Breed-Specific Hematological Phenotypes in the Dog: A Natural Resource for the Genetic Dissection of Hematological Parameters in a Mammalian Species

Remarkably little has been published on hematological phenotypes of the domestic dog, the most polymorphic species on the planet. Information on the signalment and complete blood cell count of all dogs with normal red and white blood cell parameters judged by existing reference intervals was extracted from a veterinary database. Normal hematological profiles were available for 6046 dogs, 5447 of which also had machine platelet concentrations within the reference interval. Seventy-five pure breeds plus a mixed breed control group were represented by 10 or more dogs. All measured parameters except mean corpuscular hemoglobin concentration (MCHC) varied with age. Concentrations of white blood cells (WBCs), neutrophils, monocytes, lymphocytes, eosinophils and platelets, but not red blood cell parameters, all varied with sex. Neutering status had an impact on hemoglobin concentration, mean corpuscular hemoglobin (MCH), MCHC, and concentrations of WBCs, neutrophils, monocytes, lymphocytes and platelets. Principal component analysis of hematological data revealed 37 pure breeds with distinctive phenotypes. Furthermore, all hematological parameters except MCHC showed significant differences between specific individual breeds and the mixed breed group. Twenty-nine breeds had distinctive phenotypes when assessed in this way, of which 19 had already been identified by principal component analysis. Tentative breed-specific reference intervals were generated for breeds with a distinctive phenotype identified by comparative analysis. This study represents the first large-scale analysis of hematological phenotypes in the dog and underlines the important potential of this species in the elucidation of genetic determinants of hematological traits, triangulating phenotype, breed and genetic predisposition

Tumor collagenase stimulatory factor (TCSF) expression and localization in human lung and breast cancers.

Tumor cell-derived collagenase stimulatory factor (TCSF) stimulates in vitro the biosynthesis of various matrix metalloproteinases involved in tumor invasion, such as interstitial collagenase, gelatinase A, and stromelysin 1. The expression of TCSF mRNAs was studied in vivo, using in situ hybridization and Northern blotting analysis, in seven normal tissues and in 22 squamous cell carcinomas of the lung, and in seven benign proliferations and in 22 ductal carcinomas of the mammary gland. By in situ hybridization, TCSF mRNAs were detected in 40 of 44 carcinomas, in pre-invasive and invasive cancer cells of both lung and breast cancers. TCSF mRNAs and gelatinase A mRNAs were both visualized in the same areas in serial sections in breast cancers, and were expressed by different cells, tumor cells, and fibroblasts. The histological results were confirmed by Northern blot analysis, which showed a higher expression of TCSF mRNAs in cancers than in benign and normal tissues. These observations support the hypothesis that TCSF is an important factor in lung and breast tumor progression

The P-Loop Domain of Yeast Clp1 Mediates Interactions Between CF IA and CPF Factors in Pre-mRNA 3′ End Formation

Cleavage factor IA (CF IA), cleavage and polyadenylation factor (CPF), constitute major protein complexes required for pre-mRNA 3′ end formation in yeast. The Clp1 protein associates with Pcf11, Rna15 and Rna14 in CF IA but its functional role remained unclear. Clp1 carries an evolutionarily conserved P-loop motif that was previously shown to bind ATP. Interestingly, human and archaean Clp1 homologues, but not the yeast protein, carry 5′ RNA kinase activity. We show that depletion of Clp1 in yeast promoted defective 3′ end formation and RNA polymerase II termination; however, cells expressing Clp1 with mutant P-loops displayed only minor defects in gene expression. Similarly, purified and reconstituted mutant CF IA factors that interfered with ATP binding complemented CF IA depleted extracts in coupled in vitro transcription/3′ end processing reactions. We found that Clp1 was required to assemble recombinant CF IA and that certain P-loop mutants failed to interact with the CF IA subunit Pcf11. In contrast, mutations in Clp1 enhanced binding to the 3′ endonuclease Ysh1 that is a component of CPF. Our results support a structural role for the Clp1 P-loop motif. ATP binding by Clp1 likely contributes to CF IA formation and cross-factor interactions during the dynamic process of 3′ end formation

