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Bunch et al. describe a new assay to rapidly evaluate binding of cMyBP-C to F-actin (with or without tropomyosin) in solution. Changes in labeled actin fluorescence lifetime due to cMyBP-C phosphorylation and/or HCM mutations were consistent with measurements obtained using cosedimentation assays., Binding properties of actin-binding proteins are typically evaluated by cosedimentation assays. However, this method is time-consuming, involves multiple steps, and has a limited throughput. These shortcomings preclude its use in screening for drugs that modulate actin-binding proteins relevant to human disease. To develop a simple, quantitative, and scalable F-actin–binding assay, we attached fluorescent probes to actin's Cys-374 and assessed changes in fluorescence lifetime upon binding to the N-terminal region (domains C0–C2) of human cardiac myosin-binding protein C (cMyBP-C). The lifetime of all five probes tested decreased upon incubation with cMyBP-C C0–C2, as measured by time-resolved fluorescence (TR-F), with IAEDANS being the most sensitive probe that yielded the smallest errors. The TR-F assay was compared with cosedimentation to evaluate in vitro changes in binding to actin and actin–tropomyosin arising from cMyBP-C mutations associated with hypertrophic cardiomyopathy (HCM) and tropomyosin binding. Lifetime changes of labeled actin with added C0–C2 were consistent with cosedimentation results. The HCM mutation L352P was confirmed to enhance actin binding, whereas PKA phosphorylation reduced binding. The HCM mutation R282W, predicted to disrupt a PKA recognition sequence, led to deficits in C0–C2 phosphorylation and altered binding. Lastly, C0–C2 binding was found to be enhanced by tropomyosin and binding capacity to be altered by mutations in a tropomyosin-binding region. These findings suggest that the TR-F assay is suitable for rapidly and accurately determining quantitative binding and for screening physiological conditions and compounds that affect cMyBP-C binding to F-actin for therapeutic discovery.

Introduction Anticoagulantion therapy (OAT) represents the cornerstone to reduce thromboembolic events for atrial fibrillation (AF). Recent studies suggest that AF catheter ablation on top of OAT may be useful to further reduce the thromboembolic risk in AF patients. The aim of the present study is to compare the long-term risk of thromboembolic events and treatment-related complications in patients with AF treated by OAT strategies and catheter ablation. Methods Pubmed, Cochrane and Google Scholar were searched for studies including >500 patients evaluating AF patients treated with OAT (VKA: vitamin K antagonist or DOAC: Direct oral anticoagulants) and/or AF ablation. Pooled incidence of stroke/year was the primary end point, while that of stroke, of all cause bleeding and of major bleeding the secondary ones. All the analyses were stratified according to the CHADS 2 score of included patients. Results Overall, 27 studies were selected, including 50,973 patients in the AF catheter ablation group; 281,595 patients in the VKA group; 54,811 patients in the DOAC group. After a mean follow-up of 2.4 (1.5–3.8) years, the overall incidence of stroke and thromboembolic events was 0.63 per 100 patients/year in AF ablation group, 2.09 per 100 patients/year in VKA group and 1.24 per 100 patients/year in DOAC group ( p 2 score, the incidence of thromboembolic events remained lower in patients included in the AF ablation, followed by DOAC and VKA respectively ( p 2 cluster. Both the incidence of all cause bleedings and major bleedings resulted lower in AF ablation group ( p p Conclusion AF catheter ablation significantly reduces the incidence of long-term thromboembolic events compared to both VKA and DOAC. This reduction is maintained in all CHADS 2 score clusters and is strengthened by the concomitant reduction in hemorrhagic complications provided by AF ablation.

Unlike oocytes of many other mammalian species, parthenogenetically activated hamster oocytes have not been reported to develop beyond the two-cell stage. This study investigated the in vitro development into blastocysts of parthenogenetic embryos of Golden Syrian hamsters. We observed that hamster oocytes could easily be artificially activated (AA) by treatment with ionomycin plus 6-dimethylaminopurine + cycloheximide + cytochalasin B as assessed by embryo cleavage in HECM-9 (63.15%) or HECM-10 (63.82%). None of the cleaved embryos developed beyond the two-cell stage when cultured in either of the two media. However, some of the embryos overcame the two-cell block and developed to the blastocyst stage (26.45%) when they were first cultured in HECM-10 for 24 hours and then in HECM-9 (serial culture media HECM-10-9) for 72 hours. Blastocyst development was further significantly (66.2%) improved when embryos were cultured in HECM-10 supplemented with ethylenediaminetetraacetic acid for 24 hours, then in HECM-9 supplemented with glucose for 72 hours (serial culture media HECM-11a-b). Hamster oocytes activated with ionomycin, ethanol, or a combination of the two treatments would develop to the blastocyst stage in serial culture media HECM-11a-b, whereas none of the spontaneously activated oocytes cleaved (0% vs. 86.93%, p

Background Prior studies suggest that catheter ablation (ABL) for atrial fibrillation (AF) is a treatment option for patients (pts) with paroxysmal AF (PAF). Pts with persistent (Per) or long-standing persistent (LSP) were routinely excluded from most ABL based clinical trials. The effectiveness of ABL compared to drug therapy (MED) in relation to underlying AF type has not been evaluated in a large randomized clinical trial. Objective To assess the impact of AF type on clinical outcomes of ABL vs. MED in pts with AF. Methods The CABANA trial randomized 2204 pts with AF at 126 sites worldwide to ABL vs. MED with rate or rhythm control drugs. The primary endpoint was a composite of death, disabling stroke, severe bleeding, or cardiac arrest. Key secondary endpoints included mortality and recurrence of AF. Outcomes of ABL vs. MED were compared within subgroups defined by AF type using Intention-to-Treat (ITT) analyses. Results Of the 2204 pts, 946 had PAF, 1042 had Per and 215 presented with LSP. There were baseline differences among AF types in age, gender, HTN, LVH, CHF and NYHA Class. For the primary endpoint, there were no significant differences between ABL and MED in pts with PAF (hazard ratio (HR) 0.82; 95% confidence interval (CI) 0.51, 1.31), Per (HR 0.87; 95% CI 0.59, 1.28), or LSP (HR 1.01, 95% CI 0.39, 2.61). Likewise, there were no significant treatment differences in mortality; PAF (HR 0.84; 95% CI 0.46, 1.52), Per (HR 0.90; 95% CI 0.56, 1.46) and LSP (HR 0.67, 95% CI 0.23, 1.94). Post-blanking AF (time to first recurrence) was significantly reduced by ABL compared to MED across all AF types (PAF by 51%), (Per by 47%) and (LSP by 36%). Clinical Characteristics and Outcomes Clinical Outcomes Comparing ABL vs. MED (HR and 95% CI) Interaction p-value Primary Endpoint 0.82 (0.51, 1.31) 0.87 (0.59, 1.28) 1.01 (0.39, 2.61) 0.925 Mortality 0.84 (0.46, 1.52) 0.90 (0.56, 1.46) 0.67 (0.23, 1.94) 0.881 Recurrent AF 0.49 (0.38, 0.62) 0.53 (0.43, 0.65) 0.64 (0.41, 1.01) 0.564 Conclusion Pts with LSP have a lower proportion of women, and a higher proportion with manifestations of heart failure despite lower CHA2DS2VASc scores. By ITT analysis, there is no significant effect of ABL compared to MED in the primary endpoint or mortality in any AF group. However, ABL is more effective than MED for reducing recurrences of AF regardless of AF type, but with a greater effect in PAF vs Per vs LSP. Acknowledgement/Funding NIH, St Jude Medical Foundation and Corporation, Biosense Webster Inc., Medtronic Corporation, and Boston Scientific Corporation

