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BACKGROUND Little is known regarding how trotting through curves affects locomotion symmetry in Standardbred trotters. OBJECTIVES To investigate differences in objectively measured Standardbred trotter vertical motion symmetry between straight and non-banked, curved sections of oval trotting tracks during exercise warm-up, using a wireless inertial measurement unit (IMU) system. STUDY DESIGN Cross-sectional, observational study. METHODS Sixteen horses were included. Mixed models were used to assess associations between symmetry, track segment (straight vs curve) and stride duration. RESULTS Significant results for forelimb parameters were dependent on interactions between track segments and stride duration. At mean stride duration (0.611 second), during the curved track segment horses showed a lower maximum vertical position of the head after push-off of the outside forelimb (estimate -2.3 mm, P

Better methods to predict and prevent the emergence of zoonotic viruses could support future efforts to reduce the risk of epidemics. We propose a network science framework for understanding and predicting human and animal susceptibility to viral infections. Related approaches have so far helped to identify basic biological rules that govern cross-species transmission and structure the global virome. We highlight ways to make modelling both accurate and actionable, and discuss the barriers that prevent researchers from translating viral ecology into public health policies that could prevent future pandemics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Forecasting the risk of pathogen spillover from reservoir populations of wild or domestic animals is essential for the effective deployment of interventions such as wildlife vaccination or culling. Due to the sporadic nature of spillover events and limited availability of data, developing and validating robust, spatially explicit, predictions is challenging. Recent efforts have begun to make progress in this direction by capitalizing on machine learning methodologies. An important weakness of existing approaches, however, is that they generally rely on combining human and reservoir infection data during the training process and thus conflate risk attributable to the prevalence of the pathogen in the reservoir population with the risk attributed to the realized rate of spillover into the human population. Because effective planning of interventions requires that these components of risk be disentangled, we developed a multi-layer machine learning framework that separates these processes. Our approach begins by training models to predict the geographic range of the primary reservoir and the subset of this range in which the pathogen occurs. The spillover risk predicted by the product of these reservoir specific models is then fit to data on realized patterns of historical spillover into the human population. The result is a geographically specific spillover risk forecast that can be easily decomposed and used to guide effective intervention. Applying our method to Lassa virus, a zoonotic pathogen that regularly spills over into the human population across West Africa, results in a model that explains a modest but statistically significant portion of geographic variation in historical patterns of spillover. When combined with a mechanistic mathematical model of infection dynamics, our spillover risk model predicts that 897,700 humans are infected by Lassa virus each year across West Africa, with Nigeria accounting for more than half of these human infections., Author summary The 2019 emergence of SARS-CoV-2 is a grim reminder of the threat animal-borne pathogens pose to human health. Even prior to SARS-CoV-2, the spillover of pathogens from animal reservoirs was a persistent problem, with pathogens such as Ebola, Nipah, and Lassa regularly but unpredictably causing outbreaks. Machine-learning models that anticipate when and where pathogen transmission from animals to humans is likely to occur would help guide surveillance efforts and preemptive countermeasures like information campaigns or vaccination programs. We develop a novel machine learning framework that uses datasets describing the distribution of a virus within its host and the range of its animal host, along with data on spatial patterns of human immunity, to infer rates of animal-to-human transmission across a region. By training the model on data from the animal host alone, our framework allows rigorous validation of spillover predictions using human data. We apply our framework to Lassa fever, a viral disease of West Africa that is spread to humans by rodents, and use the predictions to update estimates of Lassa virus infections in humans. Our results suggest that Nigeria is most at risk for the emergence of Lassa virus, and should be prioritized for outbreak-surveillance.

Invasive alien species (IAS) are among the main causes of global biodiversity loss. Invasive brown (Rattus norvegicus) and black (R. rattus) rats, in particular, are leading drivers of extinction on islands, especially in the case of seabirds where >50% of all extinctions have been attributed to rat predation. Eradication is the primary form of invasive rat management, yet this strategy has resulted in a ~10-38% failure rate on islands globally. Genetic tools can help inform IAS management, but such applications to date have been largely reactive, time-consuming, and costly. Here, we developed a Genotyping-in-Thousands by sequencing (GT-seq) panel for rapid species identification and population assignment of invasive brown and black rats (RapidRat) in Haida Gwaii, an archipelago comprising ~150 islands off the central coast of British Columbia, Canada. We constructed an optimized panel of 443 single nucleotide polymorphisms (SNPs) using previously generated double-digest restriction-site associated DNA (ddRAD) genotypic data (27,686 SNPs) from brown (n = 295) and black rats (n = 241) sampled throughout Haida Gwaii. The informativeness of this panel for identifying individuals to species and island of origin was validated relative to the ddRAD results; in all comparisons, admixture coefficients and population assignments estimated using RapidRat were consistent. To demonstrate application, 20 individuals from novel invasions of three islands (Agglomerate, Hotspring, Ramsay) were genotyped using RapidRat, all of which were confidently assigned (>98.5% probability) to Faraday and Murchison Islands as putative source populations. These results indicated that a previous eradication on Hotspring Island was conducted at an inappropriate geographic scale; future management should expand the eradication unit to include neighboring islands to prevent re-invasion. Overall, we demonstrated that RapidRat is an effective tool for managing invasive rat populations in Haida Gwaii and provided a clear framework for GT-seq panel development for informing biodiversity conservation in other systems.

