Comparison of intravenous lidocaine/ketorolac combination to either analgesic alone for suspected renal colic pain in the ED.

STUDY OBJECTIVE: To compare analgesic efficacy and safety of intravenous lidocaine and ketorolac combination to each analgesic alone for ED patients with suspected renal colic. METHODS: We conducted a randomized, double-blind trial comparing analgesic efficacy of a combination of intravenous lidocaine (1.5 mg/kg) and ketorolac (30 mg), to ketorolac (30 mg), and to lidocaine (1.5 mg/kg) in patients aged 18-64 presenting to the ED with suspected renal colic. Primary outcome included difference in pain scores between the groups at 30 min. Secondary outcomes included a comparative reduction in pain scores in each group from baseline to 30 and 60 min as well as rates of adverse events and need for rescue analgesia at 30 and 60 min. RESULTS: We enrolled 150 subjects (50 per group). The difference in mean pain scores at 30 min between Lidocaine and Lidocaine/Ketorolac groups was -2.89 (95% CI: -4.39 to -1.39); between Ketorolac and Lidocaine/Ketorolac group was -0.92 (95% CI: -2.44 to 0.61); and between Ketorolac and Lidocaine was -1.98 (95% CI: -3.69 to -0.27). A comparative percentage of subjects in each group required rescue analgesia at 30 and 60 min. No clinically concerning changes in vital signs were observed. No serious adverse events occurred in either group. Commonly reported adverse effects were dizziness, nausea, and headache. CONCLUSION: The administration of intravenous lidocaine/ketorolac combination to ED patients with suspected renal colic results in better analgesia in comparison to lidocaine alone but provides no analgesic advantages over ketorolac alone. Clinicaltrials.gov Registration: NCT02902770.

hERG K(+) channels: structure, function, and clinical significance.

The human ether-a-go-go related gene (hERG) encodes the pore-forming subunit of the rapid component of the delayed rectifier K(+) channel, Kv11.1, which are expressed in the heart, various brain regions, smooth muscle cells, endocrine cells, and a wide range of tumor cell lines. However, it is the role that Kv11.1 channels play in the heart that has been best characterized, for two main reasons. First, it is the gene product involved in chromosome 7-associated long QT syndrome (LQTS), an inherited disorder associated with a markedly increased risk of ventricular arrhythmias and sudden cardiac death. Second, blockade of Kv11.1, by a wide range of prescription medications, causes drug-induced QT prolongation with an increase in risk of sudden cardiac arrest. In the first part of this review, the properties of Kv11.1 channels, including biogenesis, trafficking, gating, and pharmacology are discussed, while the second part focuses on the pathophysiology of Kv11.1 channels.

Intravenous subdissociative-dose ketamine versus morphine for acute geriatric pain in the Emergency Department: A randomized controlled trial.

STUDY OBJECTIVE: We compare the analgesic efficacy and safety of subdissociative intravenous-dose ketamine (SDK) versus morphine in geriatric Emergency Department (ED) patients. METHODS: This was a prospective, randomized, double-blind trial evaluating ED patients aged 65 and older experiencing moderate to severe acute abdominal, flank, musculoskeletal, or malignant pain. Patients were randomized to receive SDK at 0.3 mg/kg or morphine at 0.1 mg/kg by short intravenous infusion over 15 min. Evaluations occurred at 15, 30, 60, 90, and 120 min. Primary outcome was reduction in pain at 30 min. Secondary outcomes included overall rates of adverse effects and incidence of rescue analgesia. RESULTS: Thirty patients per group were enrolled in the study. The primary change in mean pain scores was not significantly different in the ketamine and morphine groups: 9.0 versus 8.4 at baseline (mean difference 0.6; 95% CI -0.30 to 1.43) and 4.2 versus 4.4 at 30 min (mean difference -0.2; 95% CI -1.93 to1.46). Patients in the SDK group reported higher rates of psychoperceptual adverse effects at 15, 30, and 60 min post drug administration. Two patients in the ketamine group and one in the morphine group experienced brief desaturation episodes. There were no statistically significant differences with respect to changes in vital signs and need for rescue medication. CONCLUSION: SDK administered at 0.3 mg/kg over 15 min provides analgesic efficacy comparable to morphine for short-term treatment of acute pain in the geriatric ED patients but results in higher rates of psychoperceptual adverse effects. ClinicalTrials.gov Registration #: NCT02673372.

Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay.

BACKGROUND: The ubiquitin-proteasome system (UPS) controls the stability, localization and/or activity of the proteome. However, the identification and characterization of complex individual ubiquitination cascades and their modulators remains a challenge. Here, we report a broadly applicable, multiplexed, miniaturized on-bead technique for real-time monitoring of various ubiquitination-related enzymatic activities. The assay, termed UPS-confocal fluorescence nanoscanning (UPS-CONA), employs a substrate of interest immobilized on a micro-bead and a fluorescently labeled ubiquitin which, upon enzymatic conjugation to the substrate, is quantitatively detected on the bead periphery by confocal imaging. RESULTS: UPS-CONA is suitable for studying individual enzymatic activities, including various E1, E2, and HECT-type E3 enzymes, and for monitoring multi-step reactions within ubiquitination cascades in a single experimental compartment. We demonstrate the power of the UPS-CONA technique by simultaneously following ubiquitin transfer from Ube1 through Ube2L3 to E6AP. We applied this multi-step setup to investigate the selectivity of five ubiquitination inhibitors reportedly targeting different classes of ubiquitination enzymes. Using UPS-CONA, we have identified a new activity of a small molecule E2 inhibitor, BAY 11-7082, and of a HECT E3 inhibitor, heclin, towards the Ube1 enzyme. CONCLUSIONS: As a sensitive, quantitative, flexible, and reagent-efficient method with a straightforward protocol, UPS-CONA constitutes a powerful tool for interrogation of ubiquitination-related enzymatic pathways and their chemical modulators, and is readily scalable for large experiments.

Natural products modulating the hERG channel: heartaches and hope.

Covering: 1996-December 2016The human Ether-à-go-go Related Gene (hERG) channel is a voltage-gated potassium channel playing an essential role in the normal electrical activity in the heart. It is involved in the repolarization and termination of action potentials in excitable cardiac cells. Mutations in the hERG gene and hERG channel blockage by small molecules are associated with increased risk of fatal arrhythmias. Several drugs have been withdrawn from the market due to hERG channel-related cardiotoxicity. Moreover, as a result of its notorious ligand promiscuity, this ion channel has emerged as an important antitarget in early drug discovery and development. Surprisingly, the hERG channel blocking profile of natural compounds present in frequently consumed botanicals (i.e. dietary supplements, spices, and herbal medicinal products) is not routinely assessed. This comprehensive review will address these issues and provide a critical compilation of hERG channel data for isolated natural products and extracts over the past two decades (1996-2016). In addition, the review will provide (i) a solid basis for the molecular understanding of the physiological functions of the hERG channel, (ii) the translational potential of in vitro/in vivo results to cardiotoxicity in humans, (iii) approaches for the identification of hERG channel blockers from natural sources, (iv) future perspectives for cardiac safety guidelines and their applications within phytopharmaceuticals and dietary supplements, and (v) novel applications of hERG channel modulation (e.g. as a drug target).

Convergence of models of human ventricular myocyte electrophysiology after global optimization to recapitulate clinical long QT phenotypes.

In-silico models of human cardiac electrophysiology are now being considered for prediction of cardiotoxicity as part of the preclinical assessment phase of all new drugs. We ask the question whether any of the available models are actually fit for this purpose. We tested three models of the human ventricular action potential, the O'hara-Rudy (ORD11), the Grandi-Bers (GB10) and the Ten Tusscher (TT06) models. We extracted clinical QT data for LQTS1 and LQTS2 patients with nonsense mutations that would be predicted to cause 50% loss of function in IKs and IKr respectively. We also obtained clinical QT data for LQTS3 patients. We then used a global optimization approach to improve the existing in silico models so that they reproduced all three clinical data sets more closely. We also examined the effects of adrenergic stimulation in the different LQTS subsets. All models, in their original form, produce markedly different and unrealistic predictions of QT prolongation for LQTS1, 2 and 3. After global optimization of the maximum conductances for membrane channels, all models have similar current densities during the action potential, despite differences in kinetic properties of the channels in the different models, and more closely reproduce the prolongation of repolarization seen in all LQTS subtypes. In-silico models of cardiac electrophysiology have the potential to be tremendously useful in complementing traditional preclinical drug testing studies. However, our results demonstrate they should be carefully validated and optimized to clinical data before they can be used for this purpose.

