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Compares three Ohio districts with national trends to identify rural school characteristics that foster extracurricular activity participation. Surveys students, teachers, and community members. Reports student participation levels. Examines attraction and pressure responses, parental influence, at-risk student involvement, and urban-rural differences. Concludes rural schools offer potentially significant opportunities. (TES)

Abstract Background Increasing demand for qualitative research within global health has emerged alongside increasing demand for demonstration of quality of research, in line with the evidence-based model of medicine. In quantitative health sciences research, in particular clinical trials, there exist clear and widely-recognised guidelines for conducting quality assurance of research. However, no comparable guidelines exist for qualitative research and although there are long-standing debates on what constitutes 'quality' in qualitative research, the concept of 'quality assurance' has not been explored widely. In acknowledgement of this gap, we sought to review discourses around quality assurance of qualitative research, as a first step towards developing guidance. Methods A range of databases, journals and grey literature sources were searched, and papers were included if they explicitly addressed quality assurance within a qualitative paradigm. A meta-narrative approach was used to review and synthesise the literature. Results Among the 37 papers included in the review, two dominant narratives were interpreted from the literature, reflecting contrasting approaches to quality assurance. The first focuses on demonstrating quality within research outputs; the second focuses on principles for quality practice throughout the research process. The second narrative appears to offer an approach to quality assurance that befits the values of qualitative research, emphasising the need to consider quality throughout the research process. Conclusions The paper identifies the strengths of the approaches represented in each narrative and recommend these are brought together in the development of a flexible framework to help qualitative researchers to define, apply and demonstrate principles of quality in their research.

Abstract Background There is a well established link between obesity and cancer. Emerging research is characterising this relationship further and delineating the specific role of excess visceral adiposity, as opposed to simple obesity, in promoting tumorigenesis. This review summarises the evidence from an epidemiological and pathophysiological perspective. Methods Relevant medical literature was identified from searches of PubMed and references cited in appropriate articles identified. Selection of articles was based on peer review, journal and relevance. Results Numerous epidemiological studies consistently identify increased risk of developing carcinoma in the obese. Adipose tissue, particularly viscerally located fat, is metabolically active and exerts systemic endocrine effects. Putative pathophysiological mechanisms linking obesity and carcinogenesis include the paracrine effects of adipose tissue and systemic alterations associated with obesity. Systemic changes in the obese state include chronic inflammation and alterations in adipokines and sex steroids. Insulin and the insulin-like growth factor axis influence tumorigenesis and also have a complex relationship with adiposity. There is evidence to suggest that insulin and the IGF axis play an important role in mediating obesity associated malignancy. Conclusions There is much evidence to support a role for obesity in cancer progression, however further research is warranted to determine the specific effect of excess visceral adipose tissue on tumorigenesis. Investigation of the potential mechanisms underpinning the association, including the role of insulin and the IGF axis, will improve understanding of the obesity and cancer link and may uncover targets for intervention.

Abstract Background The development of a fistula between the tracheobronchial tree and the gastric conduit post esophagectomy is a rare and often fatal complication. Case presentation A 68 year old man underwent radical esophagectomy for esophageal adenocarcinoma. On postoperative day 14 the nasogastric drainage bag dramatically filled with air, without deterioration in respiratory function or progressive sepsis. A fiberoptic bronchoscopy was performed which demonstrated a gastro-bronchial fistula in the posterior aspect of the left main bronchus. He was managed conservatively with antibiotics, enteral nutrition via jejunostomy, and non-invasive respiratory support. A follow- up bronchoscopy 60 days after the diagnostic bronchoscopy, confirmed spontaneous closure of the fistula Conclusions This is the first such case where a conservative approach with no surgery or endoprosthesis resulted in a successful outcome, with fistula closure confirmed at subsequent bronchoscopy. Our experience would suggest that in very carefully selected cases where bronchopulmonary contamination from the fistula is minimal or absent, there is no associated inflammation of the tracheobronchial tree and the patient is stable from a respiratory point of view without evidence of sepsis, there may be a role for a trial of conservative management.

Abstract We report herein a case of a synchronous presentation of an adenocarcinoma of esophagago-gastric junction type II and an ampullary tumor that was treated by combined Whipple's pancreaticoduodenectomy, total gastrectomy and esophagectomy. The magnitude of this operation was safely achieved with meticulous surgical techniques and perioperative care without any major short or long term complications. Patient returned to a good quality of life at six-month follow up with no further gastrointestinal symptoms or evidence of disease recurrence.

Abstract Esophageal perforation is associated with a significant risk of morbidity and mortality. We report herein a case of lye-induced esophageal perforation managed successfully by employing endoscopic T-tube placement with a successful outcome.

Abstract Background A complete pathological response to neo-adjuvant chemo-radiation for oesophageal cancer is associated with favourable survival. However, such a benefit is seen in the minority. If one could identify, at diagnosis, those patients who were unlikely to respond unnecessary toxicity could be avoided and alternative treatment can be considered. The aim of this review was to highlight predictive markers currently assessed and evaluate their clinical utility. Methods A systematic search of Pubmed and Google Scholar was performed using the following keywords; "neo-adjuvant", "oesophageal", "trimodality", "chemotherapy", "radiotherapy", "chemoradiation" and "predict". The original manuscripts were sourced for further articles of relevance. Results Conventional indices including tumour stage and grade seem unable to predict histological response. Immuno-histochemical markers have been extensively studied, but none has made its way into routine clinical practice. Global gene expression from fresh pre-treatment tissue using cDNA microarray has only recently been assessed, but shows considerable promise. Molecular imaging using FDG-PET seems to be able to predict response after neo-adjuvant chemoradiation has finished, but there is a paucity of data when such imaging is performed earlier. Conclusion Currently there are no clinically useful predictors of response based on standard pathological assessment and immunohistochemistry. Genomics, proteomics and molecular imaging may hold promise.