Background Lameness evaluation of Standardbred trotters can be challenging due to discrepancies in observed movement asymmetry between in-hand and track exercise, and between different trotting speeds. There are few studies on objective measurement of movement in Standardbreds, and little knowledge regarding biological variation and clinical significance of measured movement asymmetry in this breed. Objectives To quantify the prevalence and magnitude of objectively measured movement asymmetry in young Standardbred trotters, and identify associations with trainer, sex, height, track type and in-hand measurement prior to or after track trials. Study design Cross-sectional, observational study. Methods 114 Standardbred yearlings were evaluated with a wireless inertial sensor system during trot in-hand and when driven on a track. After exclusions relating to lameness or technical difficulties, 103 horses were included in the study; 77 were evaluated in-hand and on the track, 24 only in-hand and two only on the track. Results Front and/or hindlimb parameters were above asymmetry thresholds previously established for other breeds during in-hand trials for 94 (93%) horses, and during track trials for 74 (94%) horses. Most horses showed mild asymmetry. A minority of horses (20%) switched side of the asymmetry for one or more parameters between in-hand and track trials. Mixed model analyses revealed no significant effects of trial mode (in-hand or track trial, in-hand trial pre or post track trial, straight or oval track), trainer, or horse height. Females had a significant but small reduction of asymmetry in one front limb parameter (HDmax) compared to males (1.7 mm, 95% CI 0.18 to 3.28, p=0.03). Main limitations High data variability, reflected in large trial standard deviations, relating mainly to a lack of horse compliance. Conclusions A high proportion of Standardbred yearlings showed movement asymmetries. There was no group-level effect between in-hand and track trials, however considerable individual variation was observed.

Glucocorticoids are often measured in wildlife to assess physiological responses to environmental or ecological stress. Hair, blood, saliva, or fecal samples are generally used depending on the timescale of the stress response being investigated and species‐specific considerations. Here, we report the first use of hair samples to measure long‐term corticosterone levels in the climate‐sensitive American pika (Ochotona princeps). We validated an immunoassay‐based measurement of corticosterone extracted from hair samples and compared corticosterone estimates obtained from plasma, hair, and fecal samples of nine pikas. To demonstrate an ecological application of this technique, we characterized physiological stress in 49 pikas sampled and released at eight sites along two elevational transects. Microclimate variation was measured at each site using both ambient and subsurface temperature sensors. We used an information theoretic approach to compare support for linear, mixed‐effects models relating corticosterone estimates to microclimate, body size, and sex. Corticosterone was measured accurately in pika hair samples after correcting for the influence of sample mass on corticosterone extraction efficiency. Hair‐ and plasma‐based estimates of corticosterone were weakly correlated. The best‐supported model suggested that corticosterone was lower in larger, male pikas, and at locations with higher ambient temperatures in summer. Our results are consistent with a general negative relationship between body mass and glucocorticoid concentration observed across mammalian species, attributed to the higher mass‐specific metabolic rates of smaller bodied animals. The higher corticosterone levels in female pikas likely reflected the physiological demands of reproduction, as observed in a wide array of mammalian species. Additionally, we establish the first direct physiological evidence for thermal stress in the American pika through nonlethal sampling of corticosterone. Interestingly, our data suggest evidence for cold stress likely induced during the summer molting period. This technique should provide a useful tool to researchers wishing to assess chronic stress in climate‐sensitive mammals.

Background: Stratification by eosinophil and neutrophil counts increases our understanding of asthma and helps target therapy, but there is room for improvement in our accuracy in prediction of treatment responses and a need for better understanding of the underlying mechanisms. Objective: We sought to identify molecular subphenotypes of asthma defined by proteomic signatures for improved stratification. Methods: Unbiased label-free quantitative mass spectrometry and topological data analysis were used to analyze the proteomes of sputum supernatants from 246 participants (206 asthmatic patients) as a novel means of asthma stratification. Microarray analysis of sputum cells provided transcriptomics data additionally to inform on underlying mechanisms. Results: Analysis of the sputum proteome resulted in 10 clusters (ie, proteotypes) based on similarity in proteomic features, representing discrete molecular subphenotypes of asthma. Overlaying granulocyte counts onto the 10 clusters as metadata further defined 3 of these as highly eosinophilic, 3 as highly neutrophilic, and 2 as highly atopic with relatively low granulocytic inflammation. For each of these 3 phenotypes, logistic regression analysis identified candidate protein biomarkers, and matched transcriptomic data pointed to differentially activated underlying mechanisms. Conclusion: This study provides further stratification of asthma currently classified based on quantification of granulocytic inflammation and provided additional insight into their underlying mechanisms, which could become targets for novel therapies. asthma proteomics biomarkers eosinophils neutrophils

Quantitative gait analysis has revealed that a large proportion of horses in training, perceived as free from lameness by their owners, show movement asymmetries of equal magnitude to horses with mild clinical lameness. Whether these movement asymmetries are related to orthopaedic pain and/or pathology has yet to be further investigated. Therefore, the objective of this study was to determine whether movement asymmetries in riding horses in training are affected by anti-inflammatory treatment with meloxicam. In a crossover design, horses were treated with meloxicam or placebo for four days respectively, with a 14-16 day washout period between treatments. Objective movement analysis utilising body mounted accelerometers was performed on a hard and a soft surface before and on day four of each treatment. A trial mean was calculated for the differences between the two vertical displacement minima and maxima of head (HDmin, HDmax) and pelvis (PDmin, PDmax) per stride. Horses (n = 66) with trial mean asymmetries greater than 6 mm for HDmin or HDmax, or more than 3 mm for PDmin or PDmax, at baseline were included. The difference before and after each treatment in the measured movement asymmetry was assessed with linear mixed models. Treatment with meloxicam did not significantly affect the movement asymmetry in any of the models applied (all p>0.30). These results raise new questions: are the movement asymmetries in riding horses in training simply expressions of biological variation or are they related to pain/dysfunction that is non-responsive to meloxicam treatment?