BackgroundAtrial fibrillation (AF) is associated with an increased risk of dementia. It is presently unknown to what extent AF contributes to dementia onset independently from prevalent and incident cerebrovascular accidents (CVAs)/transient ischaemic attacks (TIAs).MethodsMEDLINE/PubMed and Embase databases were searched for prospective observational results, which produced risk estimates for dementia in AF patients, adjusted for prevalent and incident CVAs/TIAs.ResultsFive prospective observational studies were included, comprising 61 008 patients, having a median follow-up of 12.5 years. Meta-analysis of observational results indicates an increased risk of dementia in AF, adjusted for cerebrovascular clinical events (HR 1.28, 95% CI 1.17 to 1.41, I2=0%). Funnel plot analysis did not reveal a statistically significant asymmetry. Meta-regression analysis did not indicate statistically significant associations between baseline study-level covariates and risk estimates.ConclusionAF confers a nearly 30% increased risk of dementia, independently from CVAs/TIAs. Screening for AF and subsequent optimised management to lower risk of cranial injury could help in preventing dementia, a condition characterised by high social and healthcare costs.

Magnetic resonance imaging (MRI) in patients with left ventricular (LV) leads may cause tissue or lead heating, dislodgment, venous damage, or lead dysfunction.The purpose of this study was to determine the safety of MRI in patients with LV pacing leads.Prospective data on patients with coronary sinus LV leads undergoing clinically indicated MRI at 3 institutions were collected. Patients were not pacemaker-dependent. Scans were performed under pacing nurse, technician, radiologist, and physicist supervision using continuous vital sign, pulse oximetry, and ECG monitoring and a 1.5-T scanner with specific absorption rate1.5 W/kg. Devices were interrogated pre- and post-MRI, programmed to asynchronous or inhibition mode with tachyarrhythmia therapies off (if present), and reprogrammed to their original settings post-MRI.MRI scans (n = 42) were performed in 40 patients with non-MRI conditional LV leads between 2005 and 2013 (mean age 67 ± 9 years, n = 16 [40%] women, median lead implant duration 740 days with interquartile range 125-1173 days). MRIs were performed on the head/neck/spine (n = 35 [83%]), lower extremities (n = 4 [10%]), chest (n = 2 [5%]), and abdomen (n = 1 [2%]). There were no overall differences in pre- and post-MRI interrogation LV lead sensing (12.4 ± 6.2 mV vs 12.9 ± 6.7 mV, P = .38), impedance (724 ± 294 Ω vs 718 ± 312 Ω, P = .67), or threshold (1.4 ± 1.1 V vs 1.4 ± 1.0 V, P = .91). No individual LV lead changes required intervention.MRI scanning was performed safely in non-pacemaker-dependent patients with coronary sinus LV leads who were carefully monitored during imaging without clinically significant adverse effect on LV lead function.

Atrial fibrillation (AF) is strongly associated with dementia, including idiopathic dementias such as Alzheimer's disease. The relative risk of dementia is highest in AF patients 70 years and younger, and the burden of exposure to arrhythmia appears to underlie part of the risk. Areas covered: Anticoagulation choices and approach influence dementia risk. In warfarin patients, inadequate time spent in therapeutic range is highly associated with the increased dementia risk long-term. This risk is further accentuated with frequent over anticoagulation in patients also receiving aspirin. Direct oral anticoagulant therapies in early observational studies show that there is potential for improving long-term risk of dementia when compared to warfarin, although prospective trials are needed. AF and dementia are end manifestations of systemic disease; a systemic approach is needed with early treatment of shared risk factors to prevent disease presentation altogether. Expert commentary: In this review, we will bring together available data with regards to the link between anticoagulant use for AF and dementia. Anticoagulation initiation timing, use, and efficacy remain critical risk factors for dementia in AF patients and consequently provide opportunities to decrease risk.

Atrial fibrillation (AF) is associated with coronary artery disease (CAD) and major cardiovascular adverse events (MACE); cardiac PET imaging may be used to determine the risk of these outcomes. However, it is unclear whether having AF at the time of the PET will impact the results and subsequent

Eric S. Williams, MD, MACC, Chair Jonathan L. Halperin, MD, FACC, Co-Chair James A. Arrighi, MD, FACC Eric H. Awtry, MD, FACC Eric R. Bates, MD, FACC Salvatore Costa, MD, FACC Lori Daniels, MD, FACC Akshay Desai, MD, FACC Douglas E. Drachman, MD, FACC Susan Fernandes, LPD, PA-C Rosario

Primary cilia are chemosensors that play a dual role to either activate or repress Hedgehog signaling, depending on presence or absence of ligand, respectively. While inhibition of ciliogenesis has been shown to be characteristic of breast cancers, the functional consequence is unknown. Here, for the first time, inhibition of ciliogenesis led to earlier tumor formation, faster tumor growth rate, higher grade tumor formation, and increased metastasis in the polyoma middle T (PyMT) mouse model of breast cancer. In in vitro model systems, inhibition of ciliogenesis resulted in increased expression of Hedgehog-target genes through a mechanism involving loss of the repressor form of the GLI transcription factor (GLIR) and activation of Hedgehog target gene expression through cross-talk with TGF-alpha (TGFA) signaling. Bioinformatics analysis revealed that increased Hedgehog signaling is frequently associated with increased TGFA; signaling in patients with triple-negative breast cancers (TNBC), a particularly aggressive breast cancer subtype. These results identify a previously unrecognized role for inhibition of ciliogenesis in breast cancer progression. This study identifies inhibition of ciliogenesis as an important event for activation of Hedgehog signaling and progression of breast cancer to a more aggressive, metastatic disease. Implications: These findings change the way we understand how cancer cells turn on a critical signaling pathways and a provide rationale for developing novel therapeutic approaches to target noncanonical Hedgehog signaling for the treatment of breast cancer. Mol Cancer Res; 15(10); 1421–30. ©2017 AACR.