Temperature Effects on Kinetics of KV11.1 Drug Block Have Important Consequences for In Silico Proarrhythmic Risk Prediction.

Drug block of voltage-gated potassium channel subtype 11.1 human ether-a-go-go related gene (Kv11.1) (hERG) channels, encoded by the KCNH2 gene, is associated with reduced repolarization of the cardiac action potential and is the predominant cause of acquired long QT syndrome that can lead to fatal cardiac arrhythmias. Current safety guidelines require that potency of KV11.1 block is assessed in the preclinical phase of drug development. However, not all drugs that block KV11.1 are proarrhythmic, meaning that screening on the basis of equilibrium measures of block can result in high attrition of potentially low-risk drugs. The basis of the next generation of drug-screening approaches is set to be in silico risk prediction, informed by in vitro mechanistic descriptions of drug binding, including measures of the kinetics of block. A critical issue in this regard is characterizing the temperature dependence of drug binding. Specifically, it is important to address whether kinetics relevant to physiologic temperatures can be inferred or extrapolated from in vitro data gathered at room temperature in high-throughout systems. Here we present the first complete study of the temperature-dependent kinetics of block and unblock of a proarrhythmic drug, cisapride, to KV11.1. Our data highlight a complexity to binding that manifests at higher temperatures and can be explained by accumulation of an intermediate, non-blocking encounter-complex. These results suggest that for cisapride, physiologically relevant kinetic parameters cannot be simply extrapolated from those measured at lower temperatures; rather, data gathered at physiologic temperatures should be used to constrain in silico models that may be used for proarrhythmic risk prediction.

Rescue of protein expression defects may not be enough to abolish the pro-arrhythmic phenotype of long QT type 2 mutations.

KEY POINTS: Most missense long QT syndrome type 2 (LQTS2) mutations result in Kv11.1 channels that show reduced levels of membrane expression. Pharmacological chaperones that rescue mutant channel expression could have therapeutic potential to reduce the risk of LQTS2-associated arrhythmias and sudden cardiac death, but only if the mutant Kv11.1 channels function normally (i.e. like WT channels) after membrane expression is restored. Fewer than half of mutant channels exhibit relatively normal function after rescue by low temperature. The remaining rescued missense mutant Kv11.1 channels have perturbed gating and/or ion selectivity characteristics. Co-expression of WT subunits with gating defective missense mutations ameliorates but does not eliminate the functional abnormalities observed for most mutant channels. For patients with mutations that affect gating in addition to expression, it may be necessary to use a combination therapy to restore both normal function and normal expression of the channel protein. ABSTRACT: In the heart, Kv11.1 channels pass the rapid delayed rectifier current (IKr ) which plays critical roles in repolarization of the cardiac action potential and in the suppression of arrhythmias caused by premature stimuli. Over 500 inherited mutations in Kv11.1 are known to cause long QT syndrome type 2 (LQTS2), a cardiac electrical disorder associated with an increased risk of life threatening arrhythmias. Most missense mutations in Kv11.1 reduce the amount of channel protein expressed at the membrane and, as a consequence, there has been considerable interest in developing pharmacological agents to rescue the expression of these channels. However, pharmacological chaperones will only have clinical utility if the mutant Kv11.1 channels function normally after membrane expression is restored. The aim of this study was to characterize the gating phenotype for a subset of LQTS2 mutations to assess what proportion of mutations may be suitable for rescue. As an initial screen we used reduced temperature to rescue expression defects of mutant channels expressed in Xenopus laevis oocytes. Over half (∼56%) of Kv11.1 mutants exhibited functional gating defects that either dramatically reduced the amount of current contributing to cardiac action potential repolarization and/or reduced the amount of protective current elicited in response to premature depolarizations. Our data demonstrate that if pharmacological rescue of protein expression defects is going to have clinical utility in the treatment of LQTS2 then it will be important to assess the gating phenotype of LQTS2 mutations before attempting rescue.