Gastro-oesophageal reflux disease (GORD) and obesity are associated with frequent exacerbations and poor quality of life in asthmatics. Multiple mechanisms have been proposed for the effect of obesity, including modification of inflammation affecting epithelial cell proliferation and wound repair, while the role of GORD is poorly understood and proton pump inhibitor (PPI) are of variable efficacy. GORD might exert a deleterious effect by inducing vagal reflex, neuroinflammation and directly ( via microaspiration) triggering airway inflammation. Studies of reflux in animal models and human bronchial epithelial cell culture show varying impact on inflammation and airway remodelling. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Dr PEROTIN has nothing to disclose. Conflict of interest: Dr Schofield has nothing to disclose. Conflict of interest: Dr Wilson has nothing to disclose. Conflict of interest: Dr Ward has nothing to disclose. Conflict of interest: Dr Brandsma has nothing to disclose. Conflict of interest: Dr Strazzeri has nothing to disclose. Conflict of interest: Dr Bansal has nothing to disclose. Conflict of interest: Dr Yang has nothing to disclose. Conflict of interest: Dr Rowe reports and a full time employee and shareholder of Janssen Pharmaceutical Companies of Johnson and Johnson. Conflict of interest: Miss Corfield has nothing to disclose. Conflict of interest: Dr Lutter has nothing to disclose. Conflict of interest: Prof. Shaw reports personal fees and non-financial support from AstraZeneca, personal fees from Boehringer Ingelheim, personal fees from Novartis, personal fees from Teva, personal fees from Circassia, and a grant from GSK, outside the submitted work. Conflict of interest: Dr Bakke reports personal fees from GSK, AZ, Novartis andTeva, outside the submitted work. Conflict of interest: MC have no conflict of interest to disclose. Conflict of interest: Dr Dahlen has nothing to disclose. Conflict of interest: Dr Fowler reports personal fees and non-financial support from AstraZeneca, grants and personal fees from Boehringer Ingelheim, personal fees from Novartis, personal fees from Teva, outside the submitted work. Conflict of interest: Dr Horvath reports personal fees from Astra Zeneca, Boehringer Ingelheim, Novartis, CSL, Chiesi, Roche, GSK, Berlin-Chemie and Sandoz, outside the submitted work. Conflict of interest: Dr Howarth reports personal fees from GSK, outside the submitted work. Conflict of interest: Dr Krug has nothing to disclose. Conflict of interest: Dr Montuschi has nothing to disclose. Conflict of interest: Dr Sanak has nothing to disclose. Conflict of interest: Dr Sandstrom reports other monetary support from Boehringer Ingelheim, outside the submitted work. Conflict of interest: Dr Sun has nothing to disclose. Conflict of interest: Dr Pandis has nothing to disclose. Conflict of interest: Dr Auffray reports grants from Innovative Medicine Initiative, during the conduct of the study. Conflict of interest: Dr De Meulder reports grants from Innovative Medicine Initiative, during the conduct of the study. Conflict of interest: Ms. Lefaudeux reports grants from Innovative Medicine Initiative, grants from Innovative Medicine Initiative, during the conduct of the study. Conflict of interest: Dr Riley reports and I have shares in and I am employed by GSK. Conflict of interest: Dr Sousa has nothing to disclose. Conflict of interest: Dr Dahlen has nothing to disclose. Conflict of interest: Dr Adcock reports grants from EU-IMI, during the conduct of the study. Conflict of interest: KFC has received honoraria for participating in Advisory Board meetings of GSK, AZ, BI, Teva, Novartis and Merck regarding treatments for asthma and chronic obstructive pulmonary disease and has also been renumerated for speaking engagements. Conflict of interest: Dr Sterk reports grants from Innovative Medicines Initiative, during the conduct of the study. Conflict of interest: Dr Skipp has nothing to disclose. Conflict of interest: Dr Collins reports a patent application for use of a genetically modified Drosophila line carrying one or more mammalian genes associated with a chronic respiratory disease and uses to screen the impact of such genes. Conflict of interest: Dr Davies has nothing to disclose. Conflict of interest: Dr Djukanovic reports receiving fees for lectures at symposia organised by Novartis, AstraZeneca and TEVA, consultation for TEVA and Novartis as member of advisory boards, and participation in a scientific discussion about asthma organised by GlaxoSmithKline. He is a co-founder and current consultant, and has shares in Synairgen, a University of Southampton spin out company.

The deamination of adenosine to inosine (known as A-to-I editing) is one of the most prevalent RNA modifications that occurs in metazoans and is mediated by the Adenosine Deaminase Acting on RNA (ADAR) family of enzymes. The enzyme ADAR/ADAR1 catalyzes the majority of A-to-I editing where it has been demonstrated to effect coding sequence, miRNA function and silencing of Alu repetitive elements1. A critical function of ADAR1 is to edit double stranded RNA (dsRNA) structures that can activate the cytoplasmic nucleoside sensors MDA5 and PKR, preventing aberrant activation of an innate immune type I interferon (IFN) response2,3. Consistent with this hypothesis, mutations in ADAR1 and other enzymes involved in nucleoside metabolism and/or sensing are found in Aicardi-Goutiéres Syndrome (AGS), an interferonopathy associated with spontaneous interferon production4. Through analysis of pooled CRISPR and shRNA screening data, we have identified that cancer cell lines with elevated expression of specific type I interferon stimulated genes (ISGs) are dependent on ADAR1 for their survival. Tumor cells can display elevated intrinsic type I interferon signaling and associated dsRNA burden due to multiple factors, including chronic cytoplasmic DNA activation of STING, oncovirus infection and other proinflammatory signals. We have identified a core subset of 26 ISGs that predict a cellular dependency on ADAR1 and demonstrate elevated expression of these ISGs in a diverse range of tumor types including head and neck squamous cell carcinoma, breast cancer, esophageal cancer, and lung cancer. Utilizing an isogenic cell line with low basal type I interferon signaling and knockout of ADAR1, we demonstrate that we can induce cell death through exogenous treatment with the dsRNA mimetic poly-(I:C) or type I interferons (IFNα or IFNβ) but not the type II interferon IFNγ. We believe that chronic ISG expression and dsRNA burden creates a dependency on ADAR1 to prevent activation of MDA5 and PKR. These data suggest an ADAR1 inhibitor could be beneficial for the treatment of tumors with elevated ISG expression. Eisenberg et al., Nature Review Genetics (2018) Ahmad et al., Cell (2018) Chung et al., Cell (2018) Crow et al., Nature Review Immunology (2015) Citation Format: Alexandra K. Gardino, Cindy Collins, Maureen S. Lynes, P. Ann Boriack-Sjodin, Robert A. Copeland, Scott A. Ribich. Elevated cancer-intrinsic type I interferon signaling confers a dependency on the RNA editor ADAR1 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B130. doi:10.1158/1535-7163.TARG-19-B130

