Contribution of electrostatic and structural properties of Kv4.3 S4 arginine residues to the regulation of channel gating

AbstractPrevious work has demonstrated that replacing individual arginine (R) residues in the S4 domain of Kv4.3 with alanine (A) not only altered activation and deactivation processes, but also those of closed-state inactivation (CSI) and recovery. R → A mutants eliminated individual positive charge while substantially reducing side chain volume and hydrophilic character. Their novel effects on gating may thus have been the result of electrostatic and/or structural perturbations. To address this issue, and to gain further insights into the roles that S4 plays in the regulation of Kv4.3 gating transitions, we comparatively analyzed arginine to glutamine (R → Q) mutations at positions 290, 293, and 296. This maneuver maintained positive charge elimination of the R → A mutants, while partially restoring native side chain volume and hydrophilic properties. R → A and R → Q mutant pairs produced similar effects on the forward gating process of activation. In contrast, significant differences between the two substitutions were discovered on deactivation, CSI, and recovery, with the R → Q mutants partially restoring wild type characteristics. Our results argue that modification of individual S4 residue properties may result in altered localized interactions within unique microenvironments encountered during forward and reverse gating transitions. As such, predominant effects appear on the reverse gating transitions of deactivation and recovery. These results are consistent with the proposal that arginine residues in S4 are involved in regulating Kv4.3 CSI and recovery

Non-Native R1 Substitution in the S4 Domain Uniquely Alters Kv4.3 Channel Gating

The S4 transmembrane domain in Shaker (Kv1) voltage-sensitive potassium channels has four basic residues (R1–R4) that are responsible for carrying the majority of gating charge. In Kv4 channels, however, R1 is replaced by a neutral valine at position 287. Among other differences, Kv4 channels display prominent closed state inactivation, a mechanism which is minimal in Shaker. To determine if the absence of R1 is responsible for important variation in gating characteristics between the two channel types, we introduced the V287R mutant into Kv4.3 and analyzed its effects on several voltage sensitive gating transitions. We found that the mutant increased the voltage sensitivity of steady-state activation and altered the kinetics of activation and deactivation processes. Although the kinetics of macroscopic inactivation were minimally affected, the characteristics of closed-state inactivation and recovery from open and closed inactivated states were significantly altered. The absence of R1 can only partially account for differences in the effective voltage sensitivity of gating between Shaker and Kv4.3. These results suggest that the S4 domain serves an important functional role in Kv4 channel activation and deactivation processes, and also those of closed-state inactivation and recovery

Student Attitudes Contribute to the Effectiveness of a Genomics CURE

The Genomics Education Partnership (GEP) engages students in a course-based undergraduate research experience (CURE). To better understand the student attributes that support success in this CURE, we asked students about their attitudes using previously published scales that measure epistemic beliefs about work and science, interest in science, and grit. We found, in general, that the attitudes students bring with them into the classroom contribute to two outcome measures, namely, learning as assessed by a pre- and postquiz and perceived self-reported benefits. While the GEP CURE produces positive outcomes overall, the students with more positive attitudes toward science, particularly with respect to epistemic beliefs, showed greater gains. The findings indicate the importance of a student\u27s epistemic beliefs to achieving positive learning outcomes

Carotid endarterectomy and carotid artery stenting utilization trends over time

<p>Abstract</p> <p>Background</p> <p>Carotid endarterectomy (CEA) has been the standard in atherosclerotic stroke prevention for over 2 decades. More recently, carotid artery stenting (CAS) has emerged as a less invasive alternative for revascularization. The purpose of this study was to investigate whether an increase in stenting parallels a decrease in endarterectomy, if there are specific patient factors that influence one intervention over the other, and how these factors may have changed over time.</p> <p>Methods</p> <p>Using a nationally representative sample of US hospital discharge records, data on CEA and CAS procedures performed from 1998 to 2008 were obtained. In total, 253,651 cases of CEA and CAS were investigated for trends in utilization over time. The specific data elements of age, gender, payer source, and race were analyzed for change over the study period, and their association with type of intervention was examined by multiple logistic regression analysis.</p> <p>Results</p> <p>Rates of intervention decreased from 1998 to 2008 (P < 0.0001). Throughout the study period, endarterectomy was the much more widely employed procedure. Its use displayed a significant downward trend (P < 0.0001), with the lowest rates of intervention occurring in 2007. In contrast, carotid artery stenting displayed a significant increase in use over the study period (P < 0.0001), with the highest intervention rates occurring in 2006. Among the specific patient factors analyzed that may have altered utilization of CEA and CAS over time, the proportion of white patients who received intervention decreased significantly (P < 0.0001). In multivariate modeling, increased age, male gender, white race, and earlier in the study period were significant positive predictors of CEA use.</p> <p>Conclusions</p> <p>Rates of carotid revascularization have decreased over time, although this has been the result of a reduction in CEA despite an overall increase in CAS. Among the specific patient factors analyzed, age, gender, race, and time were significantly associated with the utilization of these two interventions.</p

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

Recovery Characteristics.