Background: Spontaneously depolarizing nodal cells comprise the pacemaker of the heart. Intracellular calcium (Ca 2+ ) plays a critical role in mediating nodal cell automaticity and understanding this so-called Ca 2+ clock is critical to understanding nodal arrhythmias. We previously demonstrated a role for Jph2 (junctophilin 2) in regulating Ca 2+ -signaling through inhibition of RyR2 (ryanodine receptor 2) Ca 2+ leak in cardiac myocytes; however, its role in pacemaker function and nodal arrhythmias remains unknown. We sought to determine whether nodal Jph2 expression silencing causes increased sinoatrial and atrioventricular nodal cell automaticity due to aberrant RyR2 Ca 2+ leak., Methods: A tamoxifen-inducible, nodal tissue-specific, knockdown mouse of Jph2 was achieved using a Cre-recombinase-triggered short RNA hairpin directed against Jph2 (Hcn4:shJph2). In vivo cardiac rhythm was monitored by surface ECG, implantable cardiac telemetry, and intracardiac electrophysiology studies. Intracellular Ca 2+ imaging was performed using confocal-based line scans of isolated nodal cells loaded with fluorescent Ca 2+ reporter Cal-520. Whole cell patch clamp was conducted on isolated nodal cells to determine action potential kinetics and sodium-calcium exchanger function., Results: Hcn4:shJph2 mice demonstrated a 40% reduction in nodal Jph2 expression, resting sinus tachycardia, and impaired heart rate response to pharmacologic stress. In vivo intracardiac electrophysiology studies and ex vivo optical mapping demonstrated accelerated junctional rhythm originating from the atrioventricular node. Hcn4:shJph2 nodal cells demonstrated increased and irregular Ca 2+ transient generation with increased Ca 2+ spark frequency and Ca 2+ leak from the sarcoplasmic reticulum. This was associated with increased nodal cell AP firing rate, faster diastolic repolarization rate, and reduced sodium-calcium exchanger activity during repolarized states compared to control. Phenome-wide association studies of the JPH2 locus identified an association with sinoatrial nodal disease and atrioventricular nodal block., Conclusions: Nodal-specific Jph2 knockdown causes increased nodal automaticity through increased Ca 2+ leak from intracellular stores. Dysregulated intracellular Ca 2+ underlies nodal arrhythmogenesis in this mouse model.

Aims: Abnormal intracellular calcium (Ca2+) handling contributes to the progressive nature of atrial fibrillation (AF), the most common sustained cardiac arrhythmia. Evidence in mouse models suggests that activation of the nuclear factor of activated T-cell (NFAT) signalling pathway contributes to atrial remodelling. Our aim was to determine the role of NFATc2 in AF in humans and mouse models., Methods and Results: Expression levels of NFATc1-c4 isoforms were assessed by quantitative reverse transcription-polymerase chain reaction in right atrial appendages from patients with chronic AF (cAF). NFATc1 and NFATc2 mRNA levels were elevated in cAF patients compared with those in normal sinus rhythm (NSR). Western blotting revealed increased cytosolic and nuclear levels of NFATc2 in AF patients. Similar findings were obtained in CREM-IbΔC-X transgenic (CREM) mice, a model of progressive AF. Telemetry ECG recordings revealed age-dependent spontaneous AF in CREM mice, which was prevented by NFATc2 knockout in CREM:NFATc2-/- mice. Programmed electrical stimulation revealed that CREM:NFATc2-/- mice lacked an AF substrate. Morphometric analysis and histology revealed increased atrial weight and atrial fibrosis in CREM mice compared with wild-type controls, which was reversed in CREM:NFATc2-/- mice. Confocal microscopy showed an increased Ca2+ spark frequency despite a reduced sarcoplasmic reticulum (SR) Ca2+ load in CREM mice compared with controls, whereas these abnormalities were normalized in CREM:NFATc2-/- mice. Western blotting revealed that genetic inhibition of Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of S2814 on ryanodine receptor type 2 (RyR2) in CREM:RyR2-S2814A mice suppressed NFATc2 activation observed in CREM mice, suggesting that NFATc2 is activated by excessive SR Ca2+ leak via RyR2. Finally, chromatin immunoprecipitation sequencing from AF patients identified Ras and EF-hand domain-containing protein (Rasef) as a direct target of NFATc2-mediated transcription., Conclusion: Our findings reveal activation of the NFAT signalling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model., Competing Interests: Conflict of interest: X.H.T.W. is a co-founder and Scientific Advisory Board member of Elex Biotech, a drug development company focused on novel compounds for the cardiac arrhythmia disorders and heart failure. All other authors declared no conflict of interest., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Background: Atrial fibrillation (AF) is the most common type of arrhythmia. Abnormal atrial myocyte Ca 2+ handling promotes aberrant membrane excitability and remodeling that are important for atrial arrhythmogenesis. The sequence of molecular events leading to loss of normal atrial myocyte Ca 2+ homeostasis is not established. Late Na + current (I Na,L ) is increased in atrial myocytes from AF patients together with an increase in activity of Ca 2+ /calmodulin-dependent kinase II (CaMKII)., Objective: The purpose of this study was to determine whether CaMKII-dependent phosphorylation at Ser571 on Na V 1.5 increases atrial I Na,L , leading to aberrant atrial Ca 2+ cycling, altered electrophysiology, and increased AF risk., Methods: Atrial myocyte electrophysiology, Ca 2+ handling, and arrhythmia susceptibility were studied in wild-type and Scn5a knock-in mice expressing phosphomimetic (S571E) or phosphoresistant (S571A) Na V 1.5 at Ser571., Results: Atrial myocytes from S571E but not S571A mice displayed an increase in I Na,L and action potential duration, and with adrenergic stress have increased delayed afterdepolarizations. Frequency of Ca 2+ sparks and waves was increased in S571E atrial myocytes compared to wild type. S571E mice showed an increase in atrial events induced by adrenergic stress and AF inducibility in vivo. Isolated S571E atria were more susceptible to spontaneous atrial events, which were abrogated by inhibiting sarcoplasmic reticulum Ca 2+ release, CaMKII, or the Na + /Ca 2+ exchanger. Expression of phospho-Na V 1.5 at Ser571 and autophosphorylated CaMKII were increased in atrial samples from human AF patients., Conclusion: This study identified CaMKII-dependent regulation of Na V 1.5 as an important upstream event in Ca 2+ handling defects and abnormal impulse generation in the setting of AF., (Copyright © 2019 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Background: Abnormal calcium (Ca 2+ ) release from the sarcoplasmic reticulum (SR) contributes to the pathogenesis of atrial fibrillation (AF). Increased phosphorylation of 2 proteins essential for normal SR-Ca 2+ cycling, the type-2 ryanodine receptor (RyR2) and phospholamban (PLN), enhances the susceptibility to AF, but the underlying mechanisms remain unclear. Protein phosphatase 1 (PP1) limits steady-state phosphorylation of both RyR2 and PLN. Proteomic analysis uncovered a novel PP1-regulatory subunit (PPP1R3A [PP1 regulatory subunit type 3A]) in the RyR2 macromolecular channel complex that has been previously shown to mediate PP1 targeting to PLN. We tested the hypothesis that reduced PPP1R3A levels contribute to AF pathogenesis by reducing PP1 binding to both RyR2 and PLN., Methods: Immunoprecipitation, mass spectrometry, and complexome profiling were performed from the atrial tissue of patients with AF and from cardiac lysates of wild-type and Pln-knockout mice. Ppp1r3a-knockout mice were generated by CRISPR-mediated deletion of exons 2 to 3. Ppp1r3a-knockout mice and wild-type littermates were subjected to in vivo programmed electrical stimulation to determine AF susceptibility. Isolated atrial cardiomyocytes were used for Stimulated Emission Depletion superresolution microscopy and confocal Ca 2+ imaging., Results: Proteomics identified the PP1-regulatory subunit PPP1R3A as a novel RyR2-binding partner, and coimmunoprecipitation confirmed PPP1R3A binding to RyR2 and PLN. Complexome profiling and Stimulated Emission Depletion imaging revealed that PLN is present in the PPP1R3A-RyR2 interaction, suggesting the existence of a previously unknown SR nanodomain composed of both RyR2 and PLN/sarco/endoplasmic reticulum calcium ATPase-2a macromolecular complexes. This novel RyR2/PLN/sarco/endoplasmic reticulum calcium ATPase-2a complex was also identified in human atria. Genetic ablation of Ppp1r3a in mice impaired binding of PP1 to both RyR2 and PLN. Reduced PP1 targeting was associated with increased phosphorylation of RyR2 and PLN, aberrant SR-Ca 2+ release in atrial cardiomyocytes, and enhanced susceptibility to pacing-induced AF. Finally, PPP1R3A was progressively downregulated in the atria of patients with paroxysmal and persistent (chronic) AF., Conclusions: PPP1R3A is a novel PP1-regulatory subunit within the RyR2 channel complex. Reduced PPP1R3A levels impair PP1 targeting and increase phosphorylation of both RyR2 and PLN. PPP1R3A deficiency promotes abnormal SR-Ca 2+ release and increases AF susceptibility in mice. Given that PPP1R3A is downregulated in patients with AF, this regulatory subunit may represent a new target for AF therapeutic strategies.