CARM1 is an arginine methyltransferase with diverse histone and non-histone substrates implicated in the regulation of cellular processes including transcriptional co-activation and RNA processing. CARM1 overexpression has been reported in multiple cancer types and has been shown to modulate oncogenic pathways in in vitro studies. Detailed understanding of the mechanism of action of CARM1 in oncogenesis has been limited by a lack of selective tool compounds, particularly for in vivo studies. We describe the identification and characterization of, to our knowledge, the first potent and selective inhibitor of CARM1 that exhibits anti-proliferative effects both in vitro and in vivo and, to our knowledge, the first demonstration of a role for CARM1 in multiple myeloma (MM). EZM2302 (GSK3359088) is an inhibitor of CARM1 enzymatic activity in biochemical assays (IC50 = 6 nM) with broad selectivity against other histone methyltransferases. Treatment of MM cell lines with EZM2302 leads to inhibition of PABP1 and SMB methylation and cell stasis with IC50 values in the nanomolar range. Oral dosing of EZM2302 demonstrates dose-dependent in vivo CARM1 inhibition and anti-tumor activity in an MM xenograft model. EZM2302 is a validated chemical probe suitable for further understanding the biological role CARM1 plays in cancer and other diseases.

SMYD3 has been implicated in a range of cancers; however, until now no potent selective small molecule inhibitors have been available for target validation studies. A novel oxindole series of SMYD3 inhibitors was identified through screening of the Epizyme proprietary histone methyltransferase-biased library. Potency optimization afforded two tool compounds, sulfonamide EPZ031686 and sulfamide EPZ030456, with cellular potency at a level sufficient to probe the in vitro biology of SMYD3 inhibition. EPZ031686 shows good bioavailability following oral dosing in mice making it a suitable tool for potential in vivo target validation studies.

Bacterial NAD+-dependent DNA ligase (LigA) is an essential enzyme involved in the repair, replication, and recombination of DNA in all bacteria, making it an attractive target in antibacterial drug discovery. A novel series of LigA inhibitors was discovered by combining information from fragment-based lead generation, X-ray crystal structures of previously discovered inhibitors bound to the adenosine binding site of LigA bound to and established structure–activity relationships of LigA inhibitors. The compounds described here were evaluated in a variety of Gram-positive and Gram-negative pathogens as well as evaluation of physical properties.

Rearrangements of the MLL gene define a genetically distinct subset of acute leukemias with poor prognosis. Current treatment options are of limited effectiveness; thus, there is a pressing need for new therapies for this disease. Genetic and small molecule inhibitor studies have demonstrated that the histone methyltransferase DOT1L is required for the development and maintenance of MLL-rearranged leukemia in model systems. Here we describe the characterization of EPZ-5676, a potent and selective aminonucleoside inhibitor of DOT1L histone methyltransferase activity. The compound has an inhibition constant value of 80 pM, and demonstrates 37 000-fold selectivity over all other methyltransferases tested. In cellular studies, EPZ-5676 inhibited H3K79 methylation and MLL-fusion target gene expression and demonstrated potent cell killing that was selective for acute leukemia lines bearing MLL translocations. Continuous IV infusion of EPZ-5676 in a rat xenograft model of MLL-rearranged leukemia caused complete tumor regressions that were sustained well beyond the compound infusion period with no significant weight loss or signs of toxicity. EPZ-5676 is therefore a potential treatment of MLL-rearranged leukemia and is under clinical investigation.

DNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC 50 ) of 3 μM. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated in Staphylococcus aureus by plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of the gyrB genes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated against Streptococcus pneumoniae in a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications.

Interruption of TGFbeta signaling through inhibition of the TGFbetaR1 kinase domain may prove to have beneficial effect in both fibrotic and oncological diseases. Herein we describe the SAR of a novel series of TGFbetaR1 kinase inhibitors containing a pyrazolone core. Most TGFbetaR1 kinase inhibitors described to date contain a core five-membered ring bearing N as H-bond acceptor. Described herein is a novel strategy to replace the core structure with pyrazolone ring, in which the carbonyl group is designed as an H-bond acceptor to interact with catalytic Lys 232.

Objective— TGF-β plays a significant role in vascular injury-induced stenosis. This study evaluates the efficacy of a novel, small molecule inhibitor of ALK5/ALK4 kinase, in the rat carotid injury model of vascular fibrosis. Methods and Results— The small molecule, SM16, was shown to bind with high affinity to ALK5 kinase ATP binding site using a competitive binding assay and biacore analysis. SM16 blocked TGF-β and activin-induced Smad2/3 phosphorylation and TGF-β-induced plasminogen activator inhibitor (PAI)-luciferase activity in cells. Good overall selectivity was demonstrated in a large panel of kinase assays, but SM16 also showed nanomolar inhibition of ALK4 and weak (micromolar) inhibition of Raf and p38. In the rat carotid injury model, SM16 dosed once daily orally at 15 or 30 mg/kg SM16 for 14 days caused significant inhibition of neointimal thickening and lumenal narrowing. SM16 also prevented induction of adventitial smooth muscle α-actin–positive myofibroblasts and the production of intimal collagen, but did not decrease the percentage of proliferative cells. Conclusion— These results are the first to demonstrate the efficacy of an orally active, small-molecule ALK5/ALK4 inhibitor in a vascular fibrosis model and suggest the potential therapeutic application of these inhibitors in vascular fibrosis.

B cell activating factor (BAFF), a ligand belonging to the tumor necrosis factor (TNF) family, plays a critical role in regulating survival and activation of peripheral B cell populations and has been associated with autoimmune disease. BAFF is known to interact with three receptors, BCMA, TACI and BAFF-R, that have distant similarities with other receptors of the TNF family. We have determined the crystal structure of the TNF-homologous domain of BAFF at 2.8 A resolution. The structure reveals significant differences when compared to other TNF family members, including an unusually long D-E loop that participates in the formation of a deep, concave and negatively charged region in the putative receptor binding site. The BAFF structure was further used to generate a homology model of APRIL, a closely related TNF family ligand that also binds to BCMA and TACI, but not BAFF-R. Analysis of the putative receptor binding sites of BAFF and APRIL suggests that differences in the D-E loop structure and electrostatic surface potentials may be important for determining binding specificities for BCMA, TACI and BAFF-R.

