Effects of Isoproterenol on IhERG during K+ changes in HEK293 cells

noIntroduction:The human ether-a-go-go related gene (hERG) encodes the pore forming protein which mediates the rapid delayed rectifier K+ current in the heart (IKr). Together with other ion channels hERG determines the cardiac action potential and regulates the heart beating. Dysfuction of the hERG ion channel will lead to acquired long QT syndrome (LQTS). Therefore, new drug candidates must pass the test for a potential inhibitory effect on the hERG current as a first step in a nonclinical testing strategy. Arrhythmias in patients with LQTS are typically triggered during physical or emotional stress, suggesting a link between sympathetic stimulation and arrhythmias. It is well known that potassium level can affect the QT interval through affecting IhERG both in vivo and in vitro.In this study, we try to find out whether the trigger effect still exist when K+ changes violently in a short time period. In other words, whether the risk of TdP aggravate when patients suffer from acute water electrolyte balance disorder, which is a common symptom in hot weather. Methods: HEK293 Cell line stably expressing hERG channel were cultured in DMEM supplemented with 10% of fetal bovine serum.Whole-cell patch-clamp method was applied for ionic current recordings. The compositions of pipette was (in mM) 125 KCl, 5 MgCl2, 5 EGTA-K, 10 HEPES-K and 5 Na-ATP adjusted to pH 7.2 with KOH. The bath solutions for recording the IhERG currents was 136 NaCl, 4 KCl, 1 MgCl2, 10 HEPES-Na, 1.8 CaCl2 and 10 glucose, pH 7.4 with NaOH. The low extracellular K+ solution was 115 KCl, 5 MgCl2, 5 EGTA-K, 10 HEPES-K and 10 Na-ATP adjusted to pH 7.2 with NaOH. Patch-clamp experiments were performed at room temperature (22 ± 1°C). The recording of low K+ current was carried out immediately after the original normal K+ solution has been totally replaced. Isoproterenol (ISO) 100nM was added into both kinds of K+ solution to apply the effect of β1-AR stimulation. Results: We found that low K+ solution increased IhERG from 907.39±18.68to 1620.08±249.44pA(n=30,P<0.05); Low K+also shifted the I-V curve to the left. IC50 in control is 10.31±5.52 mV, low K+ is -6.15±1.58 mV. When adding ISO 100nM to extracellular solution, same effects were shown for both groups.ISO decreased Imax for both group. In control group, Imax reduced from 907.39±18.68to493.16±54.41pA (n=30, P<0.01), while in low K+ group, I max decreased Imax from 1620.08±29.44to 488.48±81.87pA(n=30,P<0.05). At the same time, ISO shifts the I-V curve to the right for the control group and shift the curve to the left for low K+ group. IC50 in control when added ISO is 22.25±3.80 mV, while IC50 in low K+ group after adding 100nM ISO is -31.00±5.73 mV. Conclusion: The results from this study is contradict to those in our previous study where low K+ combined with ISO can lead to temporarily increase of QT interval in vivo.It is reported that an increase in net outward repolarizing current, due to a relatively large increase of IKs, is responsible for the changes of QT interval in response to beta-adrenergic stimulation in vivo(2). Therefore future studies need to co-transfect IKs channel to confirm this. References: 1. Guo J, Massaeli H, Xu J, Jia Z, Wigle JT, Mesaeli N, et al. Extracellular K+ concentration controls cell surface density of IKr in rabbit hearts and of the HERG channel in human cell lines. The Journal of clinical investigation. 2009;119(9):2745- 57. 2. Shimizu W, Antzelevitch C. Differential effects of beta-adrenergic agonists and antagonists in LQT1, LQT2 and LQT3 models of the long QT syndrome. Journal of the American College of Cardiology. 2000;35(3):778-86

