Investigating the viability of yeast expressing histone from extrachromosomal DNA : progress in construction of a plasmid bearing one copy of each yeast histone gene

Much of the current information on histones has been based on in vitro studies. The original goal of this research was to construct a mutant strain of Saccharomyces cerevisiae containing all chromosomal copies of the yeast histone genes made nonfunctional. This project would demonstrate whether such a strain could be rescued by a plasmid carrying the wild-type copies of the four core histone genes. Furthermore, this yeast strain construct would allow future investigations to take advantage of histone mutant analysis in vivo. The critical step in beginning this work was the construction of a plasmid which contained the genes coding for histones H2A, H2B, H3, and H4. Construction of this plasmid proved difficult to complete due to such problems as using a nontransformable bacterial single colony, utilizing a plasmid which was subsequently found to be in fact another plasmid. This thesis describes specific methods used for transformation, partial digestion and DNA recovery techniques which were attempted and the results of these attempts. The approaches developed should simplify future construction of the plasmid and contribute to further studies investigating the viability of yeast using histone expressed from extrachromosomal DNA

Dual mode laser velocimeter

Described is a laser Doppler velocimeter (LDV) which is capable of operating with a small focus diameter for analyzing fluid flows at low velocity with high spatial resolution, or with a larger focus diameter to measure fluid flows at higher velocities accurately. More particularly, this is an LDV in which a simple reversal of a lens pair will allow it to operate in the two focus diameter modes

SARS-CoV and emergent coronaviruses: viral determinants of interspecies transmission

Most new emerging viruses are derived from strains circulating in zoonotic reservoirs. Coronaviruses, which had an established potential for cross-species transmission within domesticated animals, suddenly became relevant with the unexpected emergence of the highly pathogenic human SARS-CoV strain from zoonotic reservoirs in 2002. SARS-CoV infected approximately 8000 people worldwide before public health measures halted the epidemic. Supported by robust time-ordered sequence variation, structural biology, well-characterized patient pools, and biological data, the emergence of SARS-CoV represents one of the best studied natural models of viral disease emergence from zoonotic sources. This review article summarizes previous and more recent advances into the molecular and structural characteristics, with particular emphasis on host-receptor interactions, that drove this remarkable virus disease outbreak in human populations

A decade after SARS: strategies for controlling emerging coronaviruses

Two novel coronaviruses have emerged in humans in the 21st century, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome human coronavirus (MERS-CoV), both of which cause acute respiratory distress syndrome (ARDS) and have high mortality rates. There are no clinically approved vaccines or antiviral drugs available for either of these infections; thus, a priority in the field is the development of effective therapeutic and preventive strategies that can be readily applied to new emergent strains. This review will: describe the emergence and identification of novel human coronaviruses over the last 10 years; review their key biological features, including tropism and receptor use; and summarize approaches to develop broadly effective vaccines

A response-initiated differential reinforcement of low rates schedule of reinforcement

The purpose of the present research was to study the effects of timing and motivational variables on a two-response DRL schedule. The primary question was whether or not timing behavior and the response-strengthening effects of reinforcement are confounded in DRL performance. The subjects were albino rats maintained at 80% of their free-feeding body weights, 8nd the reinforcing event was dipper presentation of a sucrose solution. A procedure was used in which two responses were required for the reinforcing event to occur. The minimum latency contingency was imposed only on the latency of the second response. When the DRL value was varied the timing interval matched the DRL requirement more closely than in one-response DRL performance. When the subjects were pre-fed prior to the sessions to decrease the severity of deprivation, the frequency of timing behavior decreased tut the accuracy of the timing intervals remained unaffected. In extinction the accuracy of timing was maintained even as the frequency of the timing behavior reached zero

Comparing and Contrasting a Program versus System Approach to Evaluation: The Example of a Cardiac Care System

This paper focuses on the application of systems thinking concepts to evaluate systems.  The terms systems and systems thinking concepts are first defined.  The use of systems thinking concepts in program and system evaluation are then highlighted.  It is noted that while there are methods available to assist evaluation practitioners in applying systems thinking concepts to program evaluation, there is a need for similar guidance in applying systems thinking concepts to evaluating systems.  System Evaluation Theory (SET) is then reviewed as one evaluation theory designed to apply systems thinking concepts to evaluating systems. A case illustration is presented to help teach practitioners how to apply SET’s three steps.  The discussion focuses on comparing the differences between evaluation questions answered by applying systems thinking concepts versus those using program logic models