We argue that within-sex variation resulting from the prenatal organizational and adult activational effects of gonadal steroid hormones has the potential to obscure between sex differences in cognitive performance and functional cerebral asymmetry. Two putative markers for prenatal testosterone, finger ridge count (FRC) asymmetry and the 2D:4D finger length ratio, have been linked to within-sex variation in cognitive performance. In particular, FRC allows the identification of men and women who show a reversal of the typical sex-related pattern of task performance. Three paradigms for the study of activational effects—seasonal, menstrual, and diurnal hormonal cycles—have evaluated changes in task performance and functional cerebral asymmetry. The performance of sex-dimorphic, but not sex-neutral, tasks changes with estrogen across the menstrual cycle and with testosterone across its seasonal and diurnal cycles. Functional cerebral asymmetry also changes systematically across both the menstrual cycle and the diurnal testosterone cycle in such a way that suggests left hemisphere performance increases as testosterone levels decline whereas right hemisphere performance increases as estrogen levels decline. In studies of sex differences, such correlates of within-sex hormone-related differences are rarely measured or controlled. Whatever the explanation for the associations of putative markers and hormone cycles with differences in cognitive abilities and cerebral asymmetry, it is clear that these relationships have the potential to contribute to the elusive nature of sex differences in cognition and functional brain organization.

The canonical Wnt pathway plays an important role in embryonic development, adult tissue homeostasis, and cancer. Germline mutations of several Wnt pathway components, such as Axin, APC, and ß-catenin, can lead to oncogenesis. Inhibition of the poly(ADP-ribose) polymerase (PARP) catalytic domain of the tankyrases (TNKS1 and TNKS2) is known to inhibit the Wnt pathway via increased stabilization of Axin. In order to explore the consequences of tankyrase and Wnt pathway inhibition in preclinical models of cancer and its impact on normal tissue, we sought a small molecule inhibitor of TNKS1/2 with suitable physicochemical properties and pharmacokinetics for hypothesis testing in vivo. Starting from a 2-phenyl quinazolinone hit (compound 1), we discovered the pyrrolopyrimidinone compound 25 (AZ6102), which is a potent TNKS1/2 inhibitor that has 100-fold selectivity against other PARP family enzymes and shows 5 nM Wnt pathway inhibition in DLD-1 cells. Moreover, compound 25 can be formulated well in a clinically relevant intravenous solution at 20 mg/mL, has demonstrated good pharmacokinetics in preclinical species, and shows low Caco2 efflux to avoid possible tumor resistance mechanisms.

Carbonic anhydrase IV (CAIV) is a membrane-associated enzyme anchored to plasma membrane surfaces by a phosphatidylinositol glycan linkage. We have determined the 2.8-angstroms resolution crystal structure of a truncated, soluble form of recombinant murine CAIV. We have also determined the structure of its complex with a drug used for glaucoma therapy, the sulfonamide inhibitor brinzolamide (Azopt). The overall structure of murine CAIV is generally similar to that of human CAIV; however, some local structural differences are found in the active site resulting from amino acid sequence differences in the "130's segment" and the residue-63 loop (these may affect the nearby catalytic proton shuttle, His-64). Similar to human CAIV, the C-terminus of murine CAIV is surrounded by a substantial electropositive surface potential that may stabilize the interaction with the phospholipid membrane. Binding interactions observed for brinzolamide rationalize the generally weaker affinity of inhibitors used in glaucoma therapy toward CAIV compared with CAII.

Background: Endocrine disrupting chemicals (EDCs) may increase the risk of autism, possibly by perturbing fetal hormone signaling or metabolism, but this complex exposure mixture makes identifying ...

In an attempt to identify novel inhibitors of NAD(+)-dependent DNA ligase (LigA) that are not affected by a known resistance mutation in the adenosine binding pocket, a detailed analysis of the binding sites of a variety of bacterial ligases was performed. This analysis revealed several similarities to the adenine binding region of kinases, which enabled a virtual screen of known kinase inhibitors. From this screen, a thienopyridine scaffold was identified that was shown to inhibit bacterial ligase. Further characterization through structure and enzymology revealed the compound was not affected by a previously disclosed resistance mutation in Streptococcus pneumoniae LigA, Leu75Phe. A subsequent medicinal chemistry program identified substitutions that resulted in an inhibitor with moderate activity across various Gram-positive bacterial LigA enzymes.

There is an urgent need for new antibacterials that pinpoint novel targets and thereby avoid existing resistance mechanisms. We have created novel synthetic antibacterials through structure-based drug design that specifically target bacterial thymidylate kinase (TMK), a nucleotide kinase essential in the DNA synthesis pathway. A high-resolution structure shows compound TK-666 binding partly in the thymidine monophosphate substrate site, but also forming new induced-fit interactions that give picomolar affinity. TK-666 has potent, broad-spectrum Gram-positive microbiological activity (including activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus), bactericidal action with rapid killing kinetics, excellent target selectivity over the human ortholog, and low resistance rates. We demonstrate in vivo efficacy against S. aureus in a murine infected-thigh model. This work presents the first validation of TMK as a compelling antibacterial target and provides a rationale for pursuing novel clinical candidates for treating Gram-positive infections through TMK.

We present the discovery and optimization of a novel series of bacterial topoisomerase inhibitors. Starting from a virtual screening hit, activity was optimized through a combination of structure-based design and physical property optimization. Synthesis of fewer than a dozen compounds was required to achieve inhibition of the growth of methicillin-resistant Staphyloccus aureus (MRSA) at compound concentrations of 1.56 μM. These compounds simultaneously inhibit DNA gyrase and Topoisomerase IV at similar nanomolar concentrations, reducing the likelihood of the spontaneous occurrence of target-based mutations resulting in antibiotic resistance, an increasing threat in the treatment of serious infections.