Getting to the heart of hERG K(+) channel gating.

Potassium ion channels encoded by the human ether-a-go-go related gene (hERG) form the ion-conducting subunit of the rapid delayed rectifier potassium current (IKr ). Although hERG channels exhibit a widespread tissue distribution they play a particularly important role in the heart. There has been considerable interest in hERG K(+) channels for three main reasons. First, they have very unusual gating kinetics, most notably rapid and voltage-dependent inactivation coupled to slow deactivation, which has led to the suggestion that they may play a specific role in the suppression of arrhythmias. Second, mutations in hERG are the cause of 30-40% of cases of congenital long QT syndrome (LQTS), the commonest inherited primary arrhythmia syndrome. Third, hERG is the molecular target for the vast majority of drugs that cause drug-induced LQTS, the commonest cause of drug-induced arrhythmias and cardiac death. Drug-induced LQTS has now been reported for a large range of both cardiac and non-cardiac drugs, in which this side effect is entirely undesired. In recent years there have been comprehensive reviews published on hERG K(+) channels (Vandenberg et al. 2012) and we will not re-cover this ground. Rather, we focus on more recent work on the structural basis and dynamics of hERG gating with an emphasis on how the latest developments may facilitate translational research in the area of stratifying risk of arrhythmias.

Genetic variation in the two-pore domain potassium channel, TASK-1, may contribute to an atrial substrate for arrhythmogenesis.

The two-pore domain potassium channel, K2P3.1 (TASK-1) modulates background conductance in isolated human atrial cardiomyocytes and has been proposed as a potential drug target for atrial fibrillation (AF). TASK-1 knockout mice have a predominantly ventricular phenotype however, and effects of TASK-1 inactivation on atrial structure and function have yet to be demonstrated in vivo. The extent to which genetic variation in KCNK3, that encodes TASK-1, might be a determinant of susceptibility to AF is also unknown. To address these questions, we first evaluated the effects of transient knockdown of the zebrafish kcnk3a and kcnk3b genes and cardiac phenotypes were evaluated using videomicroscopy. Combined kcnk3a and kcnk3b knockdown in 72 hour post fertilization embryos resulted in lower heart rate (p<0.001), marked increase in atrial diameter (p<0.001), and mild increase in end-diastolic ventricular diameter (p=0.01) when compared with control-injected embryos. We next performed genetic screening of KCNK3 in two independent AF cohorts (373 subjects) and identified three novel KCNK3 variants. Two of these variants, present in one proband with familial AF, were located at adjacent nucleotides in the Kozak sequence and reduced expression of an engineered reporter. A third missense variant, V123L, in a patient with lone AF, reduced resting membrane potential and altered pH sensitivity in patch-clamp experiments, with structural modeling predicting instability in the vicinity of the TASK-1 pore. These in vitro data suggest that the double Kozak variants and V123L will have loss-of-function effects on ITASK. Cardiac action potential modeling predicted that reduced ITASK prolongs atrial action potential duration, and that this is potentiated by reciprocal changes in activity of other ion channel currents. Our findings demonstrate the functional importance of ITASK in the atrium and suggest that inactivation of TASK-1 may have diverse effects on atrial size and electrophysiological properties that can contribute to an arrhythmogenic substrate.

Kinetics of drug interaction with the Kv11.1 potassium channel.

The Kv11.1 potassium channel is the molecular target for the majority of drugs implicated in acquired long QT syndrome, the most common cause of drug-induced sudden cardiac death, and a common reason for drug restriction or withdrawal from the market. While the IC50 for block of Kv11.1 is commonly used to estimate the risk of acquired long QT syndrome, this approach is crude, and it is widely accepted that the kinetics of drug interactions with the channel are a critical component in understanding their mechanism of action and risk profiles. In this study we report the first directly measured kinetics of block and unblock of Kv11.1 by a QT prolonging drug: the antipsychotic clozapine. Our data show that clozapine binding to Kv11.1 is complex. There are at least two kinetically distinct components to both block and unblock, while the kinetics of unblock are dependent on the dose or duration of drug application. Based on these observations, we have proposed a model incorporating kinetically distinct binding to the open and inactivated states of Kv11.1 that can describe the observed kinetic features of clozapine block and correctly predict the overall affinity and apparent nonstate-dependent interaction of clozapine with Kv11.1. Mechanistic insights into drug block of Kv11.1 gained though detailed kinetic analyses such as this have a potential role in development of drugs targeted to specific channel states to reduce unwanted side effects, as well as in the design of better high-throughput preclinical tests for assessing the proarrhythmic effects of QT prolonging drugs.