In vitro synergistic cytoreductive effects of zoledronic acid and radiation on breast cancer cells

INTRODUCTION: Bisphosphonates are mostly used in the treatment of bone metastases. They have been shown to act synergistically with other chemotherapeutic agents. It is not known, however, whether similar synergistic effects exist with radiation on breast cancer cells. METHODS: Human MCF-7 breast cancer cells were treated with up to 100 μM zoledronic acid, were irradiated with up to 800 cGy or were exposed to combinations of both treatments to determine the antiproliferative effects of zoledronic acid and radiation. RESULTS: Zoledronic acid and radiation caused a dose-dependent and time-dependent decrease in cell viability (approximate 50% growth inhibition values were 48 μM and 20 μM for 24 hours and 72 hours, respectively, for zoledronic acid and 500 cGy for radiation). A synergistic cytotoxic effect of the combination of zoledronic acid and radiation was confirmed by isobologram analysis. CONCLUSION: These data constitute the first in vitro evidence for synergistic effects between zoledronic acid and radiation. This combination therapy might thus be expected to be more effective than either treatment alone in patients with metastatic breast carcinoma

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Human Pentraxin 3 Binds to the Complement Regulator C4b-Binding Protein

The long pentraxin 3 (PTX3) is a soluble recognition molecule with multiple functions including innate immune defense against certain microbes and the clearance of apoptotic cells. PTX3 interacts with recognition molecules of the classical and lectin complement pathways and thus initiates complement activation. In addition, binding of PTX3 to the alternative complement pathway regulator factor H was shown. Here, we show that PTX3 binds to the classical and lectin pathway regulator C4b-binding protein (C4BP). A PTX3-binding site was identified within short consensus repeats 1–3 of the C4BP α-chain. PTX3 did not interfere with the cofactor activity of C4BP in the fluid phase and C4BP maintained its complement regulatory activity when bound to PTX3 on surfaces. While C4BP and factor H did not compete for PTX3 binding, the interaction of C4BP with PTX3 was inhibited by C1q and by L-ficolin. PTX3 bound to human fibroblast- and endothelial cell-derived extracellular matrices and recruited functionally active C4BP to these surfaces. Whereas PTX3 enhanced the activation of the classical/lectin pathway and caused enhanced C3 deposition on extracellular matrix, deposition of terminal pathway components and the generation of the inflammatory mediator C5a were not increased. Furthermore, PTX3 enhanced the binding of C4BP to late apoptotic cells, which resulted in an increased rate of inactivation of cell surface bound C4b and a reduction in the deposition of C5b-9. Thus, in addition to complement activators, PTX3 interacts with complement inhibitors including C4BP. This balanced interaction on extracellular matrix and on apoptotic cells may prevent excessive local complement activation that would otherwise lead to inflammation and host tissue damage

Rhythmicity in Mice Selected for Extremes in Stress Reactivity: Behavioural, Endocrine and Sleep Changes Resembling Endophenotypes of Major Depression