Method for filling missing data of mine ventilation parameters

The intelligent mine ventilation system is very important for the intelligent construction of coal mines. In order to solve the problem of missing mine ventilation parameter data caused by the lack of measurement conditions, instrument signal interference, uneven wind speed of roadway section, improper manual operation and other restrictive factors during actual measurement of mine ventilation parameters, a method for filling the missing data of mine ventilation parameters based on the multiple imputation method of random forest-chained equation was proposed. Multiple imputation with chained equations is used to generate n filled values for each missing attribute value by iterations, resulting in n complete datasets, and a final complete dataset is obtained by analyzing and optimizing the n complete datasets. In order to improve the filling accuracy of missing values, the influence of the uncertainty of missing data of mine ventilation parameters on the analysis process is reasonably considered, and the missing data is filled in the prediction task of random forest in combination with the prediction mean matching model. Taking the Luxin No.2 Mine as an experimental example, the intelligent mine ventilation simulation system IMVS was used to preprocess the original data set of ventilation parameters of the Luxin No.2 Mine to obtain a complete and accurate complete dataset of mine ventilation parameters. Comparative experiments with different missing attributes, different data missing rates, and different number of iterations were conducted separately for the complete data set. The effectiveness of the model was evaluated by a variety of model evaluation indicators. The results show that the complete data set formed by the multiple imputation method of random forest-chained equation has good similarity with the original data set. Results of filling experiments with different missing columns show that the filling model can easily handle mixed data types, autonomously learning the correlations between parameters and thus reducing filling complexity. The n datasets formed after iterations are combined into a final dataset by analysis, which improves the filling accuracy. Experiments with different iterations on the complete data set after initial filling show that the data correlation will converge after a certain number of iterations

Strain Differences in Developmental Vulnerability to Alcohol Exposure Via Embryo Culture in Mice

Background Prenatal alcohol exposure can result in varying degrees of neurodevelopmental deficits, growth retardation, and facial dysmorphology. Variation in these adverse outcomes not only depends on the dose and pattern of alcohol exposure but also on less well understood interactions among environmental, genetic, and maternal factors. The current study tested the hypothesis that fetal genotype is an important determinant of ethanol teratogenesis by evaluating effects of ethanol exposure via embryo culture in three inbred strains of mice known to differ in the vulnerability of prenatal alcohol exposure in vivo. Methods and results Three strains of mice, C57BL/6N (B6), DBA/2 (D2), and 129S6/SvEvTac (129S6) were assessed in a whole embryo culture beginning on embryonic day 8.25 (E8.25), with or without alcohol administration at 88mM for 6 hours followed by 42 hrs culture in ethanol-free media. Contrasting strain differences in susceptibility were observed for the brain, the face, and other organ systems using the Maele-Fabry and Picard scoring system. The forebrain, midbrain, hindbrain, heart, optic vesicle, caudal neural tube, and hindlimbs of the B6 mice were severely delayed in growth, whereas compared to the respective controls, only the forebrain and optic vesicle were delayed in the D2 mice, and no effects were found in the 129S6 mice. A large number of cleaved(c)-caspase3 positive (+) cells were found in regions of the brain, optic vesicles, cranial nerve nuclei V, VII, VIII, and IX as well as the craniofacial primordial; only a few were found in corresponding regions of the B6 controls. In contrast, only a small number of c-caspase 3-im cells were found in either the alcohol-treated or the controls of the D2 embryos and in 129S6 embryos. The independent apoptotic markers TUNEL and Nile blue staining further confirmed the strain differences in apoptotic responses in both the neural tube and craniofacial primordia. Conclusions Under embryo culture conditions, in which alcohol exposure factors and fetal developmental staging were controlled, and maternal and intrauterine factors were eliminated, the degree of growth retardation and the extent and type of neurodevelopmental teratogenesis varied significantly across strains. Notably, the 129S6 strain was remarkably resistant to alcohol-induced growth deficits, confirming a previous in vivo study, and the D2 strain was also significantly less affected than the B6 strain. These findings demonstrate that fetal genotype is an important factor that can contribute to the variation in fetal alcohol spectrum disorder

Accelerating K-12 computational thinking using scaffolding, staging, and abstraction

We describe a three-stage model of computing instruction beginning with a simple, highly scaffolded programming en-vironment (Kodu) and progressing to more challenging frame-works (Alice and Lego NXT-G). In moving between frame-works, students explore the similarities and differences in how concepts such as variables, conditionals, and looping are realized. This can potentially lead to a deeper under-standing of programming, bringing students closer to true computational thinking. Some novel strategies for teach-ing with Kodu are outlined. Finally, we briefly report on our methodology and select preliminary results from a pi-lot study using this curriculum with students ages 10–17, including several with disabilities

Learning From Peers: A Survey of Perception and Utilization of Online Peer Support Among Informal Dementia Caregivers