Trafficking defects in PAS domain mutant Kv11.1 channels: roles of reduced domain stability and altered domain-domain interactions.

Loss of Kv11.1 potassium channel function is the underlying cause of pathology in long-QT syndrome type 2, one of the commonest causes of sudden cardiac death in the young. Previous studies have identified the cytosolic PAS (Per/Arnt/Sim) domain as a hotspot for mutations that cause Kv11.1 trafficking defects. To investigate the underlying basis of this observation, we have quantified the effect of mutants on domain folding as well as interactions between the PAS domain and the remainder of the channel. Apart from R56Q, all mutants impaired the thermostability of the isolated PAS domain. Six mutants, located in the vicinity of a hydrophobic patch on the PAS domain surface, also affected binding of the isolated PAS domain to an N-terminal truncated hERG (human ether-a-go-go-related gene) channel. Conversely, four other surface mutants (C64Y, T65P, A78P and I96T) and one buried mutant (L86R) did not prevent the isolated PAS domain binding to the truncated channels. Our results highlight a critical role for interactions between the PAS domain and the remainder of the channel in the hERG assembly and that mutants that affect PAS domain interactions with the remainder of the channel have a more severe trafficking defect than that caused by domain unfolding alone.

A transgenic zebrafish model of a human cardiac sodium channel mutation exhibits bradycardia, conduction-system abnormalities and early death.

The recent exponential increase in human genetic studies due to the advances of next generation sequencing has generated unprecedented numbers of new gene variants. Determining which of these are causative of human disease is a major challenge. In-vitro studies and murine models have been used to study inherited cardiac arrhythmias but have several limitations. Zebrafish models provide an attractive alternative for modeling human heart disease due to similarities in cardiac electrophysiology and contraction, together with ease of genetic manipulation, external development and optical transparency. Although zebrafish cardiac mutants and morphants have been widely used to study loss and knockdown of zebrafish gene function, the phenotypic effects of human dominant-negative gene mutations expressed in transgenic zebrafish have not been evaluated. The aim of this study was to generate and characterize a transgenic zebrafish arrhythmia model harboring the pathogenic human cardiac sodium channel mutation SCN5A-D1275N, that has been robustly associated with a range of cardiac phenotypes, including conduction disease, sinus node dysfunction, atrial and ventricular arrhythmias, and dilated cardiomyopathy in humans and in mice. Stable transgenic fish with cardiac expression of human SCN5A were generated using Tol2-mediated transgenesis and cardiac phenotypes were analyzed using video microscopy and ECG. Here we show that transgenic zebrafish expressing the SCN5A-D1275N mutation, but not wild-type SCN5A, exhibit bradycardia, conduction-system abnormalities and premature death. We furthermore show that SCN5A-WT, and to a lesser degree SCN5A-D1275N, are able to compensate the loss of endogenous zebrafish cardiac sodium channels, indicating that the basic pathways, through which SCN5A acts, are conserved in teleosts. This proof-of-principle study suggests that zebrafish may be highly useful in vivo models to differentiate functional from benign human genetic variants in cardiac ion channel genes in a time- and cost-efficient manner. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".

Quantifying the origins of population variability in cardiac electrical activity through sensitivity analysis of the electrocardiogram.

