The role of genotype in Saccharomyces cerevisiae resistance to 4-methylcyclohexanemethanol

The chemical 4-methylcyclohexanemethanol (MCHM) spilled in the Elk River near Charleston, WV in January 2014, impacting the drinking water of 300,000 residents. Initial studies on MCHM following the spill attempted to characterize its toxicological effects in animals, but largely ignored characterization of its effects on cellular pathways and metabolism. In this study, I used this chemical as a novel stressor for the model eukaryote S. cerevisiae to explore the relationship of genotype and stress phenotypes. Initial exploration characterized the stress phenotype of MCHM in yeast through growth assays, cell cycle analysis, and transcriptomic data. MCHM exposure caused cells to arrest growth in G1, activating a well characterized yeast process called the environmental stress response. Further exploration was carried out using a genetic screen of approximately 5000 haploid gene knockout strains, which combined with the transcriptomic data, revealed that the causes of the stress response in yeast were nutrient deprivation related to amino acid biosynthesis and reactive oxygen species production. A QTL analysis of standing variation between two parental strains with variable resistance uncovered the role of zinc homeostasis and its interaction with the hydrotrope chemical properties of MCHM in protein aggregation as a contributor to resistant phenotypes. Finally, an In-Lab evolution study to produce resistant strains for variant analysis showed mutations in the pleiotropic drug response transcription factor PDR3 cause a reproducible induction of MCHM resistance. These studies combined to characterize cellular changes from MCHM, identify genes required for tolerance, and explore both standing and evolved variation in genotypes that contribute to and produce an MCHM resistant phenotype

Oxidative Stress Responses and Nutrient Starvation in MCHM Treated Saccharomyces cerevisiae

In 2014, the coal cleaning chemical 4-methylcyclohexane methanol (MCHM) spilled into the water supply for 300,000 West Virginians. Initial toxicology tests showed relatively mild results, but the underlying effects on cellular biology were underexplored. Treated wildtype yeast cells grew poorly, but there was only a small decrease in cell viability. Cell cycle analysis revealed an absence of cells in S phase within thirty minutes of treatment. Cells accumulated in G1 over a six-hour time course, indicating arrest instead of death. A genetic screen of the haploid knockout collection revealed 329 high confidence genes required for optimal growth in MCHM. These genes encode three major cell processes: mitochondrial gene expression/translation, the vacuolar ATPase, and aromatic amino acid biosynthesis. The transcriptome showed an upregulation of pleiotropic drug response genes and amino acid biosynthetic genes and downregulation in ribosome biosynthesis. Analysis of these datasets pointed to environmental stress response activation upon treatment. Overlap in datasets included the aromatic amino acid genes ARO1, ARO3, and four of the five TRP genes. This implicated nutrient deprivation as the signal for stress response. Excess supplementation of nutrients and amino acids did not improve growth on MCHM, so the source of nutrient deprivation signal is still unclear. Reactive oxygen species and DNA damage were directly detected with MCHM treatment, but timepoints showed these accumulated slower than cells arrested. We propose that wildtype cells arrest from nutrient deprivation and survive, accumulating oxidative damage through the implementation of robust environmental stress responses

Transcriptional Profiling of Saccharomyces cerevisiae Reveals the Impact of Variation of a Single Transcription Factor on Differential Gene Expression in 4NQO, Fermentable, and Nonfermentable Carbon Sources