Informal dementia caregivers are those who care for a person living with dementia (PLWD) without receiving payment (e.g., family members, friends, or other unpaid caregivers). These informal caregivers are subject to substantial mental, physical, and financial burdens. Online communities enable these caregivers to exchange caregiving strategies and communicate experiences with other caregivers whom they generally do not know in real life. Research has demonstrated the benefits of peer support in online communities, but they are limited in focusing merely on caregivers who are already online users. In this paper, we designed and administered a survey to investigate the perception and utilization of online peer support from 140 informal dementia caregivers (with 100 online-community caregivers). Our findings show that the behavior to access any online community is only significantly associated with their belief in the value of online peer support (p = 0.006). Moreover, 33 (83%) of the 40 non-online-community caregivers had a belief score above 24, a score assigned when a neutral option is selected for each belief question. The reasons most articulated for not accessing any online community were no time to do so (14; 10%), and insufficient online information searching skills (9; 6%). Our findings suggest that online peer support is valuable, but practical strategies are needed to assist informal dementia caregivers who have limited time or searching skills

Size dependent effects of gold nanoparticles in ISO-induced hyperthyroid rats

YesIn this study, we applied different sizes of gold nanoparticles (Au-NPs) to isoproterenol (ISO)-induced hyperthyroid heart disease rats (HHD rats). Single dose of 5, 40, 100 nm Au-NPs were injected intravenously. Cardiac safety tests were evaluated by cardiac marker enzymes in serum and cardiac accumulation of Au-NPs were measured by ICP-MS. Our results showed that size-dependent cardiac effects of Au-NPs in ISO-induced hyperthyroid rats. 5 nm Au-NPs had some cardiac protective effect but little accumulation in heart, probably due to smaller size Au-NPs can adapt to whole body easily in vivo. Histological analysis and TUNEL staining showed that Au-NPs can induce pathological alterations including cardiac fibrosis, apoptosis in control groups, however they can protect HHD groups from these harmful effects. Furthermore, transmission electron microscopy and western blotting employed on H9C2 cells showed that autophagy presented in Au-NPs treated cells and that Au-NPs can decrease LC3 II turning to LC3 I and decrease APG7 and caspase 12 in the process in HHD groups, while opposite effects on control groups were presented, which could be an adaptive inflammation reacts. As there are few animal studies about using nanoparticles in the treatment of heart disease, our in vivo and in vitro studies would provide valuable information before they can be considered for clinical use in general

Shape Defect Effect in Perpendicular Shape Anisotropy Nanodots

Perpendicular shape anisotropy spin-transfer-torque magnetic random-access memory (PSA-STT-MRAM) demonstrates high thermal stability when size is reduced to 20 nm, which gives a new way to improve the integrity of electronic devices. This long and narrow device also poses challenges in the device fabrication process, such as sample tilt and etching defects. We used a micromagnetic simulation method to investigate the relationship between those defects and device performance. The coercivity and critical switching current density of PSA-MRAM have been calculated and analyzed with micromagnetic simulation, a three-dimensional Stoner–Wohlfarth model, and spin-filter theory. Our results demonstrate how shape defects affect the performance of the PSA-MRAM and provide guidelines for practical realization of nanoscale PSA-MRAM

ADAR2 increases in exercised heart and protects against myocardial infarction and doxorubicin-induced cardiotoxicity

Exercise training benefits the heart. The knowledge of post-transcription regulation, especially RNA editing, in hearts remain rare. ADAR2 is an enzyme that edits adenosine to inosine nucleotides in double-stranded RNA, and RNA editing is associated with many human diseases. We found that ADAR2 was upregulated in hearts during exercise training. AAV9-mediated cardiac-specific ADAR2 overexpression attenuated acute myocardial infarction (AMI), MI remodeling, and doxorubicin (DOX)-induced cardiotoxicity. In vitro, overexpression of ADAR2 inhibited DOX-induced cardiomyocyte (CM) apoptosis. but it could also induce neonatal rat CM proliferation. Mechanistically, ADAR2 could regulate the abundance of mature miR-34a in CMs. Regulations of miR-34a or its target genes (Sirt1, Cyclin D1, and Bcl2) could affect the pro-proliferation and anti-apoptosis effects of ADAR2 on CMs. These data demonstrated that exercise-induced ADAR2 protects the heart from MI and DOX-induced cardiotoxicity. Our work suggests that ADAR2 overexpression or a post-transcriptional associated RNA editing via ADAR2 may be a promising therapeutic strategy for heart diseases