New Findings: What is the central question of this study? We have evaluated changes in cardiovascular physiology using echocardiography in an experimental model of lung fibrosis. What is the main finding and its importance? Remarkably, we report changes in cardiovascular function as early as day 7, concomitant with evidence of vascular remodelling. We also report that isolated pulmonary arteries were hypercontractile in response to a thromboxane A2 agonist. These findings are significant because the development of pulmonary hypertension is one of the most significant predictors of mortality in patients with lung fibrosis, where there are no available therapies and a lack of animal models., Abstract: Group III pulmonary hypertension is observed in patients with chronic lung diseases such as chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis. Pulmonary hypertension (PH) develops as a result of extensive pulmonary vascular remodelling and resultant changes in vascular tone that can lead to right ventricle hypertrophy. This eventually leads to right heart failure, which is the leading indicator of mortality in patients with idiopathic pulmonary fibrosis. Treatments for group III PH are not available, in part owing to a lack of viable animal models. Here, we have evaluated the cardiovascular changes in a model of lung fibrosis and PH. Data obtained from this study indicated that structural alterations in the right heart, such as right ventricular wall hypertrophy, occurred as early as day 14, and similar increases in right ventricle chamber size were seen between days 21 and 28. These structural changes were correlated with decreases in the systolic function of the right ventricle and right ventricular cardiac output, which also occurred between the same time points. Characterization of pulmonary artery dynamics also highlighted that PH might be occurring as early as day 21, indicated by reductions in the velocity-time integral; however, evidence for PH is apparent as early as day 7, indicated by the significant reduction in pulmonary acceleration time values. These changes are consistent with evidence of vascular remodelling observed histologically starting on day 7. In addition, we report hyperactivity of bleomycin-exposed pulmonary arteries to a thromboxane A2 receptor (Tbxa2r) agonist., (© 2019 The Authors. Experimental Physiology © 2019 The Physiological Society.)

We have previously shown Twik-2 -/- mice develop pulmonary hypertension and vascular remodeling. We hypothesized that distal pulmonary arteries (D-PAs) of the Twik-2 -/- mice are hypercontractile under physiological venous conditions due to altered electrophysiologic properties between the conduit and resistance vessels in the pulmonary vascular bed. We measured resting membrane potential and intracellular calcium through Fura-2 in freshly digested pulmonary artery smooth muscles (PASMCs) from both the right main (RM-PA) and D-PA (distal) regions of pulmonary artery from WT and Twik-2 -/- mice. Whole segments of RM-PAs and D-PAs from 20 to 24-week-old wildtype (WT) and Twik-2 -/- mice were also pressurized between two glass micropipettes and bathed in buffer with either arterial or venous conditions. Abluminally-applied phenylephrine (PE) and U46619 were added to the buffer at log increments and vessel diameter was measured. All values were expressed as averages with ±SEM. Vasoconstrictor responses did not differ between WT and Twik-2 -/- RM-PAs under arterial conditions. Under venous conditions, Twik-2 -/- RM-PAs showed an increased sensitivity to PE with a lower EC50 (P = 0.02). Under venous conditions, Twik-2 -/- D-PAs showed an increase maximal vasoconstrictor response to both phenylephrine and U46619 compared to the WT mice (P < 0.05). Isolated PASMCs from Twik-2 -/- D-PA were depolarized and had higher intracellular calcium levels compared to PASMCs from RM-PA of both WT and Twik-2 -/- mice. These studies suggest that hypercontractile responses and electrophysiologic properties unique to the anatomic location of the D-PAs may contribute to pulmonary hypertensive vasculopathy., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Background: Heart failure (HF) is a complex disease with a rising prevalence despite advances in treatment. Protein phosphatase 1 (PP1) has long been implicated in HF pathogenesis, but its exact role is both unclear and controversial. Most previous studies measured only the PP1 catalytic subunit (PP1c) without investigating its diverse set of interactors, which confer localization and substrate specificity to the holoenzyme. In this study, we define the PP1 interactome in cardiac tissue and test the hypothesis that this interactome becomes rearranged during HF progression at the level of specific PP1c interactors., Methods: Mice were subjected to transverse aortic constriction and grouped on the basis of ejection fraction into sham, hypertrophy, moderate HF (ejection fraction, 30%-40%), and severe HF (ejection fraction <30%). Cardiac lysates were subjected to affinity purification with anti-PP1c antibodies followed by high-resolution mass spectrometry. PP1 regulatory subunit 7 (Ppp1r7) was knocked down in mouse cardiomyocytes and HeLa cells with adeno-associated virus serotype 9 and siRNA, respectively. Calcium imaging was performed on isolated ventricular myocytes., Results: Seventy-one and 98 PP1c interactors were quantified from mouse cardiac and HeLa lysates, respectively, including many novel interactors and protein complexes. This represents the largest reproducible PP1 interactome data set ever captured from any tissue, including both primary and secondary/tertiary interactors. Nine PP1c interactors with changes in their binding to PP1c were strongly associated with HF progression, including 2 known (Ppp1r7 and Ppp1r18) and 7 novel interactors. Within the entire cardiac PP1 interactome, Ppp1r7 had the highest binding to PP1c. Cardiac-specific knockdown in mice led to cardiac dysfunction and disruption of calcium release from the sarcoplasmic reticulum., Conclusions: PP1 is best studied at the level of its interactome, which undergoes significant rearrangement during HF progression. The 9 key interactors that are associated with HF progression may represent potential targets in HF therapy. In particular, Ppp1r7 may play a central role in regulating the PP1 interactome by acting as a competitive molecular "sponge" of PP1c.

