High-throughput, quantitative analyses of genetic interactions in E. coli.

Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli

Aerobic Function and Muscle Deoxygenation Dynamics during Ramp Exercise in Children

PURPOSE:To characterise changes in deoxyhemoglobin ([HHb]) response dynamics in boys and girls during ramp incremental exercise to investigate whether the reduced peak oxygen uptake (peakV˙O2) in girls is associated with a poorer matching of muscle O2 delivery to muscle O2 utilisation, as evidenced by a more rapid increase in [HHb].METHODS:52 children (31 boys, 9.9 ± 0.6 years, 1.38 ± 0.07 m, 31.70 ± 5.78 kg) completed ramp incremental exercise on a cycle ergometer during which pulmonary gas exchange and muscle oxygenation parameters were measured.RESULTS:When muscle [HHb] was expressed against absolute work rate and V˙O2, girls had an earlier change in [HHb] as evidenced by the lower c/d parameter (Girls: 54 ± 20 W vs Boys: 67 ± 19 W, P=0.023; Girls: 0.82 ± 0.28 L·min vs. Boys: 0.95 ± 0.19 L·min, P=0.055) and plateau (Girls: 85 ± 12 W vs. Boys: 99 ± 18 W, P=0.031; Girls: 1.02 ± 0.25 L·min vs. Boys: 1.22 ± 0.28 L·min, P=0.014). However, when expressed against relative work-rate or V˙O2, there were no sex differences in [HHb] response dynamics (all P>0.20). Significant correlations were observed between absolute and fat-free mass normalised peak V˙O2 and the HHb c/d and plateau parameters when expressed against absolute work-rate or V˙O2. Furthermore, when entered into a multiple regression model, the [HHb] plateau against absolute V˙O2 contributed 12% of the variance in peak V˙O2 after adjusting for fat-free mass, gas exchange threshold, and body fatness (model R =0.81, P<0.001).CONCLUSION:The sex-difference in peak V˙O2 in 9-10 year old children is, in part, related to sex-specific changes in muscle O2 extraction dynamics during incremental exercise

The Health and economic effects of light rail lines: design, methods, and protocol for a natural experiment

Background: The health impacts of community design have been studied extensively over the past two decades. In particular, public transportation use is associated with more walking between transit stops and shops, work, home and other destinations. Change in transit access has been linked with physical activity and obesity but seldom to health outcomes and associated costs, especially within a causal framework. Health related fiscal impacts of transit investment should be a key consideration in major transit investment decisions. Methods: The Rails & Health study is a natural experiment evaluating changes in clinical measures, health care utilization and health care costs among Kaiser Permanente Northwest (KPNW) members following the opening of a new light rail transit (LRT) line in Portland, Oregon. The study is prospectively following 3036 adults exposed to the new LRT line and a similar cohort of 4386 adults who do not live close to the new line. Individual-level outcomes and covariates are extracted from the electronic medical record at KPNW, including member demographics and comorbidities, blood pressure, body mass index, lipids, glycosylated hemoglobin, and health care utilization and costs. In addition, participants are surveyed about additional demographics, travel patterns, physical activity (PA), and perceived neighborhood walkability. In a subsample of the study population, we are collecting direct measures of travel-related behavior—physical activity (accelerometry), global positioning system (GPS) tracking, and travel diaries—to document mechanisms responsible for observed changes in health outcomes and cost. Comprehensive measures of the built environment at baseline and after rail construction are also collected. Statistical analyses will (1) examine the effects of opening a new LRT line on chronic disease indicators, health care utilization, and health care costs and (2) evaluate the degree to which observed effects of the LRT line on health measures and costs are mediated by changes in total and transportation-associated PA. Discussion: The results of the Rails & Health study will provide urban planners, transportation engineers, health practitioners, developers, and decision makers with critical information needed to document how transit investments impact population health and related costs.Applied Science, Faculty ofMedicine, Faculty ofOther UBCNon UBCCommunity and Regional Planning (SCARP), School ofPopulation and Public Health (SPPH), School ofReviewedFacult

Pathway Analysis Reveals Common Pro-Survival Mechanisms of Metyrapone and Carbenoxolone after Traumatic Brain Injury

<div><p>Developing new pharmacotherapies for traumatic brain injury (TBI) requires elucidation of the neuroprotective mechanisms of many structurally and functionally diverse compounds. To test our hypothesis that diverse neuroprotective drugs similarly affect common gene targets after TBI, we compared the effects of two drugs, metyrapone (MT) and carbenoxolone (CB), which, though used clinically for noncognitive conditions, improved learning and memory in rats and humans. Although structurally different, both MT and CB inhibit a common molecular target, 11β hydroxysteroid dehydrogenase type 1, which converts inactive cortisone to cortisol, thereby effectively reducing glucocorticoid levels. We examined injury-induced signaling pathways to determine how the effects of these two compounds correlate with pro-survival effects in surviving neurons of the injured rat hippocampus. We found that treatment of TBI rats with MT or CB acutely induced in hippocampal neurons transcriptional profiles that were remarkably similar (i.e., a coordinated attenuation of gene expression across multiple injury-induced cell signaling networks). We also found, to a lesser extent, a coordinated increase in cell survival signals. Analysis of injury-induced gene expression altered by MT and CB provided additional insight into the protective effects of each. Both drugs attenuated expression of genes in the apoptosis, death receptor and stress signaling pathways, as well as multiple genes in the oxidative phosphorylation pathway such as subunits of NADH dehydrogenase (Complex1), cytochrome c oxidase (Complex IV) and ATP synthase (Complex V). This suggests an overall inhibition of mitochondrial function. Complex 1 is the primary source of reactive oxygen species in the mitochondrial oxidative phosphorylation pathway, thus linking the protective effects of these drugs to a reduction in oxidative stress. The net effect of the drug-induced transcriptional changes observed here indicates that suppressing expression of potentially harmful genes, and also, surprisingly, reduced expression of pro-survival genes may be a hallmark of neuroprotective therapeutic effects.</p> </div

Traumatic Brain Injury-Induced Dysregulation of the Circadian Clock

<div><p>Circadian rhythm disturbances are frequently reported in patients recovering from traumatic brain injury (TBI). Since circadian clock output is mediated by some of the same molecular signaling cascades that regulate memory formation (cAMP/MAPK/CREB), cognitive problems reported by TBI survivors may be related to injury-induced dysregulation of the circadian clock. In laboratory animals, aberrant circadian rhythms in the hippocampus have been linked to cognitive and memory dysfunction. Here, we addressed the hypothesis that circadian rhythm disruption after TBI is mediated by changes in expression of clock genes in the suprachiasmatic nuclei (SCN) and hippocampus. After fluid-percussion TBI or sham surgery, male Sprague-Dawley rats were euthanized at 4 h intervals, over a 48 h period for tissue collection. Expression of circadian clock genes was measured using quantitative real-time PCR in the SCN and hippocampus obtained by laser capture and manual microdissection respectively. Immunofluorescence and Western blot analysis were used to correlate TBI-induced changes in circadian gene expression with changes in protein expression. In separate groups of rats, locomotor activity was monitored for 48 h. TBI altered circadian gene expression patterns in both the SCN and the hippocampus. Dysregulated expression of key circadian clock genes, such as <em>Bmal1</em> and <em>Cry1,</em> was detected, suggesting perturbation of transcriptional-translational feedback loops that are central to circadian timing. In fact, disruption of circadian locomotor activity rhythms in injured animals occurred concurrently. These results provide an explanation for how TBI causes disruption of circadian rhythms as well as a rationale for the consideration of drugs with chronobiotic properties as part of a treatment strategy for TBI.</p> </div

Pathway-Focused PCR Array Profiling of Enriched Populations of Laser Capture Microdissected Hippocampal Cells after Traumatic Brain Injury

<div><p>Cognitive deficits in survivors of traumatic brain injury (TBI) are associated with irreversible neurodegeneration in brain regions such as the hippocampus. Comparative gene expression analysis of dying and surviving neurons could provide insight into potential therapeutic targets. We used two pathway-specific PCR arrays (RT2 Profiler Apoptosis and Neurotrophins & Receptors PCR arrays) to identify and validate TBI-induced gene expression in dying (Fluoro-Jade-positive) or surviving (Fluoro-Jade- negative) pyramidal neurons obtained by laser capture microdissection (LCM). In the Apoptosis PCR array, dying neurons showed significant increases in expression of genes associated with cell death, inflammation, and endoplasmic reticulum (ER) stress compared with adjacent, surviving neurons. Pro-survival genes with pleiotropic functions were also significantly increased in dying neurons compared to surviving neurons, suggesting that even irreversibly injured neurons are able to mount a protective response. In the Neurotrophins & Receptors PCR array, which consists of genes that are normally expected to be expressed in both groups of hippocampal neurons, only a few genes were expressed at significantly different levels between dying and surviving neurons. Immunohistochemical analysis of selected, differentially expressed proteins supported the gene expression data. This is the first demonstration of pathway-focused PCR array profiling of identified populations of dying and surviving neurons in the brain after TBI. Combining precise laser microdissection of identifiable cells with pathway-focused PCR array analysis is a practical, low-cost alternative to microarrays that provided insight into neuroprotective signals that could be therapeutically targeted to ameliorate TBI-induced neurodegeneration.</p></div

MicroRNA profiling identifies a novel compound with antidepressant properties.

Patients with traumatic brain injury (TBI) are frequently diagnosed with depression. Together, these two leading causes of death and disability significantly contribute to the global burden of healthcare costs. However, there are no drug treatments for TBI and antidepressants are considered off-label for depression in patients with TBI. In molecular profiling studies of rat hippocampus after experimental TBI, we found that TBI altered the expression of a subset of small, non-coding, microRNAs (miRNAs). One known neuroprotective compound (17β-estradiol, E2), and two experimental neuroprotective compounds (JM6 and PMI-006), reversed the effects of TBI on miRNAs. Subsequent in silico analyses revealed that the injury-altered miRNAs were predicted to regulate genes involved in depression. Thus, we hypothesized that drug-induced miRNA profiles can be used to identify compounds with antidepressant properties. To confirm this hypothesis, we examined miRNA expression in hippocampi of injured rats treated with one of three known antidepressants (imipramine, fluoxetine and sertraline). Bioinformatic analyses revealed that TBI, potentially via its effects on multiple regulatory miRNAs, dysregulated transcriptional networks involved in neuroplasticity, neurogenesis, and circadian rhythms- networks known to adversely affect mood, cognition and memory. As did E2, JM6, and PMI-006, all three antidepressants reversed the effects of TBI on multiple injury-altered miRNAs. Furthermore, JM6 reduced TBI-induced inflammation in the hippocampus and depression-like behavior in the forced swim test; these are both properties of classic antidepressant drugs. Our results support the hypothesis that miRNA expression signatures can identify neuroprotective and antidepressant properties of novel compounds and that there is substantial overlap between neuroprotection and antidepressant properties

Effects of AAV-mediated knockdown of nNOS and GPx-1 gene expression in rat hippocampus after traumatic brain injury.

Virally mediated RNA interference (RNAi) to knock down injury-induced genes could improve functional outcome after traumatic brain injury (TBI); however, little is known about the consequences of gene knockdown on downstream cell signaling pathways and how RNAi influences neurodegeneration and behavior. Here, we assessed the effects of adeno-associated virus (AAV) siRNA vectors that target two genes with opposing roles in TBI pathogenesis: the allegedly detrimental neuronal nitric oxide synthase (nNOS) and the potentially protective glutathione peroxidase 1 (GPx-1). In rat hippocampal progenitor cells, three siRNAs that target different regions of each gene (nNOS, GPx-1) effectively knocked down gene expression. However, in vivo, in our rat model of fluid percussion brain injury, the consequences of AAV-siRNA were variable. One nNOS siRNA vector significantly reduced the number of degenerating hippocampal neurons and showed a tendency to improve working memory. GPx-1 siRNA treatment did not alter TBI-induced neurodegeneration or working memory deficits. Nevertheless, microarray analysis of laser captured, virus-infected neurons showed that knockdown of nNOS or GPx-1 was specific and had broad effects on downstream genes. Since nNOS knockdown only modestly ameliorated TBI-induced working memory deficits, despite widespread genomic changes, manipulating expression levels of single genes may not be sufficient to alter functional outcome after TBI