<p>A) WT and V287R macroscopic recovery kinetics (HP = −100 mV) developed during a one second pulse to +50 mV. Mean mutant data points (n = 7) fit as a single exponential function with τ_rec = 30.10 ms. WT τ_rec = 206 ms, data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003773#pone.0003773-Skerritt1" target="_blank">[9]</a>. B) <i>Main Panel</i>: Comparison of the voltage dependence of mean τ_rec values of V287R and WT, from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003773#pone.0003773-Skerritt1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003773#pone.0003773-Skeritt1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003773#pone.0003773-Amadi1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003773#pone.0003773-Patel2" target="_blank">[25]</a>. At all HPs, macroscopic recovery kinetics were significantly faster for V287R than for WT. <i>Curve fits</i>: WT: τ_rec = 23.89e^mV/11.14+142.10 ms, V287R: τ_rec = 9.42e^mV/21.77+13.95 ms. <i>Inset</i>: Representative macroscopic recovery waveforms for V287R (P2 currents at +50 mV). Peak data points fit with a single exponential relationship with time constant = 32 ms. Calibration bar: 200 nA, 20 ms. C) Representative recordings of recovery (HP = −85 mV) from closed-state inactivation for WT (<i>upper panel</i>, fit as an “a²” formulation) and V287R (<i>lower panel</i>, exponential fit). Calibration bars: WT: 1 µA, 50 ms, V287R: 500 nA, 50 ms. D) Voltage dependence of mean τ_rec,csi values for WT (hollow squares, n = 5) and V287R (solid squares, n = 7). <i>Curve fits</i>: WT: τ_rec,csi = 248.50e^mV/93.1−582.10 ms, V287R: τ_rec,csi = 5.80e^mV/19.77+34.10 ms.</p

Inactivation Characteristics.

<p>A) Isochronal one second inactivation relationship “i”. Mean data points (n = 13) fit to a single Boltzmann relationship (V_1/2 = −51.50 mV, k = 7.26 mV). WT data (V_1/2 = −60.10 mV, k = 6.20 mV) from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003773#pone.0003773-Skerritt1" target="_blank">[9]</a>. B) Comparative overlays of “a⁴” and “i” relationships for WT and V287R channels. Both expression conditions displayed significant closed-state inactivation, with V287R stabilizing non-inactivated closed states. The mutant channel also produced a greater depolarizing shift in “a⁴” than in “i”. C) V287R mean τ_inact−V_m curves (n = 12). Curve fits: τ_fast = 86.20e^{10−mV/12.23}+23.50 ms; τ_slow = 99.70e^{10−mV/33.97}+201.90 ms. WT data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003773#pone.0003773-Skeritt1" target="_blank">[10]</a>. D) <i>Main Panel</i>: V287R kinetics of closed-state inactivation. A fixed one second pulse to +50 mV (P2) was preceded by a pulse of progressively increasing duration (P1) at each of the potentials indicated. The decline of P2 current as a function of P1 duration was used to determine τ_csi. V287R curve fit: τ_csi = 725.68e^{−60+mV/10.23}+91.50 ms. WT data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003773#pone.0003773-Patel2" target="_blank">[25]</a>. <i>Inset</i>: Development of CSI at −40 mV for V287R, τ_csi = 202 ms. Calibration Bar: 100 nA, 150 ms.</p

Summary of all Kv4.3 S4 mutant data collected to date: “i” V_1/2 values as a function of “a” V_1/2 values.

<p>The solid black line (centered on WT) is a linear relationship with slope = 1.0, as predicted from previous work on Shaker channels <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003773#pone.0003773-Papazian1" target="_blank">[26]</a>. The solid gray line is a best fit to all mutant data points (WT excluded, slope = 0.65, mean R→A/Q data points from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003773#pone.0003773-Skerritt1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003773#pone.0003773-Skeritt1" target="_blank">[10]</a>). All ΔV_1/2 shifts were less than those predicted from Shaker.</p