SMYD3 (Set and Mynd Domain containing 3) is a lysine methyltransferase overexpressed in several cancer types including breast, prostrate, pancreatic, and lung, and this overexpression is associated with poor clinical prognosis. Genetic knockdown of SMYD3 by shRNA has been shown to decrease proliferation in a range of cancer cell lines suggesting that inhibition of SMYD3 may have therapeutic utility. In this presentation we describe the discovery and optimization of a novel series of oxindole sulfonamides and sulfamides with SMYD3 inhibitory activity. One of these compounds, EPZ030456, has a SMYD3 biochemical IC50 of 4 nM and is active in cells with an IC50 of 48 nM in a trimethyl MAP3K2 (MEKK2) in-cell western (ICW) assay. The crystal structure of this compound was solved with SMYD3 and the nucleotide substrate, S-adenosylmethionine and shows the oxindole portion of the molecule extends into the SMYD3 lysine binding channel. EPZ030456 shows < 30% inhibition at a 10 uM screening concentration against 17 histone methyltransferase targets tested, including SMYD2. Further optimization within the series resulted in EPZ031686 which has similar potency to EPZ030456 with a biochemical IC50 of 3 nM and an ICW IC50 of 36 nM and in addition exhibits good bioavailability following oral dosing in mice. Hence, EPZ031686 is a suitable tool to study the role of SMYD3 in cancer and other therapeutic areas, using both in vitro and in vivo models. Citation Format: Lorna H. Mitchell, Paula A. Boriack-Sjodin, Sherri Smith, Michael Thomenius, Nathalie Rioux, Michael Munchhof, James E. Mills, Christine Klaus, Jennifer Totman, Thomas V. Riera, Alejandra Raimondi, Suzanne L. Jacques, Megan Foley, Nigel J. Waters, Kevin W. Kuntz, Tim J. Wigle, Margaret Porter Scott, Robert A. Copeland, Jesse J. Smith, Richard Chesworth. Identification of a novel potent selective SMYD3 inhibitor with oral bioavailability. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C85.

Background: Deregulation of platelet-derived growth factor (PDGF) signaling is a hallmark of malignant glioma. Two alternatively spliced PDGF-A mRNAs have been described, corresponding to a long (L) and a short (S) isoform of PDGF-A. In contrast to PDGF-A(S), the PDGF-A(L) isoform has a lysine and arginine rich carboxy-terminal extension that acts as an extracellular matrix retention motif. However, the exact role of PDGF-A(L) and how it functionally differs from the shorter isoform is not well understood. Methodology/Principal Findings: We overexpressed PDGF-A(L) as a transgene under control of the glial fibrillary acidic protein (GFAP) promoter in the mouse brain. This directs expression of the transgene to astrocytic cells and GFAP expressing neural stem cells throughout the developing and adult central nervous system. Transgenic mice exhibited a phenotype with enlarged skull at approximately 6-16 weeks of age and they died between 1.5 months and 2 years of age. We detected an increased number of undifferentiated cells in all areas of transgene expression, such as in the subependymal zone around the lateral ventricle and in the cerebellar medulla. The cells stained positive for Pdgfr-alpha, Olig2 and NG2 but this population did only partially overlap with cells positive for Gfap and the transgene reporter. Interestingly, a few mice presented with overt neoplastic glioma-like lesions composed of both Olig2 and Gfap positive cell populations and with microvascular proliferation, in a wild-type p53 background. Conclusions: Our findings show that PDGF-A(L) can induce accumulation of immature cells in the mouse brain. The strong expression of NG2, Pdgfr-alpha and Olig2 in PDGF-A(L) brains suggests that a fraction of these cells are oligodendrocyte progenitors. In addition, accumulation of fluid in the subarachnoid space and skull enlargement indicate that an increased intracranial pressure contributed to the observed lethality

Udgivelsesdato: 2009-Dec OBJECTIVE: Dietary linoleic acid, an n-6 polyunsaturated fatty acid, is metabolised to arachidonic acid, a component of colonocyte membranes. Metabolites of arachidonic acid have pro-inflammatory properties and are increased in the mucosa of patients with ulcerative colitis. The aim of this investigation was to conduct the first prospective cohort study investigating if a high dietary intake of linoleic acid increases the risk of developing incident ulcerative colitis. DESIGN AND SETTING: Dietary data from food frequency questionnaires were available for 203 193 men and women aged 30-74 years, resident in the UK, Sweden, Denmark, Germany or Italy and participating in a prospective cohort study, the European Prospective Investigation into Cancer and Nutrition (EPIC). These participants were followed up for the diagnosis of ulcerative colitis. Each case was matched with four controls and the risk of disease calculated by quartile of intake of linoleic acid adjusted for gender, age, smoking, total energy intake and centre. RESULTS: A total of 126 participants developed ulcerative colitis (47% women) after a median follow-up of 4.0 years (range, 1.7-11.3 years). The highest quartile of intake of linoleic acid was associated with an increased risk of ulcerative colitis (odds ratio (OR) = 2.49, 95% confidence interval (CI) = 1.23 to 5.07, p = 0.01) with a significant trend across quartiles (OR = 1.32 per quartile increase, 95% CI = 1.04 to 1.66, p = 0.02 for trend). CONCLUSIONS: The data support a role for dietary linoleic acid in the aetiology of ulcerative colitis. An estimated 30% of cases could be attributed to having dietary intakes higher than the lowest quartile of linoleic acid intake.

We conducted a nested case-control study within two prospective cohorts, the New York University Women's Health Study and the Northern Sweden Health and Disease Study, to examine the association between prediagnostic circulating levels of 25-hydroxy vitamin D (25(OH)D) and the risk of subsequent invasive epithelial ovarian cancer (EOC). The 25(OH)D levels were measured in serum or plasma from 170 incident cases of EOC and 373 matched controls. Overall, circulating 25(OH)D levels were not associated with the risk of EOC in combined cohort analysis: adjusted OR for the top tertile versus the reference tertile, 1.09 (95% CI, 0.59–2.01). In addition, there was no evidence of an interaction effect betweenVDRSNP genotype or haplotype and circulating 25(OH)D levels in relation to ovarian cancer risk, although more complex gene-environment interactions may exist.

LFA-1 ICAM inhibitors based on ortho- and meta-phenol templates were designed and synthesized by Mitsunobu chemistry. The selection of targets was guided by X-ray co-crystal data, and led to compounds which showed an up to 30-fold increase in potency over reference compound 1 in the LFA-1/ICAM1-Ig assay. The most active compound exploited a new hydrogen bond to the I-domain and exhibited subnanomolar potency.