Rationale: Autosomal-dominant mutations in ryanodine receptor type 2 ( RYR2) are responsible for ≈60% of all catecholaminergic polymorphic ventricular tachycardia. Dysfunctional RyR2 subunits trigger inappropriate calcium leak from the tetrameric channel resulting in potentially lethal ventricular tachycardia. In vivo CRISPR/Cas9-mediated gene editing is a promising strategy that could be used to eliminate the disease-causing Ryr2 allele and hence rescue catecholaminergic polymorphic ventricular tachycardia., Objective: To determine if somatic in vivo genome editing using the CRISPR/Cas9 system delivered by adeno-associated viral (AAV) vectors could correct catecholaminergic polymorphic ventricular tachycardia arrhythmias in mice heterozygous for RyR2 mutation R176Q (R176Q/+)., Methods and Results: Guide RNAs were designed to specifically disrupt the R176Q allele in the R176Q/+ mice using the SaCas9 ( Staphylococcus aureus Cas9) genome editing system. AAV serotype 9 was used to deliver Cas9 and guide RNA to neonatal mice by single subcutaneous injection at postnatal day 10. Strikingly, none of the R176Q/+ mice treated with AAV-CRISPR developed arrhythmias, compared with 71% of R176Q/+ mice receiving control AAV serotype 9. Total Ryr2 mRNA and protein levels were significantly reduced in R176Q/+ mice, but not in wild-type littermates. Targeted deep sequencing confirmed successful and highly specific editing of the disease-causing R176Q allele. No detectable off-target mutagenesis was observed in the wild-type Ryr2 allele or the predicted putative off-target site, confirming high specificity for SaCas9 in vivo. In addition, confocal imaging revealed that gene editing normalized the enhanced Ca 2+ spark frequency observed in untreated R176Q/+ mice without affecting systolic Ca 2+ transients., Conclusions: AAV serotype 9-based delivery of the SaCas9 system can efficiently disrupt a disease-causing allele in cardiomyocytes in vivo. This work highlights the potential of somatic genome editing approaches for the treatment of lethal autosomal-dominant inherited cardiac disorders, such as catecholaminergic polymorphic ventricular tachycardia.

Introduction: Pulmonary hypertension (PH) carries significant associated morbidity and mortality and the underlying molecular mechanisms of PH are not well understood. Loss-of-function mutations in TASK-1 potassium channels are associated with PH in humans. Although TASK-1 has been considered in the development of PH for over a decade, characterization of TASK-1 knockout mice has been limited to in vitro studies or in vivo studies in room air at isolated time points. The purpose of this study was twofold. First, we sought to determine if TASK -/- male and female mice developed PH over the span of one year. Second, we sought to determine the effect of chronic hypoxia, a stimulus for PH, and its recovery on PH in TASK-1 -/- mice., Methods: We measured right ventricular systolic pressure (RVSP) and vascular remodeling in male and female C57BL/6 WT and TASK-1 -/- mice at separate time points: 20-24 weeks and 1 year of age. Additionally, we measured RVSP and vascular remodeling in TASK-1 -/- and wild-type mice between 13 and 16 weeks of age exposed to 10% hypoxia for 3 weeks followed by recovery to room air conditions for an additional 6 weeks., Results: RVSP was similar between WT and TASK -/- mice. Male and female WT and TASK-1 -/- mice all demonstrated age-related increases in RVSP, which correlated to age-related vascular remodeling in male mice but not in female mice. Male TASK-1 -/- and WT mice exposed to chronic hypoxia demonstrated increased RVSP, which decreased following room air recovery. WT and TASK-1 -/- male mice demonstrated vascular remodeling upon exposure to hypoxia that persisted in room air recovery., Conclusion: Female and male TASK-1 -/- mice do not develop hemodynamic or vascular evidence for PH, but RVSP rises in an age-dependent manner independent of genotype. TASK-1 -/- and WT male mice develop hypoxia-induced elevations in RVSP that decrease to baseline after recovery in room air. TASK-1 -/- and WT male mice demonstrate vascular remodeling after exposure to hypoxia that persists despite recovery to room air conditions and does not correlate with RVSP normalization.

Background: Hypertrophic cardiomyopathy (HCM), defined as asymmetric left ventricular hypertrophy, is a leading cause of cardiac death in the young. Perturbations in calcium (Ca 2+ ) handling proteins have been implicated in the pathogenesis of HCM. JPH2 -encoded junctophilin 2 is a major component of the junctional membrane complex, the subcellular microdomain involved in excitation-contraction coupling. We hypothesized that a novel JPH2 mutation identified in patients with HCM is causally linked to HCM, and alters intracellular Ca 2+ signaling in a pro-hypertrophic manner., Objectives: To determine using a transgenic mouse model whether a JPH2 mutation found in a HCM patient is responsible for disease development., Methods: Genetic interrogation of a large cohort of HCM cases was conducted for all coding exons of JPH2 . Pseudo-knock-in (PKI) mice containing a novel JPH2 variant were subjected to echocardiography, cardiac MRI, hemodynamic analysis, and histology., Results: A novel JPH2 mutation, A405S, was identified in a genotype-negative proband with significant basal septal hypertrophy. Although initially underappreciated by traditional echocardiographic imaging, PKI mice with this JPH2 mutation (residue A399S in mice) were found to exhibit similar basal hypertrophy using a newly developed echo imaging plane, and this was confirmed using cardiac MRI. Histological analysis demonstrated cardiomyocyte hypertrophy and disarray consistent with HCM., Conclusions: Variant A405S is a novel HCM-associated mutation in JPH2 found in a proband negative for mutations in the canonical HCM-associated genes. Studies in the analogous mouse model demonstrated for the first time a causal link between a JPH2 defect and HCM. Moreover, novel imaging approaches identified subvalvular septal hypertrophy, specific findings also reported in the human JPH2 mutation carrier.

Rationale: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal arrhythmic disorder caused by mutations in the type-2 ryanodine receptor (RyR2). Mutant RyR2 cause abnormal Ca 2+ leak from the sarcoplasmic reticulum (SR), which is associated with the development of arrhythmias., Objective: To determine whether derivatives of tetracaine, a local anesthetic drug with known RyR2 inhibiting action, could prevent CPVT induction by suppression of RyR2-mediated SR Ca 2+ leak., Methods and Results: Confocal microscopy was used to assess the effects of tetracaine and 9 derivatives (EL1-EL9) on spontaneous Ca 2+ sparks in ventricular myocytes isolated from RyR2-R176Q/+ mice with CPVT. Whereas each derivative suppressed the Ca 2+ spark frequency, derivative EL9 was most effective at the screening dose of 500nmol/L. At this high dose, the Ca 2+ transient amplitude was not affected in myocytes from WT or R176Q/+ mice. The IC 50 of EL9 was determined to be 13nmol/L, which is about 400× time lower than known RyR2 stabilizer K201. EL9 prevented the induction of ventricular tachycardia observed in placebo-treated R176Q/+ mice, without affecting heart rate or cardiac contractility., Conclusions: Tetracaine derivatives represent a novel class of RyR2 stabilizing drugs that could be used for the treatment of the potentially fatal disorder catecholaminergic polymorphic ventricular tachycardia., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Rationale: Junctional membrane complexes (JMCs) in myocytes are critical microdomains, in which excitation-contraction coupling occurs. Structural and functional disruption of JMCs underlies contractile dysfunction in failing hearts. However, the role of newly identified JMC protein SPEG (striated muscle preferentially expressed protein kinase) remains unclear., Objective: To determine the role of SPEG in healthy and failing adult hearts., Methods and Results: Proteomic analysis of immunoprecipitated JMC proteins ryanodine receptor type 2 and junctophilin-2 (JPH2) followed by mass spectrometry identified the serine-threonine kinase SPEG as the only novel binding partner for both proteins. Real-time polymerase chain reaction revealed the downregulation of SPEG mRNA levels in failing human hearts. A novel cardiac myocyte-specific Speg conditional knockout (MCM-Speg fl/fl ) model revealed that adult-onset SPEG deficiency results in heart failure (HF). Calcium (Ca 2+ ) and transverse-tubule imaging of ventricular myocytes from MCM-Speg fl/fl mice post HF revealed both increased sarcoplasmic reticulum Ca 2+ spark frequency and disrupted JMC integrity. Additional studies revealed that transverse-tubule disruption precedes the development of HF development in MCM-Speg fl/fl mice. Although total JPH2 levels were unaltered, JPH2 phosphorylation levels were found to be reduced in MCM-Speg fl/fl mice, suggesting that loss of SPEG phosphorylation of JPH2 led to transverse-tubule disruption, a precursor of HF development in SPEG-deficient mice., Conclusions: The novel JMC protein SPEG is downregulated in human failing hearts. Acute loss of SPEG in mouse hearts causes JPH2 dephosphorylation and transverse-tubule loss associated with downstream Ca 2+ mishandling leading to HF. Our study suggests that SPEG could be a novel target for the treatment of HF., (© 2016 American Heart Association, Inc.)

Background: Junctophilin-2 (JPH2) is the primary structural protein for the coupling of transverse (T)-tubule associated cardiac L-type Ca channels and type-2 ryanodine receptors on the sarcoplasmic reticulum within junctional membrane complexes (JMCs) in cardiomyocytes. Effective signaling between these channels ensures adequate Ca-induced Ca release required for normal cardiac contractility. Disruption of JMC subcellular domains, a common feature of failing hearts, has been attributed to JPH2 downregulation. Here, we tested the hypothesis that adeno-associated virus type 9 (AAV9) mediated overexpression of JPH2 could halt the development of heart failure in a mouse model of transverse aortic constriction (TAC)., Methods and Results: Following TAC, a progressive decrease in ejection fraction was paralleled by a progressive decrease of cardiac JPH2 levels. AAV9-mediated expression of JPH2 rescued cardiac contractility in mice subjected to TAC. AAV9-JPH2 also preserved T-tubule structure. Moreover, the Ca 2+ spark frequency was reduced and the Ca 2+ transient amplitude was increased in AAV9-JPH2 mice following TAC, consistent with JPH2-mediated normalization of SR Ca 2+ handling., Conclusions: This study demonstrates that AAV9-mediated JPH2 gene therapy maintained cardiac function in mice with early stage heart failure. Moreover, restoration of JPH2 levels prevented loss of T-tubules and suppressed abnormal SR Ca 2+ leak associated with contractile failure following TAC. These findings suggest that targeting JPH2 might be an attractive therapeutic approach for treating pathological cardiac remodeling during heart failure., Competing Interests: 6. CONFLICT OF INTEREST None declared., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Entropion is an inward rolling of the eyelid allowing contact between the eyelashes and cornea that may lead to blindness if not corrected. Although many mammalian species, including humans and dogs, are afflicted by congenital entropion, no specific genes or gene regions related to development of entropion have been reported in any mammalian species to date. Entropion in domestic sheep is known to have a genetic component therefore, we used domestic sheep as a model system to identify genomic regions containing genes associated with entropion. A genome-wide association was conducted with congenital entropion in 998 Columbia, Polypay, and Rambouillet sheep genotyped with 50,000 SNP markers. Prevalence of entropion was 6.01%, with all breeds represented. Logistic regression was performed in PLINK with additive allelic, recessive, dominant, and genotypic inheritance models. Two genome-wide significant (empirical P<0.05) SNP were identified, specifically markers in SLC2A9 (empirical P = 0.007; genotypic model) and near NLN (empirical P = 0.026; dominance model). Six additional genome-wide suggestive SNP (nominal P<1x10(-5)) were identified including markers in or near PIK3CB (P = 2.22x10(-6); additive model), KCNB1 (P = 2.93x10(-6); dominance model), ZC3H12C (P = 3.25x10(-6); genotypic model), JPH1 (P = 4.68x20(-6); genotypic model), and MYO3B (P = 5.74x10(-6); recessive model). This is the first report of specific gene regions associated with congenital entropion in any mammalian species, to our knowledge. Further, none of these genes have previously been associated with any eyelid traits. These results represent the first genome-wide analysis of gene regions associated with entropion and provide target regions for the development of sheep genetic markers for marker-assisted selection.

TWIK-2 (KCNK6) is a member of the 2-pore domain (K2P) family of potassium channels, which are highly expressed in the vascular system. We tested the hypothesis that TWIK-2 deficiency leads to pulmonary hypertension. TWIK-2 knockout mice and their wildtype littermates at 8 weeks of age had similar mean right ventricular systolic pressures (24±3 and 21±3 mm Hg, respectively.) Significantly, by 20 weeks of age, the mean right ventricular systolic pressures in TWIK-2 knockout mice increased to 35±3 mm Hg (P≤0.036), whereas mean right ventricular systolic pressures in wildtype littermates remained at 22±3 mm Hg. Elevated mean right ventricular systolic pressures in the TWIK-2 knockout mice was accompanied by pulmonary vascular remodeling as determined by a 25% increase in the cross-sectional area of the vessels occupied by the vessel wall. Additionally, secondary branches of the pulmonary artery from 20-week-old TWIK-2 knockout mice showed an enhanced contractile response to U46619 (10(-6) moles/L), a thromboxane A2 mimetic, which was completely abolished with the Rho-kinase inhibitor, Y27632 (10(-6) and 10(-5) moles/L). Treatment of TWIK-2 knockout mice with the Rho-kinase inhibitor, fasudil, in the drinking water for 12 weeks, abolished the development of pulmonary hypertension and attenuated the vessel remodeling. We concluded that mice deficient in the TWIK-2 channel develop pulmonary hypertension between 8 and 20 weeks of age through a mechanism involving Rho-kinase. Our results suggest that downregulation of TWIK-2 in the pulmonary vasculature may be an underlying mechanism in the development of pulmonary hypertension., (© 2014 American Heart Association, Inc.)

Background: Long-term exposure to microgravity during space flight may lead to cardiac remodeling and rhythm disturbances. In mice, hindlimb unloading (HU) mimics the effects of microgravity and stimulates physiological adaptations, including cardiovascular deconditioning. Recent studies have demonstrated an important role played by changes in intracellular Ca handling in the pathogenesis of heart failure and arrhythmia. In this study, we tested the hypothesis that cardiac remodeling following HU in mice involves abnormal intracellular Ca regulation through the cardiac ryanodine receptor (RyR2)., Methods and Results: Mice were subjected to HU by tail suspension for 28 to 56 days in order to induce cardiac remodeling (n=15). Control mice (n=19) were treated equally, with the exception of tail suspension. Echocardiography revealed cardiac enlargement and depressed contractility starting at 28 days post-HU versus control. Moreover, mice were more susceptible to pacing-induced ventricular arrhythmias after HU. Ventricular myocytes isolated from HU mice exhibited an increased frequency of spontaneous sarcoplasmic reticulum (SR) Ca release events and enhanced SR Ca leak via RyR2. Western blotting revealed increased RyR2 phosphorylation at S2814, and increased CaMKII auto-phosphorylation at T287, suggesting that CaMKII activation of RyR2 might underlie enhanced SR Ca release in HU mice., Conclusion: These data suggest that abnormal intracellular Ca handling, likely due to increased CaMKII phosphorylation of RyR2, plays a role in cardiac remodeling following simulated microgravity in mice., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Small ruminant lentivirus (SRLV), also called ovine progressive pneumonia virus or maedi-visna, is present in 24% of US sheep. Like human immunodeficiency virus, SRLV is a macrophage-tropic lentivirus that causes lifelong infection. The production impacts from SRLV are due to a range of disease symptoms, including pneumonia, arthritis, mastitis, body condition wasting and encephalitis. There is no cure and no effective vaccine for preventing SRLV infection. However, breed differences in prevalence and proviral concentration indicate a genetic basis for susceptibility to SRLV. Animals with high blood proviral concentration show increased tissue lesion severity, so proviral concentration represents a live animal test for control post-infection in terms of proviral replication and disease severity. Recently, it was found that sheep with two copies of TMEM154 haplotype 1 (encoding lysine at position 35) had lower odds of SRLV infection. In this study, we examined the relationship between SRLV control post-infection and variants in two genes, TMEM154 and CCR5, in four flocks containing 1403 SRLV-positive sheep. We found two copies of TMEM154 haplotype 1 were associated with lower SRLV proviral concentration in one flock (P < 0.02). This identified the same favorable diplotype for SRLV control post-infection as for odds of infection. However, frequencies of haplotypes 2 and 3 were too low in the other three flocks to test. The CCR5 promoter deletion did not have consistent association with SRLV proviral concentration. Future work in flocks with more balanced allele frequencies is needed to confirm or refute TMEM154 association with control of SRLV post-infection., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.)

Aims: Altered Ca(2+) handling in atrial fibrillation (AF) has been associated with dysregulated protein phosphatase 1 (PP1) and subcellular heterogeneities in protein phosphorylation, but the underlying mechanisms remain unclear. This is due to a lack of investigation into the local, rather than global, regulation of PP1 on different subcellular targets such as ryanodine receptor type 2 (RyR2), especially in AF., Methods and Results: We tested the hypothesis that impaired local regulation of PP1 causes RyR2 hyperphosphorylation thereby promoting AF susceptibility. To specifically disrupt PP1's local regulation of RyR2, we used the spinophilin knockout (Sp(-/-)) mice (Mus musculus) since PP1 is targeted to RyR2 via spinophilin. Without spinophilin, the interaction between PP1 and RyR2 was reduced by 64%, while RyR2 phosphorylation was increased by 43% at serine (S)2814 but unchanged at S2808. Lipid bilayer experiments revealed that single RyR2 channels isolated from Sp(-/-) hearts had an increased open probability. Likewise, Ca(2+) spark frequency normalized to sarcoplasmic reticulum Ca(2+) content was also enhanced in Sp(-/-) atrial myocytes, but normalized by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitors KN-93 and AIP and also by genetic inhibition of RyR2 S2814 phosphorylation. Finally, Sp(-/-) mice exhibited increased atrial ectopy and susceptibility to pacing-induced AF, both of which were also prevented by the RyR2 S2814A mutation., Conclusion: PP1 regulates RyR2 locally by counteracting CaMKII phosphorylation of RyR2. Decreased local PP1 regulation of RyR2 contributes to RyR2 hyperactivity and promotes AF susceptibility. This represents a novel mechanism for subcellular modulation of calcium channels and may represent a potential drug target of AF., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Ovine lentivirus (OvLV) is a macrophage-tropic lentivirus found in many countries that causes interstitial pneumonia, mastitis, arthritis and cachexia in sheep. There is no preventive vaccine and no cure, but breed differences suggest marker-assisted selective breeding might improve odds of infection and control of OvLV post-infection. Although variants in TMEM154 have consistent association with odds of infection, no variant in any gene has been associated with host control of OvLV post-infection in multiple animal sets. Proviral concentration is a live-animal diagnostic measure of OvLV control post-infection related to severity of OvLV-induced lesions. A recent genome-wide association study identified a region including four zinc finger genes associated with proviral concentration in one Rambouillet flock. To refine this region, we tested additional variants and identified a small insertion/deletion variant near ZNF389 that showed consistent association with proviral concentration in three animal sets (P < 0.05). These animal sets contained Rambouillet, Polypay and crossbred sheep from multiple locations and management conditions. Strikingly, one flock had exceptionally high prevalence (>87%, including yearlings) and mean proviral concentration (>950 copies/μg), possibly due to needle sharing. The best estimate of proviral concentration by genotype, obtained from all 1310 OvLV-positive animals tested, showed insertion homozygotes had less than half the proviral concentration of other genotypes (P < 0.0001). Future work will test additional breeds, management conditions and viral subtypes, and identify functional properties of the haplotype this deletion variant tracks. To our knowledge, this is the first genetic variant consistently associated with host control of OvLV post-infection in multiple sheep flocks., (Published 2013. This article is a U.S. Government work and is in the public domain in the USA. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.)

Purpose: The purpose of this study was to determine if magnetization transfer contrast (MTC) imaging could be used to detect early macromolecular accumulation in a mouse model of early Alzheimer's disease., Methods: We obtained MTC images at 9.4 T at three different age points in the Tg2576 mouse model of Alzheimer's disease. The Tg2576 mouse exhibits increased amyloid beta deposition that eventually progresses into amyloid beta plaque formation, increased hyper-phosphorylated tau but does not exhibit neurodegeneration., Results: Our results show an increase in the MTC signal that predates plaque formation and reported learning and memory deficits in the Tg2576 mouse. This increase in the MTC signal was reversed in a model of antioxidant therapy., Conclusion: MTC magnetic resonance imaging can be used to detect early macromolecular changes in the Tg2576 mouse model of Alzheimer's disease. The source of the MTC contrast is likely complex and warrants further investigation in additional preclinical models that represent early and late stage Alzheimer's disease pathologies., (Copyright © 2013 Wiley Periodicals, Inc.)

Aims: Transverse tubules (TTs) provide the basic subcellular structures that facilitate excitation-contraction (EC) coupling, the essential process that underlies normal cardiac contractility. Previous studies have shown that TTs develop within the first few weeks of life in mammals but the molecular determinants of this development have remained elusive. This study aims to elucidate the role of junctophilin-2 (JPH2), a junctional membrane complex protein, in the maturation of TTs in cardiomyocytes., Methods and Results: Using a novel cardiac-specific short-hairpin-RNA-mediated JPH2 knockdown mouse model (Mus musculus; αMHC-shJPH2), we assessed the effects of the loss of JPH2 on the maturation of the ventricular TT structure. Between embryonic day (E) 10.5 and postnatal day (P) 10, JPH2 mRNA and protein levels were reduced by >70% in αMHC-shJPH2 mice. At P8 and P10, knockdown of JPH2 significantly inhibited the maturation of TTs, while expression levels of other genes implicated in TT development remained mostly unchanged. At the same time, intracellular Ca(2+) handling was disrupted in ventricular myocytes from αMHC- shJPH2 mice, which developed heart failure by P10 marked by reduced ejection fraction, ventricular dilation, and premature death. In contrast, JPH2 transgenic mice exhibited accelerated TT maturation by P8., Conclusion: Our findings suggest that JPH2 is necessary for TT maturation during postnatal cardiac development in mice. In particular, JPH2 may be critical in anchoring the invaginating sarcolemma to the sarcoplasmic reticulum, thereby enabling the maturation of the TT network.

A genome-wide association study (GWAS) was performed to investigate seven red blood cell (RBC) phenotypes in over 500 domestic sheep (Ovis aries) from three breeds (Columbia, Polypay, and Rambouillet). A single nucleotide polymorphism (SNP) showed genome-wide significant association with increased mean corpuscular hemoglobin concentration (MCHC, P = 6.2×10(-14)) and genome-wide suggestive association with decreased mean corpuscular volume (MCV, P = 2.5×10(-6)). The ovine HapMap project found the same genomic region and the same peak SNP has been under extreme historical selective pressure, demonstrating the importance of this region for survival, reproduction, and/or artificially selected traits. We observed a large (>50 kb) variant haplotype sequence containing a full-length divergent artiodactyl MYADM-like repeat in strong linkage disequilibrium with the associated SNP. MYADM gene family members play roles in membrane organization and formation in myeloid cells. However, to our knowledge, no member of the MYADM gene family has been identified in development of morphologically variant RBCs. The specific RBC differences may be indicative of alterations in morphology. Additionally, erythrocytes with altered morphological structure often exhibit increased structural fragility, leading to increased RBC turnover and energy expenditure. The divergent artiodactyl MYADM-like repeat was also associated with increased ewe lifetime kilograms of lamb weaned (P = 2×10(-4)). This suggests selection for normal RBCs might increase lamb weights, although further validation is required before implementation in marker-assisted selection. These results provide clues to explain the strong selection on the artiodactyl MYADM-like repeat locus in sheep, and suggest MYADM family members may be important for RBC morphology in other mammals.

Scrapie is the transmissible spongiform encephalopathy (TSE) of sheep and goats, and scrapie eradication in sheep is based in part on strong genetic resistance to classical scrapie. Goats may serve as a scrapie reservoir, and to date there has been no experimental inoculation confirming strong genetic resistance in goats. Two prion protein variants (amino acid substitutions S146 and K222) in goats have been significantly underrepresented in scrapie cases though present in scrapie-exposed flocks, and have demonstrated low cell-free protein conversion efficiency to the disease form (PrP(D)). To test degree of genetic resistance conferred in live animals with consistent exposure, we performed the first oral scrapie challenge of goats singly heterozygous for either PRNP S146 or K222. All N146-Q222 homozygotes became clinically scrapie positive by an average of 24months, but all S146 and K222 heterozygotes remain scrapie negative by both rectal biopsy and clinical signs at significantly longer incubation times (P<0.0001 for both comparisons). Recent reports indicate small numbers of S146 and K222 heterozygous goats have become naturally infected with scrapie, suggesting these alleles do not confer complete resistance in the heterozygous state but rather extend incubation. The oral challenge results presented here confirm extended incubation observed in a recent intracerebral challenge of K222 heterozygotes, and to our knowledge provide the first demonstration of extended incubation in S146 heterozygotes. These results suggest longer relevant trace-back histories in scrapie-eradication programs for animals bearing these alleles and strengthen the case for additional challenge experiments in both homozygotes to assess potential scrapie resistance., (Published by Elsevier B.V.)

