Approaches to overcome flow cytometry limitations in the analysis of cells from veterinary relevant species

BACKGROUND: Flow cytometry is a powerful tool for the multiparameter analysis of leukocyte subsets on the single cell level. Recent advances have greatly increased the number of fluorochrome-labeled antibodies in flow cytometry. In particular, an increase in available fluorochromes with distinct excitation and emission spectra combined with novel multicolor flow cytometers with several lasers have enhanced the generation of multidimensional expression data for leukocytes and other cell types. However, these advances have mainly benefited the analysis of human or mouse cell samples given the lack of reagents for most animal species. The flow cytometric analysis of important veterinary, agricultural, wildlife, and other animal species is still hampered by several technical limitations, even though animal species other than the mouse can serve as more accurate models of specific human physiology and diseases. RESULTS: Here we present time-tested approaches that our laboratory regularly uses in the multiparameter flow cytometric analysis of ovine leukocytes. The discussed approaches will be applicable to the analysis of cells from most animal species and include direct modification of antibodies by covalent conjugation or Fc-directed labeling (Zenon™ technology), labeled secondary antibodies and other second step reagents, labeled receptor ligands, and antibodies with species cross-reactivity. CONCLUSIONS: Using refined technical approaches, the number of parameters analyzed by flow cytometry per cell sample can be greatly increased, enabling multidimensional analysis of rare samples and giving critical insight into veterinary and other less commonly analyzed species. By maximizing information from each cell sample, multicolor flow cytometry can reduce the required number of animals used in a study

Using integrative taxonomy to distinguish cryptic halfbeak species and interpret distribution patterns, fisheries landings, and speciation

Context. Species classification disputes can be resolved using integrative taxonomy, which involves the use of both phenotypic and genetic information to determine species boundaries. Aims. Our aim was to clarify species boundaries of two commercially important cryptic species of halfbeak (Hemiramphidae), whose distributions overlap in south-eastern Australia, and assist fisheries management. Methods. We applied an integrative taxonomic approach to clarify species boundaries and assist fisheries management. Key results. Mitochondrial DNA and morphological data exhibited significant differences between the two species. The low level of mitochondrial DNA divergence, coupled with the lack of difference in the nuclear DNA, suggests that these species diverged relatively recently (c. 500 000 years ago) when compared with other species within the Hyporhamphus genus (>2.4 million years ago). Genetic differences between the species were accompanied by differences in modal gill raker counts, mean upper- jaw and preorbital length, and otolith shape. Conclusions. On the basis of these genetic and morphological differences, as well as the lack of morphological intergradation between species along the overlapping boundaries of their geographical distributions, we propose that Hyporhamphus australis and Hyporhamphus melanochir remain valid species. Implications. This study has illustrated the need for an integrative taxonomic approach when assessing species boundaries and has provided a methodological framework for studying other cryptic fish species in a management context

Development of a Method for Measuring Carbon Balance in Chemical Sequestration of CO2

Anthropogenic CO2 released from fossil fuel combustion is a primary greenhouse gas which contributes to “global warming.” It is estimated that stationary power generation contributes over one-third of total CO2 emissions. Reducing CO2 in the atmosphere can be accomplished either by decreasing the rate at which CO2 is emitted into the atmosphere or by increasing the rate at which it is removed from it. Extensive research has been conducted on determining a fast and inexpensive method to sequester carbon dioxide. These methods can be classified into two categories, CO2 fixation by natural sink process for CO2, or direct CO2 sequestration by artificial processes. In direct sequestration, CO2 produced from sources such as coal-fired power plants, would be captured from the exhausted gases. CO2 from a combustion exhaust gas is absorbed with an aqueous ammonia solution through scrubbing. The captured CO2 is then used to synthesize ammonium bicarbonate (ABC or NH4HCO3), an economical source of nitrogen fertilizer. In this work, we studied the carbon distribution after fertilizer is synthesized from CO2. The synthesized fertilizer in laboratory is used as a “CO2 carrier” to “transport” CO2 from the atmosphere to crops. After biological assimilation and metabolism in crops treated with ABC, a considerable amount of the carbon source is absorbed by the plants with increased biomass production. The majority of the unused carbon source percolates into the soil as carbonates, such as calcium carbonate (CaCO3) and magnesium carbonate (MgCO3). These carbonates are environmentally benign. As insoluble salts, they are found in normal rocks and can be stored safely and permanently in soil. This investigation mainly focuses on the carbon distribution after the synthesized fertilizer is applied to soil. Quantitative examination of carbon distribution in an ecosystem is a challenging task since the carbon in the soil may come from various sources. Therefore synthesized 14C tagged NH4HCO3 (ABC) was used. Products of ammonium bicarbonate (ABC) or long-term effect ammonium bicarbonate (LEABC) were tagged with 14C when they were synthesized in the laboratory. An indoor greenhouse was built and wheat was chosen as the plant to study in this ecosystem. The investigated ecosystem consists of plant (wheat), soils with three different pH values (alkaline, neutral and acid), and three types of underground water (different Ca2+ and Mg2+ concentrations). After biological assimilation and metabolism in wheat receiving ABC or LEABC, it was found that a considerable amount (up to 10%) of the carbon source is absorbed by the wheat with increased biomass production. The majority of the unused carbon source (up to 76%) percolated into the soil as carbonates, such as environmentally benign calcium carbonate (CaCO3). Generally speaking, alkaline soil has a higher capability to capture and store carbon. For the same soil, there is no apparent difference in carbon capturing capability between ABC fertilizer and LEABC fertilizer. These findings answer the question how carbon is distributed after synthesized fertilizer is applied into the ecosystem. In addition, a separate post-experiment on fertilizer carbon forms that exist in the soil was made. It was found that the up to 88% of the trapped carbon exists in the form of insoluble salts (i.e., CaCO3) in alkaline soils. This indicates that alkaline soil has a greater potential for storing carbon after the use of the synthesized fertilizer from exhausted CO2