We have created a high-resolution time-resolved spectroscopy molecular biosensor for heart failure therapeutic discovery based on the structural dynamics of cardiac myosin-binding protein C (cMyBP-C). Drugs of interest mimic phosphorylation states of cMyBP-C, which influences myocardial contractility. β-adrenergic stimulation enhances contractility in myocardium, in part due to Protein Kinase A (PKA)-mediated phosphorylation of cMyBP-C. Recent structural and functional results have provided an understanding for how cMyBP-C physiologically regulates inotropy and lusiotropy in the heart by modulating actin-myosin interactions. Phosphorylation introduces a structural change within the regulatory M domain of cMyBP-C, leading to altered actin-myosin binding, and regulation of myocardial contraction. Previous molecular dynamics simulations suggested a specific rotation within the M domain upon phosphorylation that facilitates bending and exposes a putative binding site that is only accessible in the phosphorylated molecule. We hypothesized that these structural rearrangements in nanometer spatial orientation could be measured spectroscopically in mechanistic studies via introduction of site-directed probes within the M domain of cMyBP-C using time-resolved fluorescence resonance energy transfer (TR-FRET). Moreover, we hypothesized that this fluorescently-labeled recombinant muscle protein could be used as a TR-FRET biosensor for detecting potential drugs that alter the M domain structure in specific ways. These drugs will be particularly relevant to enhancement of myocardial contractility and represent novel therapeutics for heart failure and cardiomyopathy. Here, we engineered pairs of cysteine residues within the M domain for FRET labeling and tested the effects of phosphorylation to influence the tertiary structure and dynamics of cMyBP-C. We have characterized cMyBP-C’s molecular basis for fine-tuning contraction and our biosensor is well aligned for high-throughput spectroscopic screens for drugs that perturb structure specific to phosphorylation state. Compound discovery using this assay is applicable to development of novel cardiac disease therapies by enhancing contractility to alleviate dysfunction.

Dementia is a chronic, progressive syndrome resulting in deterioration of memory, cognition, thinking, and function that significantly impacts quality of life. The World Health Organization estimates that 47.5 million people suffer from dementia and 7.7 million new cases develop annually. Alzheimers disease comprises nearly 60–70% of dementia cases [1]. Epidemiologic evidence supports many risk factors that continue to increase in the community that raise risk of dementia such as aging, diabetes, high blood pressure, dyslipidemia, mid-life and late-life dementia and social isolation, vascular disease, poor diets, sedentary lifestyles, and physical inactivity [2]. As a consequence of these community trends, dementia is a health trouble that progressively burdens physicianʼs practices and intimately impacts people throughout the world. HMG-CoA reductase inhibitors (statins) are most often used to lower cholesterol, in particular LDL cholesterol, for the primary and secondary prevention of cardiovascular disease. However, the benefit of statin therapy in regard to cardiovascular disease prevention and treatment extends beyond lipid lowering alone. These benefits include antioxidant effects, anti-inflammatory effects, and improved endothelial function. Since cerebral vascular disease as well as dementia shares many common risk factors with cardiovascular

BackgroundOral anticoagulation (OAC) therapy guidelines recommend using CHA2DS2-VASc to determine OAC need in atrial fibrillation (AF). A usable tool, CHA2DS2-VASc is challenged by its predictive ability. Applying components of the complete blood count and basic metabolic profile, the Intermountain Mortality Risk Score (IMRS) has been extensively validated. This study evaluated whether use of IMRS with CHA2DS2-VASc in patients with AF improves prediction.MethodsPatients with AF undergoing cardiac catheterisation (N=10 077) were followed for non-fatal stroke and mortality (mean 5.8±4.1 years, maximum 19 years). CHA2DS2-VASc and IMRS were calculated at baseline. IMRS categories were defined based on previously defined criteria. Cox regression was adjusted for demographic, clinical and treatment variables not included in IMRS or CHA2DS2-VASc.ResultsIn women (n=4122, mean age 71±12 years), the composite of non-fatal stroke/mortality was stratified (all p-trend 2DS2-VASc (1: 12.6%, 2: 22.8%, >2: 48.1%) and IMRS (low: 17.8%, moderate: 40.9%, high risk: 64.5%), as it was for men (n=5955, mean age 68±12 years) by CHA2DS2-VASc (2: 51.8%) and IMRS (low: 19.0%, moderate: 42.0%, high risk: 65.9%). IMRS stratified stroke/mortality (all p-trend 2DS2-VASc category.ConclusionsUsing IMRS jointly with CHA2DS2-VASc in patients with AF improved the prediction of stroke and mortality. For example, in patients at the OAC treatment threshold (CHA2DS2 -VASc = 2), IMRS provided ≈4-fold separation between low and high risk. IMRS provides an enhancing marker for risk in patients with AF that reflects the underlying systemic nature of this disease that may be considered in combination with the CHA2DS2-VASc score.

Surrogate electro-anatomic-derived geometries are used as the three-dimensional (3D) basis for mapping of cardiac arrhythmias. While merged computed tomography (CT) imaging may provide stellar pulmonary vein (PV) and left atrial (LA) anatomy, the applied scans must be obtained prior to ablation, and may not reflect physiologic conditions at the time of intervention. Patient-specific, ultrasound-derived 3D imaging has been developed as an alternative basis for new generation electro-anatomic mapping. An electro-anatomic sensor positioned at the tip of the phased-array intracardiac ultrasound catheter, provides the means to specify both location and orientation of each image as the 'context' for creating the 3D volumes for co-registration with electro-anatomic mapping. Specific anatomic details such as the pulmonary veins, membranous fossa, papillary muscles, or valve structures derived from real-time imaging can also be integrated into each segmented volume. This presentation reviews the basis and methods for this novel multi-modality image fusion for the creation of robust, nearly real-time anatomic images for guiding electro-anatomic mapping and ablation without requiring pre-acquired CT image sets, with accompanying limitations.

At fertilization, the sperm triggers intracellular calcium oscillations, which are pivotal to oocyte activation and development. A working hypothesis for the interaction between the sperm and the oocyte is that disintegrin ligands on the inner acrosomal membrane of the sperm bind to integrin receptors on the oocyte vitelline membrane. The aim of these experiments was to find and identify the sperm protein ligands involved in bovine sperm-oocyte interactions. In situ fluorescent labeling of proteins and 2-D gel electrophoresis were used to identify specific sperm membrane proteins that interact with proteins in the oocyte vitelline membrane. Sperm were labeled with a fluorescent dye and used to fertilize zona-free oocytes. Sperm-oocyte complexes were either lysed immediately, or following covalent cross-linking of proteins with dibromobimane. The cross-linking reagent serves the critical function of covalently linking proteins together so that they will remain as a unit through lysis of the cells and 2-D gel analysis, and which can be subsequently identified by mass spectrometry. Lysates were electrophoretically run on the same 2-D gel. The comparison of uncross-linked and cross-linked protein spots revealed that some proteins shifted position based on binding. These spots were picked and proteins identified by mass spectrometry. These results provide a list of specific sperm proteins that interact with oocyte membrane proteins and establish a group of candidate ligands, one or more of which may be responsible for induction of outside-in signaling resulting in oocyte activation and fusion of the gametes.

