Discovery of sarolaner:A novel, Orally administered, broad-spectrum, Isoxazoline ectoparasiticide for dogs

AbstractThe novel isoxazoline ectoparasiticide, sarolaner, was identified during a lead optimization program for an orally-active compound with efficacy against fleas and ticks on dogs. The aim of the discovery program was to identify a novel isoxazoline specifically for use in companion animals, beginning with de novo synthesis in the Zoetis research laboratories. The sarolaner molecule has unique structural features important for its potency and pharmacokinetic (PK) properties, including spiroazetidine and sulfone moieties. The flea and tick activity resides in the chirally pure S-enantiomer, which was purified to alleviate potential off-target effects from the inactive enantiomer. The mechanism of action was established in electrophysiology assays using CHO-K1 cell lines stably expressing cat flea (Ctenocephalides felis) RDL (resistance-to-dieldrin) genes for assessment of GABA-gated chloride channel (GABACls) pharmacology. As expected, sarolaner inhibited GABA-elicited currents at both susceptible (CfRDL-A285) and resistant (CfRDL-S285) flea GABACls with similar potency. Initial whole organism screening was conducted in vitro using a blood feeding assay against C. felis. Compounds which demonstrated robust activity in the flea feed assay were subsequently tested in an in vitro ingestion assay against the soft tick, Ornithodoros turicata. Efficacious compounds which were confirmed safe in rodents at doses up to 30mg/kg were progressed to safety, PK and efficacy studies in dogs. In vitro sarolaner demonstrated an LC80 of 0.3μg/mL against C. felis and an LC100 of 0.003μg/mL against O. turicata. In a head-to-head comparative in vitro assay with both afoxolaner and fluralaner, sarolaner demonstrated superior flea and tick potency. In exploratory safety studies in dogs, sarolaner demonstrated safety in dogs≥8 weeks of age upon repeated monthly dosing at up to 20mg/kg. Sarolaner was rapidly and well absorbed following oral dosing. Time to maximum plasma concentration occurred within the first day post-dose. Bioavailability for sarolaner was calculated at >85% and the compound was highly protein bound (>99.9%). The half-life for sarolaner was calculated at 11–12 days. Sarolaner plasma concentrations indicated dose proportionality over the range 1.25–5mg/kg, and these same doses provided robust efficacy (>99%) for ≥35days against both fleas (C. felis) and multiple species of ticks (Rhipicephalus sanguineus, Ixodes ricinus and Dermacentor reticulatus) after oral administration to dogs. As a result of these exploratory investigations, sarolaner was progressed for development as an oral monthly dose for treatment and control of fleas and ticks on dogs

Acidosis Maintains the Function of Brain Mitochondria in Hypoxia-Tolerant Triplefin Fish: A Strategy to Survive Acute Hypoxic Exposure?

The vertebrate brain is generally very sensitive to acidosis, so a hypoxia-induced decrease in pH is likely to have an effect on brain mitochondria (mt). Mitochondrial respiration (JO2) is required to generate an electrical gradient (ΔΨm) and a pH gradient to power ATP synthesis, yet the impact of pH modulation on brain mt function remains largely unexplored. As intertidal fishes within rock pools routinely experience hypoxia and reoxygenation, they would most likely experience changes in cellular pH. We hence compared four New Zealand triplefin fish species ranging from intertidal hypoxia-tolerant species (HTS) to subtidal hypoxia-sensitive species (HSS). We predicted that HTS would tolerate acidosis better than HSS in terms of sustaining mt structure and function. Using respirometers coupled to fluorimeters and pH electrodes, we titrated lactic-acid to decrease the pH of the media, and simultaneously recorded JO2, ΔΨm, and H+ buffering capacities within permeabilized brain and swelling of mt isolated from non-permeabilized brains. We then measured ATP synthesis rates in the most HTS (Bellapiscus medius) and the HSS (Forsterygion varium) at pH 7.25 and 6.65. Mitochondria from HTS brain did have greater H+ buffering capacities than HSS mt (∼10 mU pH.mgprotein-1). HTS mt swelled by 40% when exposed to a decrease of 1.5 pH units, and JO2 was depressed by up to 15% in HTS. However, HTS were able to maintain ΔΨm near -120 mV. Estimates of work, in terms of charges moved across the mt inner-membrane, suggested that with acidosis, HTS mt may in part harness extra-mt H+ to maintain ΔΨm, and could therefore support ATP production. This was confirmed with elevated ATP synthesis rates and enhanced P:O ratios at pH 6.65 relative to pH 7.25. In contrast, mt volumes and ΔΨm decreased downward pH 6.9 in HSS mt and paradoxically, JO2 increased (∼25%) but ATP synthesis and P:O ratios were depressed at pH 6.65. This indicates a loss of coupling in the HSS with acidosis. Overall, the mt of these intertidal fish have adaptations that enhance ATP synthesis efficiency under acidic conditions such as those that occur in hypoxic or reoxygenated brain