Altered function of ion channels in the heart can increase the risk of sudden arrhythmic death. Hundreds of genetic variants exist in these cardiac ion channel genes. The challenge is how to interpret the effects of multiple conductance perturbations on the complex multi-variable cardiac electrical system? In theory, sensitivity analysis can address this question. However, to date this approach has been restricted by computational overheads to analysis of isolated cells, which has limited extrapolation to physiologically relevant scales. The goal of this study was to extend existing sensitivity analyses to electrocardiogram (ECG) signals derived from multicellular systems and quantify the contribution of ionic conductances to emergent properties of the ECG. To achieve this, we have developed a highly parallelised simulation environment using unconventional high performance computing architectures to analyse the emergent electrical properties of a multicellular system. This has permitted the first systematic analysis of the molecular basis of the T wave amplitude, revealing important but distinct roles for delayed rectifier and inward rectifier K(+) currents. In addition to quantifying how interactions between multiple ion channels influence ECG parameters we show that these sensitivities are dynamic functions of heart rate. This study provides a significant advance in our understanding both of how individual ion conductances define ECG signals and of epistatic modification of cardiac electrical phenotypes. The parallelised simulation environment we have developed removes the computational roadblock that has limited this approach and so provides the framework for future analysis of more complex tissue and whole organ systems.

Five Shapes of Cognitive Dissonance - Using Objective Hermeneutics to Understand the Meat Paradox.

Objective Hermeneutics is a qualitative method that focuses on few sequences of texts, which helps understand single cases. It is used to explore how consumers cope with the contradiction between their enjoyment for meat and their empathy for animals without using frameworks drafted by social scientists. Five cases are analysed, which range from strong references towards the societal norm of meat eating to a feeling of uncertainty in the face of the animals' death. None of the cases, however, sees the necessity to find a rationale for their own meat consumption. The study concludes by raising the question about the persistence of a societal consensus for meat consumption.

Udder health, veterinary costs, and antibiotic usage in free stall compared with tie stall dairy housing systems: An optimized matching approach in Switzerland.

Observational studies are important in livestock science. As treatment is not assigned randomly in such studies, selection bias can be a problem. This is often addressed by matching methods. However, if treatment and control groups differ considerably in their characteristics, it might be necessary to additionally prune observations that lack overlap in the opposite group. "Matching Frontier" method was developed because pruning observations manually often results in suboptimal solutions. The feasibility of the approach for animal health and welfare issues was tested in an observational study evaluating the effect of free stall housing and increased lying comfort on udder health, veterinary costs, and antibiotic usage in Swiss dairy farming. Data were collected in a survey with 1835 Swiss dairy farmers (response rate 28.3%). The treatment group (n = 179) comprised farmers participating in a voluntary animal welfare program that, in addition to free stall housing, required increased lying comfort. Farmers in the control group (n = 229) kept their cows in tie stalls. Using the Matching Frontier method, treated units were matched to control units based on five confounders. Subsequently, observations were pruned to achieve sufficient balance and overlap between the two groups. The effect of the program on the eight outcome variables was finally estimated using linear regression. Farmers in the treatment group had a lower incidence of clinical mastitis (-3.66 per 100 cow-years, -25%, p < 0.05), a lower incidence of culled cows due to udder health problems (-1.61 per 100 cow-years, -30%, p < 0.05), fewer veterinary costs (-42.44 per cow-year, -22%, p < 0.05), a lower incidence of total intramammary antibiotic treatments (-15.88 per 100 cow-years, -23%, p < 0.01), a lower incidence of intramammary antibiotic treatments for mastitis therapy (-7.83 per 100 cow-years, -32%, p < 0.01), and a lower incidence of intramammary antibiotic treatments for dry-cow therapy (-8.80 per 100 cow-years, -21%, p < 0.05). No differences were found for the average somatic cell count and the number of cows with a cell count above 150.000. The results suggest that free stall housing, in combination with increased lying comfort, can have a positive effect on udder health, animal welfare, and the economic situation of the farm. Additionally, fewer antibiotic treatments can be beneficial to public health. The Matching Frontier method has proven to be a helpful tool that may also have added value for future observational studies in livestock science.

The twin arginine protein transport pathway exports multiple virulence proteins in the plant pathogen Streptomyces scabies.