The effects of nicotine on nuclear maturation and meiotic spindle dynamics of bovine oocytes and subsequent embryonic development were investigated. Maturation rates (85%-94%) derived from nicotine treatments at 0.01 to 1.0 mM were similar to the control (86%), but significantly decreased at 2.0 to 6.0 mM. Haploid complements of metaphase II oocytes in 0.01 to 1.0 mM nicotine (approximately 90%) were similar to the control, while lower (ranged from 63% to 76%, P < 0.05 or P < 0.01) haploid oocytes were observed in the 2.0 to 6.0 mM nicotine groups. The majority of the PB1-free oocytes derived from 3.0 to 6.0 mM nicotine treatments were diploidy (2n = 60). Spindle microtubules changed from characteristically being asymmetrical in the controls to being equally distributed into two separate chromosome groups in the nicotine treatments. Nicotine disorganized the microfilament organization and inhibited the movement of anaphase or telophase chromosomes to the cortical area. The inhibited two chromosome groups became two spindles that either moved close in proximity or merged entirely together resulting in diploidy within the affected oocyte. Nicotine treatment significantly reduced the rate of cleavage and blastocyst development after parthenogenetic activation. Diploidy and cell number were drastically reduced in the resultant blastocysts. In conclusion, nicotine can alter the normal process of bovine oocyte meiosis and affects subsequent embryonic development.

The Drosophila alphaPS2betaPS integrin is required for diverse development events, including muscle attachment. We characterized six unusual mutations in the alphaPS2 gene that cause a subset of the null phenotype. One mutation changes a residue in alphaPS2 that is equivalent to the residue in alphaV that contacts the arginine of RGD. This change severely reduced alphaPS2betaPS affinity for soluble ligand, abolished the ability of the integrin to recruit laminin to muscle attachment sites in the embryo and caused detachment of integrins and talin from the ECM. Three mutations that alter different parts of the alphaPS2 beta-propeller, plus a fourth that eliminated a late phase of alphaPS2 expression, all led to a strong decrease in alphaPS2betaPS at muscle ends, but, surprisingly, normal levels of talin were recruited. Thus, although talin recruitment requires alphaPS2betaPS, talin levels are not simply specified by the amount of integrin at the adhesive junction. These mutations caused detachment of talin and actin from integrins, suggesting that the integrin-talin link is weaker than the ECM-integrin link.

A method for measuring the microclustering of a class of cell surface receptors called integrins is reported. Integrins are proteins involved in bi-directional signaling across the cell membrane, and are important in cell adhesion, growth and survival. Their activity is regulated by changes in protein conformation and protein clustering. The developed in vivo clustering assay uses fluorescence resonance energy transfer (FRET), and has the benefit of requiring a single cloning step to generate FRET donors and acceptors that can be used to measure the clustering of a series of integrin mutants. The FRET reporters contain extracellular donor or acceptor fluorescent protein attached to native integrin cytoplasmic and transmembrane domains, and these are expressed along with wild-type or mutant integrins. Expression of the FRET reporters has no affect on the ligand binding properties of co-expressed integrins. FRET values are calculated for cell lines spreading on ligand coated surfaces, and these values are independent of fluorescent protein expression. No FRET is observed in cell lines expressing the reporters in the absence of integrins. Integrin dependent FRET values increase approximately 2- to 3-fold when the integrins contain mutations that result in increased ligand binding affinities.

1.1 Document Development Process #### 1.1.1 Writing Committee Organization The writing committee was selected to represent the American College of Cardiology (ACC) and the Heart Rhythm Society (HRS) and included a cardiovascular training program director, an electrophysiology (EP) program

The present retrospective study investigated pregnancy rates, the incidence of pregnancy loss and large offspring syndrome (LOS) and immune-related gene expression of sheep and goat somatic cell nuclear transfer (SCNT) pregnancies. We hypothesised that significantly higher pregnancy losses observed in sheep compared with goat SCNT pregnancies are due to the increased amounts of T-helper 1 cytokines and proinflammatory mediators at the maternal–fetal interface. Sheep and goat SCNT pregnancies were generated using the same procedure. Control pregnancies were established by natural breeding. Although SCNT pregnancy rates at 45 days were similar in both species, pregnancy losses between 45 and 60 days of gestation and the incidence of LOS were significantly greater in sheep than in goats. At term, the expression of proinflammatory genes in sheep SCNT placentas was increased, whereas that in goats was similar to that in control animals. Genes with altered expression in sheep SCNT placentas included cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin 2 receptor alpha (IL2RA), cluster of differentiation 28 (CD28), interferon gamma (IFNG), interleukin 6 (IL6), interleukin 10 (IL10), transforming growth factor beta 1 (TGFB1), tumor necrosis factor alpha (TNF-α), interleukin 1 alpha (IL1A) and chemokine (C-X-C motif) ligand 8 (CXCL8). Major histocompatibility complex-I protein expression was greater in sheep and goat SCNT placentas at term than in control pregnancies. An unfavourable immune environment is present at the maternal–fetal interface in sheep SCNT pregnancies.

This study indicated that prolonged exposure of donor cell nuclei to oocyte cytoplasm before activation results in abnormal chromatin morphology, and reduced development to compacted morula/blastocyst stagein vitro. However, after transfer of embryos to recipients, there was no difference in pregnancy rates throughout gestation. Chromatin morphology was evaluated for embryos held 2, 3, 4 and 5 h between fusion and activation. In embryos held 2 h, 15/17 (88.2%) embryos contained condensed chromosomes, while only 12/24 (50.0%) embryos held 3 h exhibited this characteristic. The proportion of embryos with elongated or fragmented chromosomes tended to increase with increased hold time. While 15/19 (78.9%) of embryos held 2 h developed a single pronucleus 6 h after activation, only 8/22 (36.4%) had one pronucleus after a 4-h hold. Embryos held 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 h cleaved at rates of 207/281 (73.7%), 142/166 (85.5%), 655/912 (71.8%), 212/368 (57.6%), 406/667 (60.9%), 362/644 (56.2%) and 120/228 (52.6%) respectively. Further development to compacted morula/blastocyst stage occurred at rates of 78/281 (27.8%), 42/166 (25.3%), 264/912 (28.9%), 79/368 (21.5%), 99/667 (14.8%), 94/644 (14.6%) and 27/228 (11.8%) respectively. Embryos held less than 2.5 h between fusion and activation established pregnancies in 18/66 (27.3%) of recipients, while embryos held over 2.5 h established pregnancies at a rate of 17/57 (29.8%). This study indicates that holding bovine nuclear transfer embryos less than 2.5 h between fusion and activation results in improved nuclear morphology and increased development to compacted morula/blastocyst stage, and results in pregnancy rates equivalent to embryos held over 2.5 h.