Theoretical Formulation of Principal Components Analysis to Detect and Correct for Population Stratification

The Eigenstrat method, based on principal components analysis (PCA), is commonly used both to quantify population relationships in population genetics and to correct for population stratification in genome-wide association studies. However, it can be difficult to make appropriate inference about population relationships from the principal component (PC) scatter plot. Here, to better understand the working mechanism of the Eigenstrat method, we consider its theoretical or “population” formulation. The eigen-equation for samples from an arbitrary number () of populations is reduced to that of a matrix of dimension , the elements of which are determined by the variance-covariance matrix for the random vector of the allele frequencies. Solving the reduced eigen-equation is numerically trivial and yields eigenvectors that are the axes of variation required for differentiating the populations. Using the reduced eigen-equation, we investigate the within-population fluctuations around the axes of variation on the PC scatter plot for simulated datasets. Specifically, we show that there exists an asymptotically stable pattern of the PC plot for large sample size. Our results provide theoretical guidance for interpreting the pattern of PC plot in terms of population relationships. For applications in genetic association tests, we demonstrate that, as a method of correcting for population stratification, regressing out the theoretical PCs corresponding to the axes of variation is equivalent to simply removing the population mean of allele counts and works as well as or better than the Eigenstrat method

Meta-analysis of gene expression microarrays with missing replicates

<p>Abstract</p> <p>Background</p> <p>Many different microarray experiments are publicly available today. It is natural to ask whether different experiments for the same phenotypic conditions can be combined using meta-analysis, in order to increase the overall sample size. However, some genes are not measured in all experiments, hence they cannot be included or their statistical significance cannot be appropriately estimated in traditional meta-analysis. Nonetheless, these genes, which we refer to as <it>incomplete genes</it>, may also be informative and useful.</p> <p>Results</p> <p>We propose a meta-analysis framework, called "Incomplete Gene Meta-analysis", which can include incomplete genes by imputing the significance of missing replicates, and computing a meta-score for every gene across all datasets. We demonstrate that the incomplete genes are worthy of being included and our method is able to appropriately estimate their significance in two groups of experiments. We first apply the <it>Incomplete Gene Meta-analysis </it>and several comparable methods to five breast cancer datasets with an identical set of probes. We simulate incomplete genes by randomly removing a subset of probes from each dataset and demonstrate that our method consistently outperforms two other methods in terms of their false discovery rate. We also apply the methods to three gastric cancer datasets for the purpose of discriminating diffuse and intestinal subtypes.</p> <p>Conclusions</p> <p>Meta-analysis is an effective approach that identifies more robust sets of differentially expressed genes from multiple studies. The incomplete genes that mainly arise from the use of different platforms may also have statistical and biological importance but are ignored or are not appropriately involved by previous studies. Our Incomplete Gene Meta-analysis is able to incorporate the incomplete genes by estimating their significance. The results on both breast and gastric cancer datasets suggest that the highly ranked genes and associated GO terms produced by our method are more significant and biologically meaningful according to the previous literature.</p

Expression of cytokine and chemokine mRNA and secretion of tumor necrosis factor-α by gallbladder epithelial cells: Response to bacterial lipopolysaccharides

BACKGROUND: In addition to immune cells, many other cell types are known to produce cytokines. Cultured normal mouse gallbladder epithelial cells, used as a model system for gallbladder epithelium, were examined for their ability to express the mRNA of various cytokines and chemokines in response to bacterial lipopolysaccharide. The synthesis and secretion of the tumor necrosis factor-α (TNF-α) protein by these cells was also measured. RESULTS: Untreated mouse gallbladder cells expressed mRNA for TNF-α, RANTES, and macrophage inflammatory protein-2 (MIP-2). Upon treatment with lipopolysaccharide, these cells now produced mRNA for Interleukin-1β (IL-1β), IL-6, monocyte chemoattractant protein-1 (MCP-1), and showed increased expression of TNF-α and MIP-2 mRNA. Untreated mouse gallbladder cells did not synthesize TNF-α protein; however, they did synthesize and secrete TNF-α upon treatment with lipopolysaccharide. METHODS: Cells were treated with lipopolysaccharides from 3 strains of bacteria. Qualitative and semi-quantitative RT-PCR, using cytokine or chemokine-specific primers, was used to measure mRNA levels of TNFα, IL-1β, IL-6, IL-10, KC, RANTES, MCP-1, and MIP-2. TNF-α protein was measured by immunoassays. CONCLUSION: This research demonstrates that gallbladder epithelial cells in response to lipopolysaccharide exposure can alter their cytokine and chemokine RNA expression pattern and can synthesize and secrete TNFα protein. This suggests a mechanism whereby gallbladder epithelial cells in vivo may mediate gallbladder secretory function, inflammation and diseases in an autocrine/paracrine fashion by producing and secreting cytokines and/or chemokines during sepsis