Establishment of an Environmental Control Technology Laboratory With a Circulating Fluidized-Bed Combustion System Quarterly Report

This report presents the progress made on the project ''Establishment of an Environmental Control Technology Laboratory (ECTL) with a Circulating Fluidized-Bed Combustion (CFBC) System'' during the quarter April 1--June 30, 2004. The following tasks have been completed. First, the final specifications for the renovation of the new Combustion Laboratory and the construction of the CFB Combustor Building have been delivered to the architect, and invitations for construction bids for the two tasks have been released. Second, the component parts of the CFBC system have been designed after the design work for assembly parts of the CFBC system was completed. Third, the literature pertaining to Polychlorinated Dibenzo-p-Dioxins (PCDD) and Polychlorinated Dibenzofurans (PCDF) released during the incineration of solid waste, including municipal solid waste (MSW) and refuse-derived fuel (RDF) have been reviewed, and an experimental plan for fundamental research of MSW incineration on a simulated fluidized-bed combustion (FBC) facility has been prepared. Finally, the proposed work for the next quarter has been outlined in this report

Initial Laboratory-Scale Melter Test Results for Combined Fission Product Waste

This report describes the methods and results used to vitrify a baseline glass, CSLNTM-C-2.5 in support of the AFCI (Advanced Fuel Cycle Initiative) using a Quartz Crucible Scale Melter at the Pacific Northwest National Laboratory. Document number AFCI-WAST-PMO-MI-DV-2009-000184

Maternal inflammation at midgestation impairs subsequent fetal myoblast function and skeletal muscle growth in rats, resulting in intrauterine growth restriction at term

Maternal inflammation induces intrauterine growth restriction (MI-IUGR) of the fetus, which compromises metabolic health in human offspring and reduces value in livestock. The objective of this study was to determine the effect of maternal inflammation at midgestation on fetal skeletal muscle growth and myoblast profiles at term. Pregnant Sprague-Dawley rats were injected daily with bacterial endotoxin (MI-IUGR) or saline (controls) from the 9th to the 11th day of gestational age (dGA; term = 21 dGA). At necropsy on dGA 20, average fetal mass and upper hindlimb cross-sectional areas were reduced (P \u3c 0.05) in MI-IUGR fetuses compared with controls. MyoD+ and myf5+ myoblasts were less abundant (P \u3c 0.05), and myogenin+ myoblasts were more abundant (P \u3c 0.05) in MI-IUGR hindlimb skeletal muscle compared with controls, indicating precocious myoblast differentiation. Type I and Type II hindlimb muscle fibers were smaller (P \u3c 0.05) in MI-IUGR fetuses than in controls, but fiber type proportions did not differ between experimental groups. Fetal blood plasma TNFα concentrations were below detectable amounts in both experimental groups, but skeletal muscle gene expression for the cytokine receptors TNFR1, IL6R, and FN14 was greater (P \u3c 0.05) in MI-IUGR fetuses than controls, perhaps indicating enhanced sensitivity to these cytokines. Maternal blood glucose concentrations at term did not differ between experimental groups, but MI-IUGR fetal blood contained less (P \u3c 0.05) glucose, cholesterol, and triglycerides. Fetal-to-maternal blood glucose ratios were also reduced (P \u3c 0.05), which is indicative of placental insufficiency. Indicators of protein catabolism, including blood plasma urea nitrogen and creatine kinase, were greater (P \u3c 0.05) in MI-IUGR fetuses than in controls. From these findings, we conclude that maternal inflammation at midgestation causes muscle-centric fetal programming that impairs myoblast function, increases protein catabolism, and reduces skeletal muscle growth near term. Fetal muscle sensitivity to inflammatory cytokines appeared to be enhanced after maternal inflammation, which may represent a mechanistic target for improving these outcomes in MI-IUGR fetuses