The ability of synthetic arginine-glycine-aspartic acid (RGD)-containing peptides to induce intracellular calcium transients similar to those observed at fertilization by spermatozoa in the bovine has been reported (Campbell et al., 2000: Biol Reprod 62:1702-1709; Sessions et al., 2006. Mol Reprod Dev). These results also indicated the ability of synthetic RGD-containing peptides to induce activation and subsequent parthenogenetic development to the blastocyst stage, although, at numbers lower than observed with control in vitro fertilization (IVF). Evidence has been provided indicating the important effect of surrounding regions on the biological activity of the RGD sequence (Zhu and Evans, 2002; Sessions et al., 2006). The current experiments were designed to use natural RGD-containing sequences (disintegrins) to understand their effects. A total of three RGD-containing snake venom peptides (Kistrin (K), Elegantin (Ele), and Echistatin (Ech)) and one nonRGD-containing venom (Erabutoxin B (EB; control) were used at three concentrations (0.1, 1, and 10 micro g /ml) to induce parthenogenetic development to the blastocyst stage and in conjunction (1.0, 5.0, and 10 micro g/ml) with spermatozoa to evaluate competitive inhibition of fertilization and subsequent development. A (P0.01) higher number of bovine oocytes developed to the blastocyst stage after incubation with K, Ele and Ech at 1.0 micro g/ml, and was not different (P0.01) from IVF control. Fertilization was significantly reduced (P0.01) at all concentrations of K, Ele and Ech as compared to IVF control. No reduction (P0.05) was observed in EB (nonRGD) treated oocytes. These results support the involvement of a disintegrin-integrin interaction at fertilization in the bovine resulting in activation and subsequent development.

The effects of cumulus cell removal and centrifugation of maturing bovine oocytes on nuclear maturation and subsequent embryo development after parthenogenetic activation and nuclear transfer were examined. Removal of cumulus cells at 4, 8, and 15 hr after in vitro maturation (IVM) or the centrifugation of denuded oocytes had no effect on maturation rates. Oocytes treated at 0 hr of IVM had a lower expulsion rate (50%) of the first polar body (PB1). The removal of cumulus cells and centrifugation affected the pattern of spindle microtubule distribution and division of chromosomes. There were almost no spindle microtubules allocated to PB1 and the spindles were swollen in anaphase I and telophase I oocytes. Approximately 20% of PB1 oocytes contained tripolar or multipolar spindles. After activation, oocytes denuded with or without centrifugation at 8 hr of IVM resulted in the lowest rate of development (3.0%). Denuded oocytes at 4, 15, and 24 hr of IVM with centrifugation or not resulted in similar blastocyst development rates (9.6%-13.2%). However, centrifugation of oocytes denuded at the beginning of IVM resulted in lower blastocyst development rate (8.1%, P < 0.05) than the noncentrifuged oocytes (17.3%). After nuclear transfer, the blastocyst development rates of oocytes denuded and centrifuged at 0, 4, and 8 hr of IVM were not different when compared to the same patch of noncentrifuged oocytes. However, oocytes denuded and centrifuged at 15 hr of IVM resulted in lower (P < 0.05) blastocyst development rates than the noncentrifuged oocytes. The results of this study suggest that removal of cumulus cells and centrifugation of denuded oocytes affect the spindle pattern. Embryo development of denuded and centrifuged oocytes may differ depending on the time of removal of cumulus cells.

The idiopathic inflammatory myopathies (IIM) are systemic connective tissue diseases in which autoimmune pathology is suspected to promote chronic muscle inflammation and weakness. We have performed low to high resolution genotyping to characterize the allelic profiles of HLA-A, -B, -Cw, -DRB1, and -DQA1 loci in a large population of North American Caucasian patients with IIM representing the major clinicopathologic groups (n = 571). We confirmed that alleles of the 8.1 ancestral haplotype were important risk markers for the development of IIM, and a random forests classification analysis suggested that within this haplotype, HLA-B*0801, DRB1*0301 and/ or closely linked genes are the principal HLA risk factors. In addition, we identified several novel HLA factors associated distinctly with 1 or more clinicopathologic groups of IIM. The DQA1*0201 allele and associated peptide-binding motif (KLPLFHRL) were exclusive protective factors for the CD8+ T cell-mediated IIM forms of polymyositis (PM) and inclusion body myositis (IBM) (pc0.005). In contrast, HLA-A*68 alleles were significant risk factors for dermatomyositis (DM) (pc = 0.0021), a distinct clinical group thought to involve a humorally mediated immunopathology. While the DQA1*0301 allele was detected as a possible risk factor for IIM, PM, and DM patients (p0.05), DQA1*03 alleles were protective factors for IBM (pc = 0.0002). Myositis associated with malignancies was the most distinctive group of IIM wherein HLA Class I alleles were the only identifiable susceptibility factors and a shared HLA-Cw peptide-binding motif (AGSHTLQWM) conferred significant risk (pc = 0.019). Together, these data suggest that HLA susceptibility markers distinguish different myositis phenotypes with divergent pathogenetic mechanisms. These variations in associated HLA polymorphisms may reflect responses to unique environmental triggers resulting in the tissue pathospecificity and distinct clinicopathologic syndromes of the IIM.

alpha-Solanine and alpha-chaconine are two naturally occurring steroidal glycoalkaloids in potatoes (Solanum tuberosum), and solanidine-N-oxide is a corresponding steroidal aglycone. The objective of this research was to screen potential cyto-toxicity of these potato glycoalkaloids using bovine oocyte maturation, in vitro fertilization techniques and subsequent embryonic development as the in vitro model. A randomized complete block design with four in vitro oocyte maturation (IVM) treatments (Experiment 1) and four in vitro embryo culture (IVC) treatments (Experiment 2) was used. In Experiment 1, bovine oocytes (n=2506) were matured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVM medium only. The in vitro matured oocytes were then subject to routine IVF and IVC procedures. Results indicated that exposure of bovine oocytes to the steroidal glycoalkaloids during in vitro maturation inhibited subsequent pre-implantation embryo development. Potency of the embryo-toxicity varied between these steroidal glycoalkaloids. In Experiment 2, IVM/IVF derived bovine embryos (n=2370) were cultured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVC medium only. The results showed that the pre-implantation embryo development is inhibited by exposure to these glycoalkaloids. This effect is significant during the later pre-implantation embryo development period as indicated by fewer numbers of expanded and hatched blastocysts produced in the media containing these alkaloids. Therefore, we conclude that in vitro exposure of oocytes and fertilized ova to the steroidal glycoalkaloids from potatoes inhibits pre-implantation embryo development. Furthermore, we suggest that ingestion of Solanum species containing toxic amounts of glycoalkaloids may have negative effects on pre-implantation embryonic survival.