[Comment] Redefine statistical significance

The lack of reproducibility of scientific studies has caused growing concern over the credibility of claims of new discoveries based on “statistically significant” findings. There has been much progress toward documenting and addressing several causes of this lack of reproducibility (e.g., multiple testing, P-hacking, publication bias, and under-powered studies). However, we believe that a leading cause of non-reproducibility has not yet been adequately addressed: Statistical standards of evidence for claiming discoveries in many fields of science are simply too low. Associating “statistically significant” findings with P < 0.05 results in a high rate of false positives even in the absence of other experimental, procedural and reporting problems. For fields where the threshold for defining statistical significance is P<0.05, we propose a change to P<0.005. This simple step would immediately improve the reproducibility of scientific research in many fields. Results that would currently be called “significant” but do not meet the new threshold should instead be called “suggestive.” While statisticians have known the relative weakness of using P≈0.05 as a threshold for discovery and the proposal to lower it to 0.005 is not new (1, 2), a critical mass of researchers now endorse this change. We restrict our recommendation to claims of discovery of new effects. We do not address the appropriate threshold for confirmatory or contradictory replications of existing claims. We also do not advocate changes to discovery thresholds in fields that have already adopted more stringent standards (e.g., genomics and high-energy physics research; see Potential Objections below). We also restrict our recommendation to studies that conduct null hypothesis significance tests. We have diverse views about how best to improve reproducibility, and many of us believe that other ways of summarizing the data, such as Bayes factors or other posterior summaries based on clearly articulated model assumptions, are preferable to P-values. However, changing the P-value threshold is simple and might quickly achieve broad acceptance

Massively parallel fitness profiling reveals multiple novel enzymes in Pseudomonas putida lysine metabolism

P. putida lysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, the connection of lysine catabolism to central metabolism in P. putida remained undefined. Here, we used random barcode transposon sequencing to fill the gaps of lysine metabolism in P. putida. We describe a route of 2-oxoadipate (2OA) catabolism, which utilizes DUF1338-containing protein P. putida 5260 (PP_5260) in bacteria. Despite its prevalence in many domains of life, DUF1338-containing proteins have had no known biochemical function. We demonstrate that PP_5260 is a metalloenzyme which catalyzes an unusual route of decarboxylation of 2OA to d-2-hydroxyglutarate (d-2HG). Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results and expand the understanding of glutarate hydroxylase CsiD by showing that can it use either 2OA or 2KG as a cosubstrate. Our work demonstrated that biological novelty can be rapidly identified using unbiased experimental genetics and that RB-TnSeq can be used to rapidly validate previous results.Despite intensive study for 50 years, the biochemical and genetic links between lysine metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish these biochemical links, we leveraged random barcode transposon sequencing (RB-TnSeq), a genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple novel enzymes in both l- and d-lysine metabolism. We first describe three pathway enzymes that catabolize l-2-aminoadipate (l-2AA) to 2-ketoglutarate (2KG), connecting d-lysine to the TCA cycle. One of these enzymes, P. putida 5260 (PP_5260), contains a DUF1338 domain, representing a family with no previously described biological function. Our work also identified the recently described coenzyme A (CoA)-independent route of l-lysine degradation that results in metabolization to succinate. We expanded on previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 2-oxoacid selectivity. Proteomics of selected pathway enzymes revealed that expression of catabolic genes is highly sensitive to the presence of particular pathway metabolites, implying intensive local and global regulation. This work demonstrated the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria, as well as its utility a powerful tool for validating previous research

The Cultural Project : Formal Chronological Modelling of the Early and Middle Neolithic Sequence in Lower Alsace

Starting from questions about the nature of cultural diversity, this paper examines the pace and tempo of change and the relative importance of continuity and discontinuity. To unravel the cultural project of the past, we apply chronological modelling of radiocarbon dates within a Bayesian statistical framework, to interrogate the Neolithic cultural sequence in Lower Alsace, in the upper Rhine valley, in broad terms from the later sixth to the end of the fifth millennium cal BC. Detailed formal estimates are provided for the long succession of cultural groups, from the early Neolithic Linear Pottery culture (LBK) to the Bischheim Occidental du Rhin Supérieur (BORS) groups at the end of the Middle Neolithic, using seriation and typology of pottery as the starting point in modelling. The rate of ceramic change, as well as frequent shifts in the nature, location and density of settlements, are documented in detail, down to lifetime and generational timescales. This reveals a Neolithic world in Lower Alsace busy with comings and goings, tinkerings and adjustments, and relocations and realignments. A significant hiatus is identified between the end of the LBK and the start of the Hinkelstein group, in the early part of the fifth millennium cal BC. On the basis of modelling of existing dates for other parts of the Rhineland, this appears to be a wider phenomenon, and possible explanations are discussed; full reoccupation of the landscape is only seen in the Grossgartach phase. Radical shifts are also proposed at the end of the Middle Neolithic