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, including hyper- or hypo-activity of the stress hormone system, plays a critical role in the pathophysiology of mood disorders such as major depression (MD). Further biological hallmarks of MD are disturbances in circadian rhythms and sleep architecture. Applying a translational approach, an animal model has recently been developed, focusing on the deviation in sensitivity to stressful encounters. This so-called 'stress reactivity' (SR) mouse model consists of three separate breeding lines selected for either high (HR), intermediate (IR), or low (LR) corticosterone increase in response to stressors.In order to contribute to the validation of the SR mouse model, our study combined the analysis of behavioural and HPA axis rhythmicity with sleep-EEG recordings in the HR/IR/LR mouse lines. We found that hyper-responsiveness to stressors was associated with psychomotor alterations (increased locomotor activity and exploration towards the end of the resting period), resembling symptoms like restlessness, sleep continuity disturbances and early awakenings that are commonly observed in melancholic depression. Additionally, HR mice also showed neuroendocrine abnormalities similar to symptoms of MD patients such as reduced amplitude of the circadian glucocorticoid rhythm and elevated trough levels. The sleep-EEG analyses, furthermore, revealed changes in rapid eye movement (REM) and non-REM sleep as well as slow wave activity, indicative of reduced sleep efficacy and REM sleep disinhibition in HR mice.Thus, we could show that by selectively breeding mice for extremes in stress reactivity, clinically relevant endophenotypes of MD can be modelled. Given the importance of rhythmicity and sleep disturbances as biomarkers of MD, both animal and clinical studies on the interaction of behavioural, neuroendocrine and sleep parameters may reveal molecular pathways that ultimately lead to the discovery of new targets for antidepressant drugs tailored to match specific pathologies within MD

A Comparative Approach Linking Molecular Dynamics of Altered Peptide Ligands and MHC with In Vivo Immune Responses

The recognition of peptide in the context of MHC by T lymphocytes is a critical step in the initiation of an adaptive immune response. However, the molecular nature of the interaction between peptide and MHC and how it influences T cell responsiveness is not fully understood.We analyzed the immunological consequences of the interaction of MHC class II (I-Au) restricted 11-mer peptides of myelin basic protein with amino acid substitutions at position 4. These mutant peptides differ in MHC binding affinity, CD4+ T cell priming, and alter the severity of peptide-induced experimental allergic encephalomyelitis. Using molecular dynamics, a computational method of quantifying intrinsic movements of proteins at high resolution, we investigated conformational changes in MHC upon peptide binding. We found that irrespective of peptide binding affinity, MHC deformation appears to influence costimulation, which then leads to effective T cell priming and disease induction. Although this study compares in vivo and molecular dynamics results for three altered peptide ligands, further investigation with similar complexes is essential to determine whether spatial rearrangement of peptide-MHC and costimulatory complexes is an additional level of T cell regulation

The exchange activities of [Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) from methanogenic archaea in comparison with the exchange activities of [FeFe] and [NiFe] hydrogenases

[Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) catalyzes the reversible reduction of methenyltetrahydromethanopterin (methenyl-H4MPT+) with H2 to methylene-H4MPT, a reaction involved in methanogenesis from H2 and CO2 in many methanogenic archaea. The enzyme harbors an iron-containing cofactor, in which a low-spin iron is complexed by a pyridone, two CO and a cysteine sulfur. [Fe] hydrogenase is thus similar to [NiFe] and [FeFe] hydrogenases, in which a low-spin iron carbonyl complex, albeit in a dinuclear metal center, is also involved in H2 activation. Like the [NiFe] and [FeFe] hydrogenases, [Fe] hydrogenase catalyzes an active exchange of H2 with protons of water; however, this activity is dependent on the presence of the hydride-accepting methenyl-H4MPT+. In its absence the exchange activity is only 0.01% of that in its presence. The residual activity has been attributed to the presence of traces of methenyl-H4MPT+ in the enzyme preparations, but it could also reflect a weak binding of H2 to the iron in the absence of methenyl-H4MPT+. To test this we reinvestigated the exchange activity with [Fe] hydrogenase reconstituted from apoprotein heterologously produced in Escherichia coli and highly purified iron-containing cofactor and found that in the absence of added methenyl-H4MPT+ the exchange activity was below the detection limit of the tritium method employed (0.1 nmol min−1 mg−1). The finding reiterates that for H2 activation by [Fe] hydrogenase the presence of the hydride-accepting methenyl-H4MPT+ is essentially required. This differentiates [Fe] hydrogenase from [FeFe] and [NiFe] hydrogenases, which actively catalyze H2/H2O exchange in the absence of exogenous electron acceptors