Isocupressic acid (ICA) [15-hydroxylabda-8 (17), 13E-dien-19-oic acid], a labdane diterpene acid, isolated from ponderosa pine (Pinus ponderosa), Lodgepole pine (Pinus contorta), common juniper (Juniperus communis) and Monterey cypress (Cupressus macrocarpa), induces abortion in pregnant cows when ingested primarily during the last trimester. The objective of this study was to investigate the effects of isocupressic acid on bovine oocyte maturation (in vitro maturation (IVM)-Experiment I) and preimplantation embryo development (in vitro culture (IVC)-Experiment II) using in vitro embryo production techniques and to subsequently evaluate viability and developmental competence of ICA-cultured embryos via embryo transfer to recipient heifers (Experiment III). A complete randomized block experimental design was used. In Experiment I and II, isocupressic acid was added to IVM or IVC media at 0 (TRT1, control), 1.3 (TRT2), and 2.6 microg/ml (TRT3) Results from Experiment I and II indicated that ICA did not inhibit oocyte maturation and did not adversely affect preinpiantation embryo development. Furthermore, results from Experiment II demonstrated that isocupressic acid enhanced bovine preimplantation embryo development in vitro in a dose dependent manner. Subsequently, Day 8 (Day 0 = IVF) blastocysts cultured in vitro in the medium containing 2.6 microg/ml ICA were transferred to recipient heifers and resulted in normal pregnancies as determined by ultrasound imaging. Subsequently, all but two births were normal as evaluated by post natal veterinary examination. In conclusion, ICA showed no adverse effects on oocyte maturation and preimplantation embryo development in vitro or subsequent viability in vivo using the ICA concentrations and in vitro culture parameters of this study.

The objective of this study was to determine the efficacy of using milk estradiol-17beta (E(2)) sampled during regular milking periods as a predictor of estrus in dairy cows. Twenty-three primiparous Holstein cows received radiotelemetric transmitters on day 16 of the estrous cycle (day 0 = estrus) for continuous monitoring of behavioral estrus. Milk and blood samples were collected every 12h at each milking, from day 18 of the estrous cycle until the fourth milking after the onset of estrus, for radioimmunoassay of E(2). Onset of estrus was indicated by the first standing event identified by radiotelemetry. Ultrasound examination of ovaries was conducted daily in a subset of cows (n = 17) from day 18 until ovulation was confirmed. Statistical analysis involved utilization of Pearson correlation to observe any association of mean plasma and milk E(2) concentrations. Intervals from highest measured plasma and highest measured milk E(2) until the first expression of standing behavior and intervals from highest measured plasma and highest measured milk E(2) until ovulation were compared using Student's t-test. Repeated measures were utilized to evaluate the effect of day and time on milk E(2) concentration. Chi-square procedures were utilized to detect differences in actual time of onset of estrus compared to the expectation of random time of onset of estrus throughout the 24 h day. Mean duration from highest measured milk E(2) until onset of standing behavior was 21+/-3.7h and until ovulation was 46.7+/-5.3h. Mean duration from onset of standing behavior until ovulation was 26.4+/-4.2h. Mean milk E(2) concentrations increased (P

We have analyzed a set of new and existing strong mutations in the myospheroid gene, which encodes the betaPS integrin subunit of Drosophila. In addition to missense and other null mutations, three mutants behave as antimorphic alleles, indicative of dominant negative properties. Unlike null alleles, the three antimorphic mutants are synthetically lethal in double heterozygotes with an inflated (alphaPS2) null allele, and they fail to complement very weak, otherwise viable alleles of myospheroid. Two of the antimorphs result from identical splice site lesions, which create a frameshift in the C-terminal half of the cytoplasmic domain of betaPS. The third antimorphic mutation is caused by a stop codon just before the cytoplasmic splice site. These mutant betaPS proteins can support cell spreading in culture, especially under conditions that appear to promote integrin activation. Analyses of developing animals indicate that the dominant negative properties are not a result of inefficient surface expression, or simple competition between functional and nonfunctional proteins. These data indicate that mutations disrupting the C-terminal cytoplasmic domain of integrin beta subunits can have dominant negative effects in situ, at normal levels of expression, and that this property does not necessarily depend on a specific new protein sequence or structure. The results are discussed with respect to similar vertebrate beta subunit cytoplasmic mutations.

Background: Patients with atrial fibrillation (AF) are at higher risk of developing all forms of dementia. The mechanisms behind the association of AF and dementia are unknown. One possibility is that exposure to chronic microbleeds results in repetitive cerebral injury that is manifest by cognitive decline. We hypothesize that AF patients receiving antiplatelet therapy will display higher rates of dementia if coupled with a higher percentage of time exposed to over-anticoagulation. Methods: Chronically anticoagulated patients receiving warfarin (target INR 2-3) for AF and managed by the Intermountain Healthcare Clinical Pharmacist Anticoagulation Service (CPAS) with no history of dementia or stroke/TIA and receiving antiplatelet therapy were included. The primary outcome was the presence of dementia defined by neurology determined ICD-9 codes. Percent time with an INR above 3.0 was determined. Multivariable Cox hazard regression was utilized to determine dementia incidence by percentage categories of supratherapeutic INR. Results: A total of 1031 patients were studied. Of these, the categories of the mean percent time with an INR >3.0 were 25% (n=240). Patients with a higher percent of time with supratherapeutic INRs were more likely to have valvular heart disease, renal failure (Cr>2.0), a higher percent of CHADS 3-6 scores, and a prior bleed. Dementia was diagnosed in 2.7% of patients with supratherapeutic INR levels 25%, p=0.05. Those with a supratherapeutic INR >25% of the time were at an increased dementia risk throughout follow-up. After multivariate adjustment, those with levels >25% had significantly higher rates of dementia compared to those with levels Discussion: In AF patients receiving antiplatelet and anticoagulant therapies, the percent of time exposed to over anticoagulation increases risk of dementia. These data support the possibility of chronic cerebral injury from microbleeds as a mechanism that underlies the association of AF and dementia.

Background: Risk stratification tools are needed to better select candidates for catheter ablation of atrial fibrillation (AF). Both the CHADS2 and CHADS2-VASC scores have utility in predicting AF-related outcomes and guiding anticoagulation treatment. We sought to determine if these risk scores predict long-term outcomes after AF ablation and if one risk score provides comparative superior performance. Methods: CHADS2 and CHADS2-VASC scores were calculated in 2179 AF ablation patients enrolled into Intermountain Heart Collaborative Study. CHADS2 and CHADS2-VASC were categorized by recursive partitioning categories as CHADS2: 0-1, 2-4, and >4 and CHADS2-VASC: 0-2, 2-5, >5. Patient outcomes were analyzed over 5 years for AF/Aflutter recurrence and MACE (death, stroke, heart failure hospitalization and AF/Aflutter recurrence). Results: Average age was 65.7±10.5 years and 61.1% were male. Both scores incrementally predicted risk of AF recurrence, stroke, heart failure, and death at 5 years (Figure). Increasing CHADS2 (hazard ratio [HR] =1.19, p Conclusion: Both the CHADS2 and CHADS2-VASC scores were excellent in stratifying patients for 5-year outcomes after AF ablation. However, the CHADS2-VASC score was superior to CHADS2 when accounting for all baseline variables for predicting both AF recurrence and AF-related morbidities.