Background: Like human immunodeficiency virus (HIV), ovine lentivirus (OvLV) is macrophage-tropic and causes lifelong infection. OvLV infects one quarter of U.S. sheep and induces pneumonia and body condition wasting. There is no vaccine to prevent OvLV infection and no cost-effective treatment for infected animals. However, breed differences in prevalence and proviral concentration have indicated a genetic basis for susceptibility to OvLV. A recent study identified TMEM154 variants in OvLV susceptibility. The objective here was to identify additional loci associated with odds and/or control of OvLV infection., Methodology/principal Findings: This genome-wide association study (GWAS) included 964 sheep from Rambouillet, Polypay, and Columbia breeds with serological status and proviral concentration phenotypes. Analytic models accounted for breed and age, as well as genotype. This approach identified TMEM154 (nominal P=9.2×10(-7); empirical P=0.13), provided 12 additional genomic regions associated with odds of infection, and provided 13 regions associated with control of infection (all nominal P<1 × 10(-5)). Rapid decline of linkage disequilibrium with distance suggested many regions included few genes each. Genes in regions associated with odds of infection included DPPA2/DPPA4 (empirical P=0.006), and SYTL3 (P=0.051). Genes in regions associated with control of infection included a zinc finger cluster (ZNF192, ZSCAN16, ZNF389, and ZNF165; P=0.001), C19orf42/TMEM38A (P=0.047), and DLGAP1 (P=0.092)., Conclusions/significance: These associations provide targets for mutation discovery in sheep susceptibility to OvLV. Aside from TMEM154, these genes have not been associated previously with lentiviral infection in any species, to our knowledge. Further, data from other species suggest functional hypotheses for future testing of these genes in OvLV and other lentiviral infections. Specifically, SYTL3 binds and may regulate RAB27A, which is required for enveloped virus assembly of human cytomegalovirus. Zinc finger transcription factors have been associated with positive selection for repression of retroviral replication. DLGAP1 binds and may regulate DLG1, a known regulator of HIV infectivity.

Background: Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of cervids including white-tailed (Odocoileus virginianus) and mule deer (Odocoileus hemionus), Rocky Mountain elk (Cervus elaphus nelsoni), and moose (Alces alces). A leucine variant at position 132 (132L) in prion protein of Rocky Mountain elk confers a long incubation time with CWD, but not complete resistance. However, variants in regulatory regions outside the open reading frame of PRNP have been associated with varying degrees of susceptibility to prion disease in other species, and some variants have been observed in similar regions of Rocky Mountain elk PRNP. Thus, additional genetic variants might provide increased protection, either alone or in combination with 132L., Findings: This study provided genomic sequence of all exons for PRNP of Rocky Mountain elk. Many functional sites in and around the PRNP gene region were sequenced, and this report approximately doubled (to 75) the number of known variants in this region. A haplotype-tagging approach was used to reduce the number of genetic variants required to survey this variation in the PRNP gene region of 559 Rocky Mountain elk. Eight haplotypes were observed with frequencies over 1.0%, and one haplotype was present at 71.2% frequency, reflecting limited genetic diversity in the PRNP gene region., Conclusions: The presence of 132L cut odds of CWD by more than half (Odds Ratio = 0.43; P = 0.0031), which was similar to a previous report. However after accounting for 132L, no association with CWD was found for any additional variants in the PRNP region (P > 0.05).

In this study, the ability of the C(60) fullerene derivative DF-1 to protect radiosensitive cells from the effects of high doses of gamma irradiation was examined. Earlier reports of DF-1's lack of toxicity in these cells were confirmed, and DF-1 was also observed to protect both human lymphocytes and rat intestinal crypt cells against radiation-induced cell death. We determined that DF-1 protected both cell types against radiation-induced DNA damage, as measured by inhibition of micronucleus formation. DF-1 also reduced the levels of reactive oxygen species in the crypt cells, a unique capability of fullerenes because of their enhanced reactivity toward electron-rich species. The ability of DF-1 to protect against the cytotoxic effects of radiation was comparable to that of amifostine, another ROS-scavenging radioprotector. Interestingly, localization of fluorescently labeled DF-1 in fibroblast was observed throughout the cell. Taken together, these results suggest that DF-1 provides powerful protection against several deleterious cellular consequences of irradiation in mammalian systems including oxidative stress, DNA damage, and cell death.

Chemokine (C-C motif) Receptor 5 (CCR5) is a chemokine receptor that regulates immune cell recruitment in inflammation and serves as a coreceptor for human immunodeficiency virus (HIV). A human CCR5 coding deletion (termed delta-32) results in strong resistance to HIV infection, and sequence variants in CCR5 regulatory regions have been implicated in delayed progression to acquired immune deficiency syndrome. Both ovine progressive pneumonia virus (OPPV), also known as maedi-visna, and HIV are macrophage-tropic lentiviruses, have similar genomic structures, and cause lifelong persistent host infection, suggesting CCR5 may have a role in regulating OPPV provirus levels. Therefore, the ovine CCR5 genomic sequence was determined, and sequence variants were obtained from the open reading frame and surrounding regulatory sites. One CCR5 variant contained a 4-base deletion within a binding site for octamer transcription factors in the promoter region. A test for differential transcription from each allele in heterozygous animals showed a 3.9-fold transcription difference (P < 0.0001). OPPV proviral levels were also measured in 351 naturally exposed Rambouillet, Polypay and Columbia sheep. Deletion homozygotes showed reduced OPPV proviral levels among these animals (P < 0.01). The association of this CCR5 promoter deletion with OPPV levels will need to be validated in additional populations before the deletion can be recommended for widespread use in marker-assisted selection. However, because of the large impact on transcription and because CCR5 has roles in inflammation, recruitment of effector cells, and cell-mediated immunity, this deletion may play a role in the control of infections of many diverse pathogens of sheep.

Cell division and expansion are growth events that contribute to the developing shape, or morphogenesis, of a plant. Division and expansion are coordinated to the extent that plant organs, such as leaves, generally portray a predictable cellular pattern. To dissect the relationship between division and expansion, and to test for the role of each during morphogenesis, we have identified a recessive mutation warty-1 that produces a primary defect in cell size and shape in mutant leaves. Warty-1 mutant plants are similar to non-mutant siblings in terms of flowering time, overall plant size and leaf shape. Mature adult leaves have raised warts, consisting of excessively enlarged cells, that appear in patchy distribution throughout the blade. Cell wall deposition is abnormal or incomplete, suggesting cytokinesis is also affected, either directly or indirectly. Cells first increase in size at specific positions, which correspond to predictable cell dimensions of a developing 1 cm leaf. Once mutant cells exceed 133% normal size, cytokinesis becomes abnormal. As differentiation progresses, cells that appear normal in the mutant are actually dividing faster and are smaller than comparable cells in non-mutant siblings. These results suggest that (1) cells may compensate for growth defects by altering their cell cycle and that (2) proper execution of cytokinesis may require that cell size ratios are properly maintained.