Each of the Eight Simian Hemorrhagic Fever Virus Minor Structural Proteins is Functionally Important

The simian hemorrhagic fever virus (SHFV) genome differs from those of other members of the family Arterivirus in encoding two adjacent sets of four minor structural protein open reading frames (ORFs). A stable, full-length, infectious SHFV-LVR cDNA clone was constructed. Virus produced from this clone had replication characteristics similar to those of the parental virus. A subgenomic mRNA was identified for the SHFV ORF previously identified as 2b. As an initial means of analyzing the functional relevance of each of the SHFV minor structural proteins, a set of mutant infectious clones was generated, each with the start codon of one minor structural protein ORF mutated. Different phenotypes were observed for each ortholog of the pairs of minor glycoproteins and all of the eight minor structural proteins were required for the production of infectious extracellular virus indicating that the duplicated sets of SHFV minor structural proteins are not functionally redundant

Escape from Human Monoclonal Antibody Neutralization Affects In Vitro and In Vivo Fitness of Severe Acute Respiratory Syndrome Coronavirus

Severe Acute Respiratory Syndrome (SARS) emerged as a human disease in 2002 and detailed phylogenetic analysis and epidemiological studies have suggested that the SARS-Coronavirus (SARS-CoV) originated from animals. The Spike (S) glycoprotein has been identified as a major target of protective immunity and contains at least three regions that are targeted by neutralizing antibodies in the S1 and S2 domains. We previously characterized a panel of neutralizing human monoclonal antibodies (MAbs) but the majority of epitopes recognized by the MAbs remained unknown

Increased Antibody Affinity Confers Broad In Vitro Protection against Escape Mutants of Severe Acute Respiratory Syndrome Coronavirus

Even though the effect of antibody affinity on neutralization potency is well documented, surprisingly, its impact on neutralization breadth and escape has not been systematically determined. Here, random mutagenesis and DNA shuffling of the single-chain variable fragment of the neutralizing antibody 80R followed by bacterial display screening using anchored periplasmic expression (APEx) were used to generate a number of higher-affinity variants of the severe acute respiratory syndrome coronavirus (SARS-CoV)-neutralizing antibody 80R with equilibrium dissociation constants (KD) as low as 37 pM, a >270-fold improvement relative to that of the parental 80R single-chain variable fragment (scFv). As expected, antigen affinity was shown to correlate directly with neutralization potency toward the icUrbani strain of SARS-CoV. Additionally, the highest-affinity antibody fragment displayed 10-fold-increased broad neutralization in vitro and completely protected against several SARS-CoV strains containing substitutions associated with antibody escape. Importantly, higher affinity also led to the suppression of viral escape mutants in vitro. Escape from the highest-affinity variant required reduced selective pressure and multiple substitutions in the binding epitope. Collectively, these results support the hypothesis that engineered antibodies with picomolar dissociation constants for a neutralizing epitope can confer escape-resistant protection

Severe Acute Respiratory Syndrome Coronavirus nsp9 Dimerization Is Essential for Efficient Viral Growth

The severe acute respiratory syndrome coronavirus (SARS-CoV) devotes a significant portion of its genome to producing nonstructural proteins required for viral replication. SARS-CoV nonstructural protein 9 (nsp9) was identified as an essential protein with RNA/DNA-binding activity, and yet its biological function within the replication complex remains unknown. Nsp9 forms a dimer through the interaction of parallel α-helices containing the protein-protein interaction motif GXXXG. In order to study the role of the nsp9 dimer in viral reproduction, residues G100 and G104 at the helix interface were targeted for mutation. Multi-angle light scattering measurements indicated that G100E, G104E, and G104V mutants are monomeric in solution, thereby disrupting the dimer. However, electrophoretic mobility assays revealed that the mutants bound RNA with similar affinity. Further experiments using fluorescence anisotropy showed a 10-fold reduction in RNA binding in the G100E and G104E mutants, whereas the G104V mutant had only a 4-fold reduction. The structure of G104E nsp9 was determined to 2.6-Å resolution, revealing significant changes at the dimer interface. The nsp9 mutations were introduced into SARS-CoV using a reverse genetics approach, and the G100E and G104E mutations were found to be lethal to the virus. The G104V mutant produced highly debilitated virus and eventually reverted back to the wild-type protein sequence through a codon transversion. Together, these data indicate that dimerization of SARS-CoV nsp9 at the GXXXG motif is not critical for RNA binding but is necessary for viral replication