Les mouflons argalis des formes Dalai-lamae (Ovis ammon dalai-lamae), Gobi (O. a. darwini), Kara Tau (O. a. nigrimontana) ont en commun 2n = 56 chromosomes diploides et un caryotype comprenant 2 paires de chromosomes a deux bras et 25 paires d'autosomes acrocentriques, un grand chromosome acrocentrique X et un minuscule chromosome Y. Les motifs des bandes Giemsa de la plus grande paire de chromosomes a deux bras sont identiques a ceux de la plus grande paire de chromosomes a deux bras qu'on observe chez tous les moutons sauvages et domestiques du genre Ovis. Les motifs de la seconde paire de chromosomes a deux bras (metacentriques) sont identiques a ceux de la troisieme paire de chromosomes a deux bras observes chez les Ovis a 2n = 54 chromosomes et a la troisieme plus grande paire de chromosomes chez le mouflon des neiges de Siberie (O. nivicola) qui a pour caryotype 2n = 52 chromosomes. Les bandes G des caryotypes des mouflons des formes dalai-lamae, darwini, hodgsoni et nigrimontana sont en accord avec celles observees pour toutes les sous-especes du mouflon argali (O. ammon), a l'exception du chromosome Y qui est acrocentrique au lieu d'etre metacentrique comme c'est generalement le cas chez les mouflons sauvages de type argali et chez le mouton domestique. Le mouflon argali des formes Dalai-lamae et Tibetaine ont un collier de poils plus clairs et longs autour de la gorge, et la couleur du corps est caracteristique de l'argali originaire du plateau Tibetain, tandis que les argalis de la forme de l'extreme Ouest de Gobi, ressemblent a l'argali dans sa phase la plus foncee. Le mouflon de Kara Tau a le pelage le plus sombre parmi les argalis.

Cloning mammalian species from cell lines of adult animals has been demonstrated. Aside from its importance for cloning multiple copies of genetically valuable livestock, cloning now has the potential to salvage endangered or even extinct species. The aim of this study was to investigate the effect of the bovine and domestic (Ovis aries) ovine oocyte cytoplasm on the nucleus of an established cell line from an endangered argali wild sheep (Ovis ammon) after nuclear transplantation. A fibroblast cell line was established from skin biopsies from an adult argali ram from the People's Republic of China. Early karyotype analysis of cells between 3-6 passages revealed a normal diploid chromosome number of 56. The argali karyotype consisted of 2 pairs of biarmed and 25 pairs of acrocentric autosomes, a large acrocentric and minute biarmed Y. Bovine ovaries were collected from a local abattoir, oocytes aspirated, and immediately placed in maturation medium consisting of M-199 containing 10% fetal bovine serum, 100 IU/mL penicillin, 100 microg/mL streptomycin, 0.5 microg/mL follicle-stimulating hormone (FSH), 5.0 microg/mL luetinizing hormone (LH) and 1.0 microg/mL estradiol. Ovine (O. aries) oocytes were collected at surgery 25 hours postonset of estrus from the oviducts of superovulated donor animals. All cultures were carried out at 39 degrees C in a humidified atmosphere of 5% CO2 and air. In vitro matured MII bovine oocytes were enucleated 16-20 hours after onset of maturation and ovine oocytes within 2-3 hours after collection. Enucleation was confirmed using Hoechst 33342 and UV light. The donor argali cells were synchronized in G0-G1 phase by culturing in Dulbecco's modified Eagle's medium (DMEM) plus 0.5% fetal bovine serum for 5-10 days. Fusion of nuclear donor cell to an enucleated oocyte (cytoplast) to produce nuclear transfer (NT) embryos was induced by 2 electric pulses of 1.4 kV/cm for 30 microsc. Fused NT embryos were activated after 24 hours of maturation by exposure to ionomycin (5 microM, 4 minutes) followed by incubation in 6-dimethylaminopurine (0.2 mM, 4 hours) and cultured in microdrops of CR1aa medium. From a total of 166 constructed nuclear donor cell-bovine cytoplasm NT couples, 128 (77%) successfully fused, 100 (78%) developed to 8-16 cell stage, and 2 (1.56%) developed to the blastocyst stage. The presence of argali nuclei in 8-16 cell stage embryo clones was confirmed after observation of Hoechst 33342 stained embryos under UV light and chromosome analysis of metaphase spreads from blastomeres. A total of 127 constructed nuclear donor cell-ovine cytoplasm NT couples were produced, 101 (80%) successfully fused, 81 (80% of fused) developed to the 16- to 32-cell stage. A total of 28 hybrid (argali-sheep) and 21 sheep-sheep NT embryos were transferred into 6 recipients and 4 recipients, respectively. Two of these recipients, 1 carrying argali-sheep and 1 sheep-sheep, were confirmed pregnant at 49 days by ultrasound, but both pregnancies terminated by 59 days. The results of this study demonstrate the possibility of using xenogenic oocytes to produce early-stage embryos and pregnancies from an established fibroblast cell line of an endangered species.

We have used quantitative epifluorescence microscopy of fluorescent ATP to measure single-nucleotide turnover in skinned skeletal muscle fibers from old male and female mice. Aging causes declines in muscle strength, often leading to weakness-related health problems for the elderly. Female muscle has additional functional decrements with age due to reduced ovarian hormone production. Estradiol (E2) is the major sex hormone signal to skeletal muscle in females, and strength loss is rescued by E2 treatment in ovariectomized (OVX) mice. We previously showed that E2-mediated signaling reversibly regulates slow ATP turnover by myosin in single fibers isolated from OVX mice. To investigate E2 mechanisms on aged skeletal muscle, single fibers were isolated from young (2 months) or old (28 months) male and female mice, and were incubated with mantATP. Old female mice receiving 60-day E2 treatment were also studied. We measured the decay of mantATP fluorescence in an ATP-chase experiment to characterize the slow nucleotide turnover, called the super-relaxed state (SRX), detected in approximately one-third of the myosin heads. The SRX turnover was faster in aged female fibers compared to young female fibers. In contrast, ATP turnover in old male fibers was not significantly different than in young males, and even trended toward slower rather than faster turnover with aging. We conclude that E2-mediated signaling reversibly regulates slow ATP turnover by myosin in aging female mice. Age- and hormone-related functional deficits may be targetable at the level of myosin and other contractile protein structure/function for strategies to offset muscle weakness and metabolic changes that occur with age. This work was supported by NIH grants to DDT (R37 AG026160 and R01 AR032961), to DL (R01 AG31743), to BAC (R00 HL122397), and to KJP and BCC (T32 AR7612).