Summary Streptomyces scabies is one of a group of organisms that causes the economically important disease potato scab. Analysis of the S. scabies genome sequence indicates that it is likely to secrete many proteins via the twin arginine protein transport (Tat) pathway, including several proteins whose coding sequences may have been acquired through horizontal gene transfer and share a common ancestor with proteins in other plant pathogens. Inactivation of the S. scabies Tat pathway resulted in pleiotropic phenotypes including slower growth rate and increased permeability of the cell envelope. Comparison of the extracellular proteome of the wild type and DeltatatC strains identified 73 predicted secretory proteins that were present in reduced amounts in the tatC mutant strain, and 47 Tat substrates were verified using a Tat reporter assay. The DeltatatC strain was almost completely avirulent on Arabidopsis seedlings and was delayed in attaching to the root tip relative to the wild-type strain. Genes encoding 14 candidate Tat substrates were individually inactivated, and seven of these mutants were reduced in virulence compared with the wild-type strain. We conclude that the Tat pathway secretes multiple proteins that are required for full virulence.

Lanterns in Marrakesh.

Description This photo was taken in Marrakesh, Morocco in 2014. I decided to travel there after a few friends inspired me with their descriptions of a beautiful desert-filled country with kind people, mountains and incredibly delicious food. For me, traveling is truly food for the soul. It allows you to break out of the monotony of work-life and potentially step outside of your comfort zone. I absolutely love exploring various cultures and over the course of my life I have traveled to over thirty countries and have back-packed numerous countries solo. There's no better feeling than embarking on an adventure free of rules and schedules, meeting people from all walks of life and cultures and exploring the beauty of our planet. My days of photography really started with making films of my travels. Capturing special moments with the camera produces a memory that will live on forever. I enjoy making films and choosing music for films almost as much as I enjoy practicing medicine! Photography is a challenging art and science. It takes years to master DSLR cameras and editing programs, but nowadays you really don't need any of that to capture a special moment, all you need is your phone! I highly recommend stepping out of your comfort zone and hitting the road. And if the time is right, get the camera out so you and your family can relive all of the special moments that make up your life.

The Old Man and the Meat: On Gender Differences in Meat Consumption across Stages of Human Life.

It is not a radically new insight that men eat more meat than women do. However, one piece of the puzzle was previously missing: the development of a gender bias in total and red meat consumption across stages of human life. To identify the gender bias across stages of human life, we apply a multiple-group regression across seven age classes. Data for the empirical analysis stem from the US National Health and Nutrition Examination Survey. Regression results reveal that gender differences in meat consumption start only after the age of four and then move in some parallel with the development of biological differences, reaching a maximum between 51 and 65 years. The effect of both household income and education on meat consumption is negative and per-capita consumption of meat rises with household size.

Assessment of proarrhythmogenic risk for chloroquine and hydroxychloroquine using the CiPA concept.

Chloroquine and hydroxychloroquine have been proposed recently as therapy for SARS-CoV-2-infected patients, but during 3 months of extensive use concerns were raised related to their clinical effectiveness and arrhythmogenic risk. Therefore, we estimated for these compounds several proarrhythmogenic risk predictors according to the Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm. Experiments were performed with either CytoPatch™2 automated or manual patch-clamp setups on HEK293T cells stably or transiently transfected with hERG1, hNav1.5, hKir2.1, hKv7.1+hMinK, and on Pluricyte® cardiomyocytes (Ncardia), using physiological solutions. Dose-response plots of hERG1 inhibition fitted with Hill functions yielded IC50 values in the low micromolar range for both compounds. We found hyperpolarizing shifts of tens of mV, larger for chloroquine, in the voltage-dependent activation but not inactivation, as well as a voltage-dependent block of hERG current, larger at positive potentials. We also found inhibitory effects on peak and late INa and on IK1, with IC50 of tens of µM and larger for chloroquine. The two compounds, tested on Pluricyte® cardiomyocytes using the ß-escin-perforated method, inhibited IKr, ICaL, INa peak, but had no effect on If. In current-clamp they caused action potential prolongation. Our data and those from literature for Ito were used to compute proarrhythmogenic risk predictors Bnet (Mistry HB, 2018) and Qnet (Dutta S et al., 2017), with hERG1 blocking/unblocking rates estimated from time constants of fractional block. Although the two antimalarials are successfully used in autoimmune diseases, and chloroquine may be effective in atrial fibrillation, assays place these drugs in the intermediate proarrhythmogenic risk group.