The multifunctional cytokine, TGF-β, is often overexpressed in human tumors and in preclinical studies has been demonstrated to have autocrine and paracrine protumor activities including immune evasion, invasiveness, epithelial to mesenchymal transition, angiogenesis, tumor-stromal interactions, survival, induction of tumor interstitial pressure, and decreased drug penetration. These findings suggest that antagonism of the TGF-β pathway may be of benefit in the treatment of cancer. One attractive target, the type I TGF-β receptor (ALK5) has an intracellular serine/threonine kinase, which is required for TGF-β signaling and is amenable to inhibition by small molecule, ATP binding site-targeted kinase inhibitors.

Co-activator-associated arginine methyltransferase 1 (CARM1) is a protein arginine N-methyltransferase (PRMT) enzyme that has been implicated in a variety of cancers including AML (1) and breast (2), prostate (3), lung (4) and colorectal (5) carcinomas. CARM1 is known to methylate H3 histones and non-histone substrates including p300/CBP, AIB1/SRC-3 and PABP1 (6). To date, several crystal structures of CARM1 have been solved, including structures with small molecule inhibitors (7), but no ternary structures with nucleotide and peptide substrate have been reported. Here, the crystal structures of human CARM1 with the SAM mimic sinefungin (SFG) and three different peptide sequences from histone H3 and PAPB1 are presented, and both non-methylated and singly-methylated arginine residues are exemplified. Extensive interactions are seen with residues Glu266, Glu257, and His414 and the substrate arginine side chain. Two key hydrogen bonds are made by the side chain of Asn161 with the backbone carbonyl of the P’1 peptide residue and the backbone NH of the P’3 peptide residue. The carbonyl of the P1 peptide residue engages the protein through water mediated hydrogen bonds. These structures show how the CARM1 binding site is capable of accommodating a variety of peptide sequences. Comparisons to known CARM1 complexes with small molecules provide additional insights into inhibitor design. 1. Vu, L. P. et al, PRMT4 blocks myeloid differentiation by assembling a methyl-RUNX1-dependent repressor complex. Cell Rep 2013, 5 (6), 1625-38. 2. Al-Dhaheri, M. et al, CARM1 is an important determinant of ERalpha-dependent breast cancer cell differentiation and proliferation in breast cancer cells. Cancer Res 2011, 71 (6), 2118-28. 3. Kim, Y. R. et al, Differential CARM1 expression in prostate and colorectal cancers. BMC Cancer 2010, 10, 197. 4. Elakoum, R. et al, CARM1 and PRMT1 are dysregulated in lung cancer without hierarchical features. Biochimie 2014, 97, 210-8. 5. Ou, C. Y. et al, A coactivator role of CARM1 in the dysregulation of beta-catenin activity in colorectal cancer cell growth and gene expression. Mol Cancer Res 2011, 9 (5), 660-70. 6. Bedford M.T.; Clarke S.G. Protein arginine methylation in mammals: who, what, and why. Mol Cell 2009, 33, 1-13. 7. Sack, J.S et al, Structural basis for CARM1 inhibition by indole and pyrazole inhibitors. Biochem J 2011, 436, 331-9. Citation Format: Ann Boriack-Sjodin, Lei Jin, Suzanne L. Jacques, Allison Drew, Margaret Porter Scott, Scott Ribich, Oscar Moradei. Crystal structures of CARM1 bound to sinefungin and diverse peptide substrates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2437. doi:10.1158/1538-7445.AM2015-2437

The paper presents a method for analysis of thermal injuries in patients undergoing hyperthermia treatment of the prostate. The application of the method shown here is the analysis of a treatment session in a patient with prostatic hyperplasia. The paper also discuss investigations performed in model experiments mimicing the clinical situation.

X-ray crystal structures of carbonic anhydrase II (CAII) complexed with sulfonamide inhibitors illuminate the structural determinants of high affinity binding in the nanomolar regime. The primary binding interaction is the coordination of a primary sulfonamide group to the active site zinc ion. Secondary interactions fine-tune tight binding in regions of the active site cavity >5 A away from zinc, and this work highlights three such features: (1) advantageous conformational restraints of a bicyclic thienothiazene-6-sulfonamide-1,1-dioxide inhibitor skeleton in comparison with a monocyclic 2,5-thiophenedisulfonamide skeleton; (2) optimal substituents attached to a secondary sulfonamide group targeted to interact with hydrophobic patches defined by Phe131, Leu198, and Pro202; and (3) optimal stereochemistry and configuration at the C-4 position of bicyclic thienothiazene-6-sulfonamides; the C-4 substituent can interact with His64, the catalytic proton shuttle. Structure-activity relationships rationalize affinity trends observed during the development of brinzolamide (Azopt), the newest carbonic anhydrase inhibitor approved for the treatment of glaucoma.

Data from long-term measurements of ambient NO2 concentrations at roof level in 15 Swedish cities have been used to verify expected trends in urban traffic NOx emissions, resulting mainly from the growth in the number of threeway catalyst (TWC) cars in Sweden since the mid 1980s. The result show that, with few exceptions, all cities exhibit a highly significant downward trend in ambient NO2 concentration since the winter season 1986/1987, as regards both winter season averages and 98th percentiles of daily averages, with an average decrease in both cases of approximately 30% through the winter season 1993/1994. The same trend is also observed when meteorological variations between years are taken into account. Corrections for NO2 in background air yield an even stronger downward trend, or an average 40% decrease for the study period. Simultaneously, rough calculations indicate a 30% decrease in urban traffic NOx emissions during the study period. The conclusions are that, since emission calculations always involve a high degree of uncertainty, use of data from long-term measurements of NO2 concentrations in urban air can be very helpful in establishing real-world trends for urban traffic NOx emissions, as soon as NOx-levels are low enough for the NO+ozone reaction to become 'NOx-limited'.