Cellular metabolism can change the potency of a chemical’s tumorigenicity. 4-nitroquinoline-1-oxide (4NQO) is a tumorigenic drug widely used on animal models for cancer research. Polymorphisms of the transcription factor Yrr1 confer different levels of resistance to 4NQO in Saccharomyces cerevisiae. To study how different Yrr1 alleles regulate gene expression leading to resistance, transcriptomes of three isogenic S. cerevisiae strains carrying different Yrr1 alleles were profiled via RNA sequencing (RNA-Seq) and chromatin immunoprecipitation coupled with sequencing (ChIP-Seq) in the presence and absence of 4NQO. In response to 4NQO, all alleles of Yrr1 drove the expression of SNQ2 (a multidrug transporter), which was highest in the presence of 4NQO resistance-conferring alleles, and overexpression of SNQ2 alone was sufficient to over- come 4NQO-sensitive growth. Using shape metrics to refine the ChIP-Seq peaks, Yrr1 strongly associated with three loci including SNQ2. In addition to a known Yrr1 target SNG1, Yrr1 also bound upstream of RPL35B; however, overexpression of these genes did not confer 4NQO resistance. RNA-Seq data also implicated nucleotide synthesis pathways including the de novo purine pathway, and the ribonuclease reductase path- ways were downregulated in response to 4NQO. Conversion of a 4NQO-sensitive allele to a 4NQO-resistant allele by a single point mutation mimicked the 4NQO-resistant allele in phenotype, and while the 4NQO resistant allele increased the expression of the ADE genes in the de novo purine biosynthetic pathway, the mutant Yrr1 increased expression of ADE genes even in the absence of 4NQO. These same ADE genes were only increased in the wild-type alleles in the presence of 4NQO, indicating that the point mutation activated Yrr1 to upregulate a pathway normally only activated in response to stress. The various Yrr1 alleles also influenced growth on different carbon sources by altering the function of the mitochondria. Hence, the complement to 4NQO resistance was poor growth on nonfermentable carbon sources, which in turn varied depending on the allele of Yrr1 expressed in the isogenic yeast. The oxidation state of the yeast affected the 4NQO toxicity by altering the reactive oxygen species (ROS) generated by cellular metabolism. The integra- tion of RNA-Seq and ChIP-Seq elucidated how Yrr1 regulates global gene transcription in response to 4NQO and how various Yrr1 alleles confer differential resistance to 4NQO. This study provides guidance for further investigation into how Yrr1 regulates cellular responses to 4NQO, as well as transcriptomic resources for further analysis of transcription factor variation on carbon source utilization

Impacts of colonial waterbirds on vegetation and potential restoration of island habitats

Colonial waterbirds have impacted forested island ecosystems throughout their breeding range, changing vegetation, and soil characteristics and bird communities. Our objectives were to (1) determine effects of three levels of colonial waterbird exclusion on overall vegetation diversity and growth, and survival of a candidate restoration species (black elderberry; Sambucus nigra canadensis); (2) investigate effects of different planting techniques on survival and growth of black elderberry; and (3) determine effects of waterbird colonization on soil chemistry. In 2012, we investigated effects of three levels of waterbird exclusion (none control plots [CON]; partial, which excluded waterbirds larger than gulls [PEX]; and full which excluded all waterbirds [FEX]) on bird use, existing vegetation growth and diversity, and survival of planted black elderberry on three islands in Door County, WI, Lake Michigan. In 2013, we evaluated survival of black elderberry established with four planting treatments within three waterbird exclusion treatments on two islands in 2013.We also compared soil chemistry characteristics between islands with and without nesting waterbirds for 2 years. Overall plant growth was greater in exclosures, but elderberry survival was similar among treatments. Soil replacement and weed suppression planting treatments did not affect survival, but generally increased overall elderberry biomass. Soil from nesting islands was more acidic and had greater nutrient concentrations than reference islands. Exclusion or removal of colonial nesting waterbirds from islands may improve overall vegetation growth, but successful restoration of woody vegetation may require significant soil manipulation and planting

Diagnostic and Prognostic Plasma Biomarkers for Idiopathic Pneumonia Syndrome after Hematopoietic Cell Transplantation

Idiopathic pneumonia syndrome (IPS) is a noninfectious pulmonary complication after hematopoietic cell transplantation (HCT) and is difficult to diagnose. In 41 patients with IPS, we evaluated 6 candidate proteins in plasma samples at day 7 post-HCT and at onset of IPS to identify potential diagnostic or prognostic biomarkers for IPS. Samples at similar times from 162 HCT recipients without documented infections and 37 HCT recipients with respiratory viral pneumonia served as controls. In multivariable models, a combination of Stimulation-2 (ST2; odds ratio [OR], 2.8; P < .001) and IL-6 (OR, 1.4; P = .025) was the best panel for distinguishing IPS at diagnosis from unaffected controls, whereas tumor necrosis factor receptor 1 (TNFR1; OR, 2.9; P = .002) was the best marker when comparing patients with IPS and viral pneumonia. The areas under the curve of the receiver operating characteristic (ROC) curves for discriminating between IPS and unaffected controls at day 7 post-HCT were .8 for ST2, .75 for IL-6, and .68 for TNFR1. Using estimated sensitivity and specificity values from cutoffs determined with the ROC analysis (cutoff level: ST2, 21 ng/mL; IL-6, 61 pg/mL; TNFR1, 3421 pg/mL), we calculated positive predictive values (PPV) for a range of estimated population prevalence values of IPS. Among the 3 markers, ST2 showed the highest PPV for IPS occurrence. Based on an assumed prevalence of 8%, a positive ST2 test increased likelihood of IPS to 50%. We conclude that a prospective validation study is warranted to determine whether a plasma biomarker panel can aid the noninvasive diagnosis and prognosis of IPS

