. Participants in the conference had the added privilege of conferring with experts and seeing an operating archive as they toured the Rockefeller Archive Center.Foundations are institutions that are shaping private initiatives for the public good, so documenting this aspect of American society falls uniquely under the stewardship of the organizations themselves. Foundation documents often provide the only surviving records of the important contributions of nonprofits and foundations to civic life. These records will help to inform future judgments and ensure that the history of the field is not lost. The publication of this volume was intended to make the information shared at the conference more widely available and to provide an entry point and a primer for foundations as they begin their records and archives journey

Development of an In Vitro Compartmentalization Screen for High-Throughput Directed Evolution of [FeFe] Hydrogenases

BACKGROUND: [FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library. CONCLUSIONS/SIGNIFICANCE: [FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy