High throughput modular chambers for rapid evaluation of anesthetic sensitivity

BACKGROUND: Anesthetic sensitivity is determined by the interaction of multiple genes. Hence, a dissection of genetic contributors would be aided by precise and high throughput behavioral screens. Traditionally, anesthetic phenotyping has addressed only induction of anesthesia, evaluated with dose-response curves, while ignoring potentially important data on emergence from anesthesia. METHODS: We designed and built a controlled environment apparatus to permit rapid phenotyping of twenty-four mice simultaneously. We used the loss of righting reflex to indicate anesthetic-induced unconsciousness. After fitting the data to a sigmoidal dose-response curve with variable slope, we calculated the MAC(LORR )(EC(50)), the Hill coefficient, and the 95% confidence intervals bracketing these values. Upon termination of the anesthetic, Emergence time(RR )was determined and expressed as the mean ± standard error for each inhaled anesthetic. RESULTS: In agreement with several previously published reports we find that the MAC(LORR )of halothane, isoflurane, and sevoflurane in 8–12 week old C57BL/6J mice is 0.79% (95% confidence interval = 0.78 – 0.79%), 0.91% (95% confidence interval = 0.90 – 0.93%), and 1.96% (95% confidence interval = 1.94 – 1.97%), respectively. Hill coefficients for halothane, isoflurane, and sevoflurane are 24.7 (95% confidence interval = 19.8 – 29.7%), 19.2 (95% confidence interval = 14.0 – 24.3%), and 33.1 (95% confidence interval = 27.3 – 38.8%), respectively. After roughly 2.5 MAC(LORR )• hr exposures, mice take 16.00 ± 1.07, 6.19 ± 0.32, and 2.15 ± 0.12 minutes to emerge from halothane, isoflurane, and sevoflurane, respectively. CONCLUSION: This system enabled assessment of inhaled anesthetic responsiveness with a higher precision than that previously reported. It is broadly adaptable for delivering an inhaled therapeutic (or toxin) to a population while monitoring its vital signs, motor reflexes, and providing precise control over environmental conditions. This system is also amenable to full automation. Data presented in this manuscript prove the utility of the controlled environment chambers and should allow for subsequent phenotyping of mice with targeted mutations that are expected to alter sensitivity to induction or emergence from anesthesia

Contribution of transcriptional regulation to natural variations in Arabidopsis

BACKGROUND: Genetic control of gene transcription is a key component in genome evolution. To understand the transcriptional basis of natural variation, we have studied genome-wide variations in transcription and characterized the genetic variations in regulatory elements among Arabidopsis accessions. RESULTS: Among five accessions (Col-0, C24, Ler, WS-2, and NO-0) 7,508 probe sets with no detectable genomic sequence variations were identified on the basis of the comparative genomic hybridization to the Arabidopsis GeneChip microarray, and used for accession-specific transcriptome analysis. Two-way ANOVA analysis has identified 60 genes whose mRNA levels differed in different accession backgrounds in an organ-dependent manner. Most of these genes were involved in stress responses and late stages of plant development, such as seed development. Correlation analysis of expression patterns of these 7,508 genes between pairs of accessions identified a group of 65 highly plastic genes with distinct expression patterns in each accession. CONCLUSION: Genes that show substantial genetic variation in mRNA level are those with functions in signal transduction, transcription and stress response, suggesting the existence of variations in the regulatory mechanisms for these genes among different accessions. This is in contrast to those genes with significant polymorphisms in the coding regions identified by genomic hybridization, which include genes encoding transposon-related proteins, kinases and disease-resistance proteins. While relatively fewer sequence variations were detected on average in the coding regions of these genes, a number of differences were identified from the upstream regions, several of which alter potential cis-regulatory elements. Our results suggest that nucleotide polymorphisms in regulatory elements of genes encoding controlling factors could be primary targets of natural selection and a driving force behind the evolution of Arabidopsis accessions

HO_x chemistry during INTEX-A 2004: Observation, model calculation, and comparison with previous studies

OH and HO_2 were measured with the Airborne Tropospheric Hydrogen Oxides Sensor (ATHOS) as part of a large measurement suite from the NASA DC-8 aircraft during the Intercontinental Chemical Transport Experiment-A (INTEX-A). This mission, which was conducted mainly over North America and the western Atlantic Ocean in summer 2004, was an excellent test of atmospheric oxidation chemistry. The HOx results from INTEX-A are compared to those from previous campaigns and to results for other related measurements from INTEX-A. Throughout the troposphere, observed OH was generally 0.95 of modeled OH; below 8 km, observed HO_2 was generally 1.20 of modeled HO_2. This observed-to-modeled comparison is similar to that for TRACE-P, another midlatitude study for which the median observed-to-modeled ratio was 1.08 for OH and 1.34 for HO_2, and to that for PEM-TB, a tropical study for which the median observed-to-modeled ratio was 1.17 for OH and 0.97 for HO_2. HO_2 behavior above 8 km was markedly different. The observed-to-modeled HO_2 ratio increased from ∼1.2 at 8 km to ∼3 at 11 km with the observed-to-modeled ratio correlating with NO. Above 8 km, the observed-to-modeled HO_2 and observed NO were both considerably greater than observations from previous campaigns. In addition, the observed-to-modeled HO_2/OH, which is sensitive to cycling reactions between OH and HO_2, increased from ∼1.5 at 8 km to almost 3.5 at 11 km. These discrepancies suggest a large unknown HO_x source and additional reactants that cycle HO_x from OH to HO_2. In the continental planetary boundary layer, the observed-to-modeled OH ratio increased from 1 when isoprene was less than 0.1 ppbv to over 4 when isoprene was greater than 2 ppbv, suggesting that forests throughout the United States are emitting unknown HO_x sources. Progress in resolving these discrepancies requires a focused research activity devoted to further examination of possible unknown OH sinks and HO_x sources

HOx Observation and Model Comparison During INTEX-A 2004

OH and HO2 were measured with the Airborne Tropospheric Hydrogen Oxides Sensor (ATHOS) as part of a large measurement suite from the NASA DC-8 aircraft during the Intercontinental Chemical Transport Experiment - A (INTEX-A). This mission, which was conducted mainly over North America and the western Atlantic Ocean in summer 2004, was an excellent test of atmospheric oxidation chemistry. Throughout the troposphere, observed OH was generally 0.60 of the modeled OH; below 8 km, observed HO2 was generally 0.78 of modeled HO2. If the over-prediction of tropospheric OH is not due to an instrument calibration error, then it implied less global tropospheric oxidation capacity and longer lifetimes for gases like methane and methyl chloroform than currently thought. This discrepancy falls well outside uncertainties in both the OH measurement and rate coefficients for known reactions and points to a large unknown OH loss. If the modeled OH is forced to agree with observed values by introducing of an undefined OH loss that removed HOx (HOx=OH+HO2), the observed and modeled HO2 and HO2/OH ratios are largely reconciled within the measurement uncertainty. HO2 behavior above 8 km was markedly different. The observed-to-modeled ratio correlating with NO. The observed-to-modeled HO2 ratio increased from approximately 1 at 8 km to more than approximately 2.5 at 11 km with the observed-to-modeled ratio correlating with NO. The observed-to-modeled HO2 and NO were both considerably greater than observations from previous campaigns. In addition, the observed-to-modeled HO2/OH, which is sensitive to cycling reactions between OH and HO2, increased from approximately 1.2 at 8 km to almost 4 above 11 km. In contrast to the lower atmosphere, these discrepancies above 8 km suggest a large unknown HOx source and additional reactants that cycle HOx from OH to HO2. In the continental planetary boundary layer, the OH observed-to-modeled ratio increased from 0.6 when isoprene was less than 0.1 ppbv to over 3 when isoprene was greater than 2 ppbv, suggesting that forests throughout the United States are emitting unknown HOx sources. Progress in resolving these discrepancies requires further examinations of possible unknown OH sinks and HOx sources and a focused research activity devoted to ascertaining the accuracy of the OH and HO2 measurements

Can a “state of the art” chemistry transport model simulate Amazonian tropospheric chemistry?

We present an evaluation of a nested high-resolution Goddard Earth Observing System (GEOS)-Chem chemistry transport model simulation of tropospheric chemistry over tropical South America. The model has been constrained with two isoprene emission inventories: (1) the canopy-scale Model of Emissions of Gases and Aerosols from Nature (MEGAN) and (2) a leaf-scale algorithm coupled to the Lund-Potsdam-Jena General Ecosystem Simulator (LPJ-GUESS) dynamic vegetation model, and the model has been run using two different chemical mechanisms that contain alternative treatments of isoprene photo-oxidation. Large differences of up to 100 Tg C yr^(−1) exist between the isoprene emissions predicted by each inventory, with MEGAN emissions generally higher. Based on our simulations we estimate that tropical South America (30–85°W, 14°N–25°S) contributes about 15–35% of total global isoprene emissions. We have quantified the model sensitivity to changes in isoprene emissions, chemistry, boundary layer mixing, and soil NO_x emissions using ground-based and airborne observations. We find GEOS-Chem has difficulty reproducing several observed chemical species; typically hydroxyl concentrations are underestimated, whilst mixing ratios of isoprene and its oxidation products are overestimated. The magnitude of model formaldehyde (HCHO) columns are most sensitive to the choice of chemical mechanism and isoprene emission inventory. We find GEOS-Chem exhibits a significant positive bias (10–100%) when compared with HCHO columns from the Scanning Imaging Absorption Spectrometer for Atmospheric Chartography (SCIAMACHY) and Ozone Monitoring Instrument (OMI) for the study year 2006. Simulations that use the more detailed chemical mechanism and/or lowest isoprene emissions provide the best agreement to the satellite data, since they result in lower-HCHO columns

Accumulation of an Antidepressant in Vesiculogenic Membranes of Yeast Cells Triggers Autophagy

Many antidepressants are cationic amphipaths, which spontaneously accumulate in natural or reconstituted membranes in the absence of their specific protein targets. However, the clinical relevance of cellular membrane accumulation by antidepressants in the human brain is unknown and hotly debated. Here we take a novel, evolutionarily informed approach to studying the effects of the selective-serotonin reuptake inhibitor sertraline/Zoloft® on cell physiology in the model eukaryote Saccharomyces cerevisiae (budding yeast), which lacks a serotonin transporter entirely. We biochemically and pharmacologically characterized cellular uptake and subcellular distribution of radiolabeled sertraline, and in parallel performed a quantitative ultrastructural analysis of organellar membrane homeostasis in untreated vs. sertraline-treated cells. These experiments have revealed that sertraline enters yeast cells and then reshapes vesiculogenic membranes by a complex process. Internalization of the neutral species proceeds by simple diffusion, is accelerated by proton motive forces generated by the vacuolar H+-ATPase, but is counteracted by energy-dependent xenobiotic efflux pumps. At equilibrium, a small fraction (10–15%) of reprotonated sertraline is soluble while the bulk (90–85%) partitions into organellar membranes by adsorption to interfacial anionic sites or by intercalation into the hydrophobic phase of the bilayer. Asymmetric accumulation of sertraline in vesiculogenic membranes leads to local membrane curvature stresses that trigger an adaptive autophagic response. In mutants with altered clathrin function, this adaptive response is associated with increased lipid droplet formation. Our data not only support the notion of a serotonin transporter-independent component of antidepressant function, but also enable a conceptual framework for characterizing the physiological states associated with chronic but not acute antidepressant administration in a model eukaryote

Assessment of urbanization impacts on surface runoff and effects of green infrastructure on hydrology and water quality

Urbanization is one of the most important anthropogenic modifications of the global environment. It has significant impacts on hydrologic processes and water quantity and quality. Research related to urbanization impacts on surface runoff has focused on changes up to the watershed scale. However, quantitative assessment at a national scale is scarce. Parallel to urbanization impacts, climate is also one of the greatest challenges facing humanity today and its effects are already being felt from strengthened storms and rising sea levels to changing temperature and weather patterns. The adverse impacts of urbanization and climate can converge in synergistic ways, which may render hundreds of millions of urban residents increasingly vulnerable to floods, landslides and other natural disasters. The challenge is in finding mitigating solutions. The specific objectives of this study were to: 1) assess urbanization impacts on surface runoff of the contiguous United States using the Long-Term Hydrologic Impact Assessment (L-THIA) model (version of L-THIA Tabular Tool) based on the 2001, 2006, and 2011 National Land Cover Databases; 2) assess simulated precipitation based on an updated CLIGEN database and associated impacts on the surface runoff using the L-THIA Tabular Tool in the five states of the Great Lakes Region (WI, IL, IN, MI, and OH); and 3) evaluate the effectiveness of green infrastructure practices on hydrology and water quality in a Combined Sewer Overflows (CSO) community. National analysis results showed that: 1) urbanization occurred non-uniformly across the nation from 2001 to 2011; 2) urban expansion and intensification were the main driving forces altering surface runoff; 3) the majority of counties had long-term (50 years) normalized average annual runoff depth (NAARD) from urban land less than 17.8 mm; 4) the states with the largest NAARD values had both high precipitation and increases in urban land, while the ten states with the largest NAARD change percentages were mainly in the western U.S. with low precipitation and the NAARD values were mainly influenced by large increases in urban land; 5) nationally, average annual runoff increased by 10% (approximately 3.3 billion m3) due to urbanization from 2001 to 2011. Weather generators rely on historical meteorological records to simulate time series of synthetic weather inputs, the quality of which has direct influence on model applications. The weather generator CLIGEN’s database has recently been updated to comprise consistent historical records from 1974 to 2013 (updated CLIGEN database, UCD) compared to the current database in which records are of different lengths. In the second objective, CLIGEN’s performance in estimating precipitation using UCD and the subsequent impact on urban runoff simulations were evaluated. Generally, UCD-based precipitation could replicate observed daily precipitation up to the 99.5th percentile, but maximum precipitation was underestimated. Results from the Long-Term Hydrologic Impact Assessment model using UCD-based precipitation showed about 0.57 billion cubic meters more (14.9%) average annual runoff being simulated compared with simulations based on the current CLIGEN database. Overall, CLIGEN with the updated database was found suitable for providing precipitation estimates and for use with modeling urban runoff or urbanization effects. From Objective 3, the enhanced L-THIA-LID 2.2 model was able to simulate more detailed impervious surfaces including sidewalks, roads, driveways, and parking lots, to conduct cost calculations for these more detailed impervious surfaces, and to consider the actual suitable area for bioretention in the study area. The effectiveness of green infrastructure (GI) practices on hydrology and water quality at a combined sewer overflow urban watershed was examined in 10 simulation scenarios using 8 practices including rain barrels/cisterns, green roofs, green roofs plus rain barrels/cisterns, bioretention, porous roads, porous parking lots, porous sidewalks, and porous driveways. The annual cost and the cost effectiveness for each scenario considering a 20-year GI practices lifetime were examined. Main findings included: (1) combined implementation of GI practices performed better than applying individual practices alone; (2) the various adoption levels and combinations of GI practices could potentially reduce runoff volume by 0.2-23.5%, TSS by 0.18-30.8%, TN by 0.2-27.9%, and TP by 0.20 to 28.1%; (3) based on site characteristics, adding more GI practices did not necessarily mean that substantial runoff and pollutant reduction would be achieved; (4) the most cost-effective scenario had an associated cost of $9.21 to achieve 1 m3 runoff reduction per year and$119 to achieve 1 kg TSS reduction per year. This, however, assumes cooperation from the general public in implementing GI practices on their properties; and, (5) adoption of GI practices on all possible areas could potentially achieve the greatest runoff and pollutant load reduction, but would not be the most cost-effective option. The enhanced model from this research can be applied to various locations to support assessing the beneficial uses of GI practices. Overall, results of this research provide important information on the negative impacts of urbanization and climate, as well as the importance of green infrastructure as a sustainable development or re-development approach. This research also demonstrated the technical capability of the L-THIA Tabular Tool and the L-THIA-LID 2.2 model. The outcomes of this research will assist urban planners and decision makers to make sustainable development or re-development strategies