The three-dimensional structure of murine mitochondrial carbonic anhydrase V has been determined and refined at 2.45-A resolution (crystallographic R factor = 0.187). Significant structural differences unique to the active site of carbonic anhydrase V are responsible for differences in the mechanism of catalytic proton transfer as compared with other carbonic anhydrase isozymes. In the prototypical isozyme, carbonic anhydrase II, catalytic proton transfer occurs via the shuttle group His-64; carbonic anhydrase V has Tyr-64, which is not an efficient proton shuttle due in part to the bulky adjacent side chain of Phe-65. Based on analysis of the structure of carbonic anhydrase V, we speculate that Tyr-131 may participate in proton transfer due to its proximity to zinc-bound solvent, its solvent accessibility, and its electrostatic environment in the protein structure. Finally, the design of isozyme-specific inhibitors is discussed in view of the complex between carbonic anhydrase V and acetazolamide, a transition-state analogue. Such inhibitors may be physiologically important in the regulation of blood glucose levels.

The muscle fiber stands alongside the red blood cell and the giant axon as one of the three classical cell types that have had major application in investigating ion transport processes in cell membranes. Of these three cell types, the muscle fiber was the first to provide definite evidence for a sodium pump. The ability of the sodium pump to produce an electrical potential difference across the cell membrane was also first demonstrated in muscle fibers. This important property of the sodium pump is now known to have physiological significance in many other types of cells. In this review, electrolyte transport investigations in skeletal muscle are traced from their inception to the current state of the field. Applications of major research techniques are discussed and key results are summarized. An overview of electrolyte transport in muscle, this article emphasizes relationships between the muscle fiber membrane potential and ionic transport processes.

In dispersed acinar cells from guinea pig pancreas, cholecystokinin variants (CCK39 and CCK33) or carboxyl-terminal octapeptide of cholecystokinin (CCK-OP) caused significant increases in outflux of 45Ca, cyclic GMP, and release of amylase. In homogenates of acinar cells each peptide caused a significant increase in adenylate cyclase activity. For each function tested (1) CCK39 was equipotent with CCK33, (2) CCK39 and CCK33 were 10 to 30 times less potent than CCK-OP, (3) the efficacies of CCK39, CCK33, and CCK-OP were the same, and (4) none of these effects were altered by concentrations of atropine sufficient to abolish the action of muscarinic cholinergic agents.

The aim of the present study was to investigate whether circulating somatostatin takes part in the inhibition of gastric acid secretion induced by acidification of the antrum and the duodenal bulb. Dogs provided with chronic pouches of the antrum or the duodenal bulb, and with Pavlov pouches were used in the study. Peripheral venous somatostatin levels rose on the average to ˜36±15 pmol/1 and 20±3 pmol/1 during perfusion of the antral or bulbar pouches with 0.1 M HC1 respectively. A slight increase was also obtained on antral perfusion with solutions of pH 2.2–3 and on bulbar perfusion with solutions of pH 2.2, whereas peripheral somatostatin levels remained unchanged, when the pouches were exposed to solutions of higher pH. The increase of plasma somatostatin levels coincided in time with the release of somatostatin into the lumen of the antrum and of the duodenal bulb, which showed the same pH dependency. Also plasma gastrin levels rose during perfusion of the pouches with 0.1 M HCI, this was especially the case during perfusion of the antral pouches. Perfusion of bulbar pouches, but not of antral pouches with 0.1 M HC1 inhibited the pentagastrin (2 μg/kg/h) stimulated gastric acid secretions. Infusions of somatostatin (0.15, 0.3 and 0.6 nmol/kg/h) caused plasma somatostatin levels ˜0, 0–20 and ˜50 pmol/1 respectively. Infusions of somatostatin (0.3-1.2 nmol/kg/h) inhibited pentagastrin stimulated (2 μg/kg/h) gastric acid secretion and 0.15 nmol/kg/h inhibited the acid secretion induced by pentagastrin (1 μg/kg/h). Thus infusions of somatostatin which inhibited pentagastrin induced acid secretion gave rise ot plasma somatostatin levels of comparable size ˜(0–50 pmol/1) to those induced by acidification of the antrum or the duodenal bulb ˜(36 and 20 pmol/1). We therefore suggest that endogenously released somatostatin may account for the inhibition of gastric acid secretion observed during acidification of the duodenal bulb, thus corresponding to the postulated hormone bulbogastrone. The existence of similar inhibitory substance of antral origin (i. e. the antral chalone) has been proposed. We also suggest that this substance corresponds to somatostatin, despite the fact that it is difficult to inhibit pentagastrin-induced gastric acid secretion by acidification of the antrum. It is suggested that the inhibitory effect on gastric acid secretion exerted by somatostatin released from the antrum might be masked under the present experimental conditions. Thus, gastrin and perhaps also other peptides, which stimulate acid secretion, are also released by antral acidification and they might in part counteract the depressant effect on acid secretion induced by somatostatin. Furthermore, since the antral pouches were innervated, vagovagal reflexes induced by antral acidification might also influence the rate of acid secretion. The main physiological function of the acid induced antral somatostatin release might be to block the release of gastrin in response to various stimuli.

When frog sartorius muscles recover from Na enrichment in the presence of external K, net K entry into the fibers occurs by both passive movement and active inward transport via a K pump. Under normal conditions, it has not been possible to experimentally distinguish these processes. Fractionation into the flux components must be accomplished from inferences concerning the K conductance or permeability during a period of rapid Na extrusion. The best estimates indicate that 60-80% of the K entry occurs via the K pump. In the presence of Ba ions, the membrane permeability to K is very much reduced. Under these conditions, Na-enriched muscles underwent a normal recovery in the presence of external K, and the amount of inward K movement due to the K pump rose to over 90% of the total K entry. The characteristics of the K pump studied by this means were: (a) essentially complete inhibition by 10(-4) M ouabain, (b) inhibition by [Na]omicron, (c) activation by [K]omicron according to a rectangular hyperbola in the absence of [Na]omicron, (d) linear activation by [Na]iota over a wide range in concentration, (e) zero or undetectably low pumping rate as [Na]iota leads to 0, (f) the number of Na ions actively transported per K ion actively transported is 1.4-1.7 normally and 1.1 in the presence of Ba.