Two in vitro maturation (IVM) and fertilization (IVF) protocols were evaluated with the objective of improving upon a protocol that will optimize IVF-assisted reproduction in sheep. There was no attempt to compare possible confounding effects within protocol. The addition of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to the maturation (IVM) medium significantly enhanced ovine oocyte maturation as compared to human chorionic gonadotropin (hCG) alone. Oocytes fertilized in vitro with spermatozoa treated with calcium ionophore A23187 and caffeine in Brackett and Oliphant medium (Protocol 1) significantly improved oocyte fertilization and embryo development in vitro than with spermatozoa separated by swim-up in synthetic oviduct fluid containing estrus ewe serum (Protocol 2). Embryos derived from oocytes matured in medium containing FSH and LH and fertilized with Protocol 1 were transferred to synchronized recipients and resulted in the birth of lambs. This protocol may be useful for assisted reproduction in sheep.

Tiggrin is a novel extracellular matrix ligand for the Drosophila PS2 integrins. We have used flanking P elements to generate a precise deletion of tiggrin. Most flies lacking tiggrin die as larvae or pupae. A few adults do emerge and these appear to be relatively normal, displaying only misshapen abdomens and a low frequency of wing defects. Examination of larvae shows that muscle connections, function and morphology are defective in tiggrin mutants. Muscle contraction waves that extend the length of the larvae are much slower in tiggrin mutants. Direct examination of bodywall muscles shows defects in muscle attachment sites, where tiggrin is specifically localized, and muscles appear thinner. Transgenes expressing tiggrin are capable of rescuing tiggrin mutant phenotypes. Transgenes expressing a mutant tiggrin, whose Arg-Gly-Asp (RGD) integrin recognition sequence has been mutated to Leu-Gly-Ala (LGA) show much reduced, but significant, rescuing ability. Cell spreading assays detect no interactions of this mutant tiggrin with PS2 integrins. Therefore, while the RGD sequence is critical for PS2 interactions and full activity in the whole fly, the mutant tiggrin retains some function(s) that are probably mediated by interactions with other ECM molecules or cell surface receptors

Resazurin is a redox dye (7-hydroxy-3H-phenoxazin-3-one-10-oxide) used for assessing potential fertility of spermatozoa and functional status of eukaryotic cells. In this study, the fertilizing capacity of spermatozoa treated with resazurin and effects of resazurin on bovine embryo development in vitro was examined. Abattoir-derived bovine oocytes were collected and subjected to in vitro maturation (IVM), fertilization (IVF) and culture (IVC). In Experiment 1, bovine oocytes (n=2767) were fertilized with spermatozoa exposed to resazurin (17.6 μg/ml) for 0, 15, 30, 60 min, respectively. There was no significant (P>0.05) difference with respect to oocyte cleavage, morula and blastocyst production between treatments. In Experiment 2, oocytes (n=1671) were treated with resazurin (1.8 μg/ml) during IVM, IVF, IVC, respectively, or during the entire IVM, IVF and IVC procedures. There was no significant (P>0.05) difference in cleavage rates. However, the proportion of embryos that developed into blastocysts, expanded and hatched blastocysts in those groups in which oocytes/embryos were treated with resazurin during IVC or IVM/IVF/IVC was significantly (P

The potential of ethylene oxide (EtO) residues in exposed plastic tissue culture dishes to adversely affect bovine oocyte maturation, fertilization and subsequent embryonic development was monitored. In experiment 1, the effects of aeration time and aeration combined with washing of EtO-gassed culture dishes on the extent of residual toxicity were investigated. There was no cleavage in any treatment in which oocytes were matured and fertilized in dishes exposed to EtO. EtO residues caused functional degeneration of oocytes even when culture dishes were aerated for more than 12 days post EtO-exposure and repeatedly washed. In experiment 2, the residual toxicity of EtO gas on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) were evaluated. Cleavage rate significantly decreased and post-cleavage development was retarded in ova maintained in dishes treated with EtO either during IVM or IVF. EtO residues may be more detrimental to spermatozoa than to oocytes which may have been the primary cause of fertilization failure during IVF.

Co-culture with various cell types can enhance development of bovine embryos, especially through the transition from maternal to embryonic mRNA utilization, a stage of growth refractory to most in vitro methods. Bovine oviductal epithelial (BOE) cells have been particularly successful for culturing embryos through the refractory stage; however, Buffalo rat liver (BRL) cells are a readily available, long-lived, easy-to-care-for alternative. This study compared the embryotrophic activity of BOE to BRL cells with particular emphasis on the transition stage of growth. A total of 7158 immature bovine oocytes, matured and fertilized in vitro, were divided into 4 different culture treatments: Treatment 1: BRL conditioned medium for 72 h then BRL co-culture; Treatment 2: BRL co-culture; Treatment 3: BOE co-culture for 72 h in 5% oxygen then BRL co-culture; and Treatment 4: BOE co-culture for 72 h in 5% oxygen followed by BOE co-culture in air. Those same treatments were used to evaluate embryotrophic differences of early (4 to 5) versus late (14 to 15) passage BRL cells maintained in M-199 medium with 10% serum. Two bulls were also evaluated to determine if there exists a bull-by-culture system interaction. Treatment 3 resulted in the best development after 9 d; 9.1% of selected immature oocytes developed to expanded blastocyst. Early passage BRL cells were significantly more embryotrophic than later passage cells; this was most pronounced for Treatment 2. There was a treatment-by-bull interaction, which should be considered when comparing results among similar studies.

Abnormal Hedgehog (Hh) pathway activity has been reported in many cancers, including basal cell carcinomas, medulloblastomas, rhabdomyosarcomas, glioblastomas, and breast and prostate cancers. For this reason, the Hh pathway is a flourishing area for development of anticancer drugs such as Hh ligand antagonists (e.g., 5E1 and robotnikinin), Smo inhibitors (e.g., GDC-0449 and IPI-926), and Gli transcriptional activity inhibitors (e.g., GANT58 and GANT61). It is now clear that primary cilia are required for activation of the Hh pathway in normal vertebrate cells. It is in the primary cilium that both positive and negative effectors of the Hh pathway are processed by posttranslational modifications. In many cancers, preliminary results suggest that primary cilia are lost. As drugs that inhibit different steps of the Hh pathway are developed, it will be important to consider how these drugs will function in the context of primary cilia in the tumor environment. Here, we discuss why some of the Hh inhibitors may be ineffective if primary cilia are lost on cancer cells. Understanding the relationships between clinical inhibitors of the Hh pathway and the presence or absence of primary cilia may turn out to be critical for targeting these therapeutics to the correct population of patients and improving their efficacy. Further work is needed in this area to maximize the potential of these exciting therapeutic targets. Clin Cancer Res; 18(9); 2429–35. ©2012 AACR.

AF ablation in the patients involving PV stumps from prior lobectomy and pneumonectomy has not been described in literature. We describe a multicenter experience of all patients with residual PV stumps after lung resection undergoing AF ablation. 14 patients with lung resection (for tumors in 9

There is limited data on the impact of very low left ventricular ejection fraction (LVEF) on mortality after catheter ablation of ventricular tachycardia (VT). Multicenter, retrospective analysis of all consecutive patients undergoing VT ablation at 4 high volume centers between 2007 and 2010. We