PONV Prophylaxis Failure Disproportionately Affects Female Patients, Despite Intraoperative Computerized Decision Support Guidance

Objectives: To compare postoperative nausea and vomiting (PONV) prophylaxis treatment and outcomes based on patients’ sex, using a retrospective cohort. The setting was the operating room and post-anesthesia care unit of a tertiary care university medical center. Patients: A total of 678 adult male and female patients with American Society of Anesthesiologist (ASA) scores of 1-4 underwent surgery with general anesthesia. All patients received preoperative PONV risk assessment. PONV prophylaxis was administered at the discretion of the anesthesia care team members with guidance from a computerized decision support system. Measurements: Adequacy of prophylaxis was retrospectively determined based on individual patient risk factors and the observed treatment received, compared with guideline-based prophylaxis recommendations. Patient outcome was measured by diagnosis of PONV in recovery. Results: Comparing patients who received fewer than the guideline-recommended number of prophylactic antiemetics by sex, 94.6% were female and 5.4% were males (p \u3c 0.001). Patients who received fewer than guideline-recommended number of antiemetics had significantly higher rates of nausea or vomiting in the post-anesthesia care unit (30.4% vs 17.5%, p \u3c 0.001). Conclusion: This retrospective cohort study shows that female patients receiving general anesthesia are disproportionately affected by failure to adhere to PONV prevention guidelines

Extinction vortex dynamics of top predators isolated by urbanization

Extinction risk is elevated in small, isolated populations due to demographic and genetic interactions. Therefore, it is critical to model these processes realistically in population viability analyses (PVA) to inform local management and contribute to a greater understanding of mechanisms within the extinction vortex. We conducted PVA’s for two small mountain lion populations isolated by urbanization in southern California to predict population growth, extinction probability, and loss of genetic diversity with empirical data. Specifically, we (1) provide the first PVA for isolated mountain lions in the Santa Ana Mountains (SAM) that considers both demographic and genetic risk factors and (2) test the hypothesis that variation in abundance and mortality between the SAM and Santa Monica Mountains (SMM) result in differences in population growth, loss of heterozygosity, and extinction probability. Our models predicted 16–21% probability of local extinction in the SAM due purely to demographic processes over 50 yr with current low levels or no immigration. Our models also predicted that genetic diversity will further erode in the SAM such that concern regarding inbreeding depression is warranted unless gene flow is increased, and that if inbreeding depression occurs, rapid local extinction will be highly likely. Dynamics of the two populations were broadly similar, but they also exhibited differences driven by larger population size and higher mortality in the SAM. Density-independent scenarios predicted a rapidly increasing population in the SMM, whereas growth potential did not differ from a stable trend in the SAM. Demographic extinction probability and loss of heterozygosity were greater in the SMM for density-dependent scenarios without immigration. However, higher levels of immigration had stronger, positive influences on both demographic viability and retention of genetic diversity in the SMM driven by lower abundance and higher adult survival. Our results elucidate demographic and genetic threats to small populations within the extinction vortex, and how these vary relative to demographic structure. Importantly, simulating seemingly attainable increases in connectivity was sufficient to greatly reduce extinction probability. Our work highlights that conservation of large carnivores is achievable within urbanized landscapes, but requires land protection, connectivity, and strategies to promote coexistence with humans

Evidence for Returning Disk Radiation in the Black Hole X-Ray Binary XTE J1550–564

We explore the accretion properties of the black hole X-ray binary XTE J1550−564 during its outbursts in 1998/99 and 2000. We model the disk, corona, and reflection components of X-ray spectra taken with the Rossi X-ray Timing Explorer, using the relxill suite of reflection models. The key result of our modeling is that the reflection spectrum in the very soft state is best explained by disk self-irradiation, i.e., photons from the inner disk are bent by the strong gravity of the black hole and reflected off the disk surface. This is the first known detection of thermal disk radiation reflecting off the inner disk. There is also an apparent absorption line at ~6.9 keV, which may be evidence of an ionized disk wind. The coronal electron temperature (kT_e) is, as expected, lower in the brighter outburst of 1998/99, explained qualitatively by more efficient coronal cooling due to irradiating disk photons. The disk inner radius is consistent with being within a few times the innermost stable circular orbit throughout the bright-hard-to-soft states (10 s of r_g in gravitational units). The disk inclination is low during the hard state, disagreeing with the binary inclination value, and very close to 90° in the soft state, recovering to a lower value when adopting a blackbody spectrum as the irradiating continuum