Translating clinical training into practice in complex mental health systems: Toward opening the 'Black Box' of implementation

<p>Abstract</p> <p>Background</p> <p>Implementing clinical training in a complex health care system is challenging. This report describes two successive trainings programs in one Veterans Affairs healthcare network and the lessons we drew from their success and failures. The first training experience led us to appreciate the value of careful implementation planning while the second suggested that use of an external facilitator might be an especially effective implementation component. We also describe a third training intervention in which we expect to more rigorously test our hypothesis regarding the value of external facilitation.</p> <p>Results</p> <p>Our experiences appear to be consonant with the implementation model proposed by Fixsen. In this paper we offer a modified version of the Fixsen model with separate components related to training and implementation.</p> <p>Conclusion</p> <p>This report further reinforces what others have noted, namely that educational interventions intended to change clinical practice should employ a multilevel approach if patients are to truly benefit from new skills gained by clinicians. We utilize an implementation research model to illustrate how the aims of the second intervention were realized and sustained over the 12-month follow-up period, and to suggest directions for future implementation research. The present report attests to the validity of, and contributes to, the emerging literature on implementation research.</p

Localization and functional consequences of a direct interaction between TRIOBP-1 and hERG/KCNH2 proteins in the heart

Reduced levels of hERG protein and the corresponding repolarizing current IKr can cause arrhythmia and sudden cardiac death, but the underlying cellular mechanisms controlling hERG surface expression are not well understood. We identified TRIOBP-1, an F-actin binding protein previously associated with actin polymerization, as a putative hERG-interacting protein in a yeast-two hybrid screen of a cardiac library. We corroborated this interaction using Forster resonance energy transfer (FRET) in HEK293 cells and co-immunoprecipitation in HEK293 cells and native cardiac tissue. TRIOBP-1 overexpression reduced hERG surface expression and current density, whereas reducing TRIOBP-1 expression via shRNA knockdown resulted in increased hERG protein levels. Immunolabeling in rat cardiomyocytes showed that native TRIOBP-1 overlapped predominantly with myosin binding protein C and secondarily with rat ERG. In human stem cell-derived cardiomyocytes, TRIOBP-1 overexpression caused intracellular co-sequestration of hERG signal, reduced native IKr, and disrupted action potential repolarization. Calcium currents were also reduced to a lesser degree and cell capacitance was increased. These findings establish that TRIOBP-1 interacts directly with hERG and can affect protein levels, IKr magnitude, and cardiac membrane excitability

A five-year multicenter study of the susceptibility of the Bacteroides fragilis group isolates to cephalosporins, cephamins, penicillins, clindamycin, and metronidazole in the United States

Over 2800 clinical strains of the Bacteroides fragilis group were collected during a 5-year period from ten geographically separate sites and tested for their susceptibility to various antimicrobial agents using a broth microdilution method. Among the cephalosporins, ceftizoxime was the most active (13% resistance) and importantly exhibited relatively equal activity against both B. fragilis species and non-B. fragilis species. Cefotaxime exhibited similar activity with an overall resistance rate of 18%. Both ceftriaxone and cefoperazone were appreciably less active cephalosporins especially against non-B. fragilis species. With regard to cephamycins, cefoxitin (MIC90, 32 [mu]g/ml) was more active than cefotetan (MIC90, [ges]256 [mu]g/ml) and cefmetazole (MIC90, 64 [mu]g/ml). Non-B. fragilis species were highly resistant to cefotetan and cefmetazole. Imipenem was highly active against all strains with the exception of four strains of B. fragilis. Ampicillin-sulbactam, amoxicillin-clavulanate, piperacillin-tazobactam, and cefoperazone-sulbactam were all highly active with resistance rates &lt;2%. No resistance was detected to metronidazole, whereas 14% of isolates were resistant to clindamycin. When compared with other studies, these findings underscore the wide variability in susceptibility patterns reported nationwide and the need to continue monitoring these patterns to aid in choosing the most active compounds for therapy.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31666/1/0000601.pd